dicle university, diyarbakır, turkeymokad.org/congress.pdf · 2019-10-10 · dicle university,...

Oral presentation

1st INTERNATIONAL MULTIDISCIPLINARY

CANCER RESEARCH CONGRESS

18-22 September, 2019

Culture and Congress Centre

Dicle University, Diyarbakır, Turkey

CONGRESS ABSTRACT BOOK

The congress is a bilingual congress in Turkish and English.

www.mokad2019.com

Dear Cancer Researchers,

You are cordially invited to participate in the 1st International Multidisciplinary Cancer

Research Congress to be held in Diyarbakır, Turkey on 18 to 22 September 2019.

The Congress provides a platform for young and renowned researches and clinicians come

together to discuss, present and showcase high-quality research and exchange of ideas on a

range of fundamental and applied topics in the area of current cancer research.

As a member of EACR -European Association for Cancer Research, MOKAD committee has

decided to organize a meeting at the international level. 1st International Multidisciplinary

Cancer Research Congress is the internationally boardened version of the national MOKAD

congress series called “Multidisciplinary Cancer Reasearh Congress”.

Young researchers will have ample opportunity to present their own research during open slots

for short talks and poster sessions. Interactions with cancer field scientists, during coffee breaks

and social events will give chance for young scientist candidates to improve their research

subjects and broaden their horizon. Among the young applicants, the ones who bring excellent

research with excellent presentation skills during short talks and poster sessions, will be

awarded!

1st International Multidisciplanary Cancer Research Congress involves two days practical

course which is intended to train students on basic cell culture and cell death detection

techniques. Students attending the course will have interactive environment with the well

experienced professional trainers who can answer the raised questions about the details of

experimental process.

The congress is held in Diyarbakır, a city hosted 26 different civilisations over 5000 years, a

city the cradle of cultures. Diyarbakır has unique standing with the strong multicultural

inhabitants and spectacular landscape. We believe that Diyarbakır’s fantastic atmosphere with

rich history will inspire young and experienced researchers and create an interactive, and

fruitful platform to discuss cancer research.

We are looking forward to an exciting and memorable scientific meeting and welcome you in

Diyarbakır, Turkey.

On Behalf of Organising Committee

Local head of organizing committee Head of Congress

Prof. Dr. Beran Yokuş Prof. Dr. Engin Ulukaya

2

1st INTERNATIONAL MULTIDISCIPLINARY CANCER RESEARCH CONGRESS

18-22 September, 2019

18 September 2019 (Course in Turkish)

Kurs 1.Gün: Kursiyerler iki gruba ayrılacaklar: Grup A ve Grup B

Kurs, sitotoksisite ve hücre ölüm modu (apoptozis ve nekrozis) değerlendirme yöntemlerini

kapsamaktadır.

Grup A: 09:00 – 13:00

09:00-09:30 Dr. Öğr. Üyesi Didem Karakaş

• Genel Giriş

09:30-13:00 Dr. Öğr. Üyesi Didem Karakaş

• Dondurulmuş hücrenin aktivasyonu ve flaska ekilmesi

• Total ve canlı hücre sayımları, hücre sayılarının hesaplaması ve pasajlama

• İlaç hazırlanması, konsantrasyon hesabı ve hücrelere ilaç uygulaması

12:00-14:00 Öğle Yemeği (Lunch box)

Grup B: 13:00 – 17:00

13:00-13:30 Dr. Öğr. Üyesi Nazlıhan Aztopal

• Genel Giriş

13:30-17:00 Dr. Öğr. Üyesi Nazlıhan Aztopal

• Dondurulmuş hücrenin aktivasyonu ve flaska ekilmesi

• Total ve canlı hücre sayımları, hücre sayılarının hesaplaması ve pasajlama

• İlaç hazırlanması, konsantrasyon hesabı ve hücrelere ilaç uygulaması

19 September 2019 (Course in Turkish)

Kurs 2.Gün: Grup A+B: Dr. Öğr. Üyesi Nazlıhan Aztopal, Dr. Öğr. Üyesi Didem Karakaş

09:00-09:30

• Apoptozisin M30 (kaspazla kırılmış sitokeratin 18) ELISA testi ile tayini

09:30-11:30

• SRB canlılık testi

11:30-12:30 Öğle Yemeği (Lunch box)

12:30-13:00

• M30 ELISA testi sonuç alma

3

13:00-16:00

• Hücre canlılığı, büyüme hızı hesaplamaları, ilgili parametrelerin tanımlanmaları*

• M30 elisa testi sonuç analizi (Grafik çizimi ve değerlendirme*)

• Hücre ölüm modunun floresan boyama ile tayini (erken ve geç apoptozis ile birlikte

nekrozisin nükleus ve hücre membranı temelinde ayırt edilmesi)

16:00-17:00 Prof. Dr. Engin Ulukaya

Sitotoksisite/Hücre ölüm modu çalışmalarında önemli detaylar, yapılan hatalar!

* Öğrencilerin kişisel bilgisayarları ile katılımı önerilmektedir.

20 September 2019 Social Programme

21 September 2019 (1st Day of the Congress)

8:30-9:00 Registration

9:00 Opening Ceremony

9:15 Opening Lecture / Keynote Lecture

Chairs: Hana Alguel,

Bülent Özpolat / Development of targeted therapies in cancer – A historical perspective –

past present and future

10:00-11:00 Special Session: Munich Technical University

Chairs: Hatice Mehtap Kutlu, Engin Ulukaya

Hana Alguel / From bench to bed: novel concepts in mutant KRAS driven cancers

Proffered Paper:

Kıvanç Görgülü / Autophagy and its role in pancreatic cancer

*the speaking sessions of the invited speakers and the oral presentations will be carried out

parallelly in Congress Hall D and Congress Hall E respectively. (Davetli konuşmacı sunumları

(Salon D) ve sözlü sunumlar (Salon E) paralel olarak gerçekleşecektir.)

11:00-11:15 Coffee Break

11:15-12:15 Session 1:

Chairs: Burçin Güngör, Gamze Tanrıöver

Maryam Moghaddam-Matin / Targeted therapy in colorectal cancer

Burçin Güngör / Protein lipidation on cancer metabolism

Sema Yılmaz / Molecular diagnosis in childhood cancers, from molecular side to bed side

4

12:15-13:15 Lunch Break

13:15-14:15 Session 2:

Chairs: Süreyya Bozkurt, Konstantinos Dimas

Nihal Karakaş / Glioblastoma multiforme beyin tümörünün erken tanı ve tedavisine yönelik

otoantikor çalışmaları

Halil Kavaklı / A molecule that desablize Cryptochrome 1 enhances apoptosis in cancer cells

Ceyda Açılan Ayhan / New therapeutic approaches targeting MLL-AF9 leukemia through

epigenetic reprogramming


14:30-15:50 Session 3:

Chairs: Beran Yokuş, Ebru İnce Bostancı

Alexandre Tavartkiladze / Personalized vaccines and CAR-T cell for cancer immunotherapy

Güneş Esendağlı / Cancer cells adaptation to immune responses

Konstantinos Dimas / Mouse models for immunotherapeutic approaches

Süreyya Bozkurt / Diagnostic and prognostic importance of cytogenetics in hematologic

cancers


16:05-17:25 Session 4:

Chairs: Mehmet Küçüköner, Dilek Ülker Çakır

Abdurrahman Şengül / Antikanser Çalışmalarında Platin Koordinasyon Bileşiklerinin Rolü

Gülperi Öktem / Kanser kök hücrede güncel tedavilere yaklaşım

Ata Özçimen / Aurora Kinases and Cancer

Şeyma Demirsoy / A Parkinson-related gene in melanoma: Exploringthe role of the P5

typeATPase ATP13A2, a Parkinson disease’sassociated gene, in melanoma cell proteostasis

17:30-18:30 Poster Session

20:00 Gala Dinner (Traditional Diyarbakır Night Event)

5

22 September 2019 (2nd Day of the Congress)

10:00-11:15 Session 5 Young Stars are Talking

Chairs: Engin Ulukaya, Ceyda Açılan Ayhan

Seçil Demirkol Canlı / A novel transcriptomic based gene panel for the prognostication of

pancreatic cancer

Burcu Şengez / The Transcription Factor Elf3 is Essential for a Successful Mesenchymal to

Epithelial Transition

Nazlıhan Aztopal / Kanser kök hücre ve epigenetik yaklaşımlar

Didem Karakaş / Pankreatik Kanserde Tümör Mikroçevresinin Hedeflenmesi

Şeyma Aydınlık / Hedefe yönelik tedavide kansere özgü moleküler mekanizmaların

aydınlatılması

11.15-11.30 Coffee Break

11.30-12.50 Session 6:

Chairs: Ceyhun Bozkurt, Hasan İçen

Ebru İnce Bostancı / Yeni antikanser ilaç keşfinde genomik ve metabolomik: fırsatlar ve

zorluklar

Dilek Odacı Demirkol / Akıllı İlaç taşıyıcı sistemlerin kanser hücrelerinin hedeflenmesinde

kullanımına genel bakış

Dilek Ülker Çakır / Nanopartiküllerin biyokimyasal ve hematolojik etkileri

Hasan İçen / Hayvanlarda görülen multiple myelomalar

12.50-14.00 Lunch time

14.00-15.00 Session 7:

Chairs: Elif İkitimur Armutak, İlhan Yaylım

Mehmet Küçüköner / Kanserde çığır açan moleküler hedef tedaviler

Ferda Arı / Kanser Tedavisinde Histon Deasetilaz İnhibitorleri

Closing Lecture: Tuba Günel / Over kanserinde liquid biyopsi ile elde edilen moleküllerle

biyobelirteç çalışmaları

15.00-16.00 Award ceremony and closing

6

Congress Chair

Engin Ulukaya

Congress Co-Chair

Beran Yokus

Course Instructors

Didem Karakaş

Nazlihan Aztopal

Scientific Secreteriat

Burçin Güngör

Melda Sarıman

Selim Karahan

Organising Committee

Alexander Tavartkiladze (Georgia)

Beran Yokuş

Bircan Çeken Toptancı

Bülent Özpolat (USA)

Engin Ulukaya

Hana Alguel (Germany)

Kıvanç Görgülü (Germany)

Konstantinos Dimas (Greece)

Maryam Moghaddam-Matin (Iran)

Local Organising Committee

Beran Yokuş

Ebru İnce Bostancı

İlhan Sabancılar

Mehmet Hüseyin Alkan

Neval Berrin Arserim

Selçuk Tunik

Selim Karahan

Sevcan İzgi

Scientific Committee

Abdurrahman Şengül

Alexandre Tavartkiladze

Ata Özçimen

Azmi Yerlikaya

7

Batu Erman

Burcu Şengez

Burçin Güngör

Buse Cevatemre

Bülent Özpolat

Ceyda Açılan Ayhan

Ceyhun Bozkurt

Devrim Gözüaçık

Didem Karakaş

Dilek Ülker Çakır

Dilek Odacı Demirkol

Ebru İnce Bostancı

Egemen Dere

Elif İkitimur Armutak

Erdal Karaöz

Ferda Arı

Funda Acar Yağcı

Gülperi Öktem

Güneş Esendağlı

Halil Kavaklı

Hana Alguel

Hasan İçen

İlhan Yaylım

Kıvanç Görgülü

Konstantinos Dimas

Maryam Moghaddam-Matin

Mehmet Hüseyin Alkan

Mehmet Küçüköner

Mehmet Sarımahmut

Meryem Alagöz

Milica Pesic

Nazlıhan Aztopal

Neval Berrin Arserim

Nihal Karakaş

Nuray Erin

Seçil Demirkol Canlı

Selçuk Tunik

Sema Yılmaz

Serap Çelikler

Sonja Levenat

Süreyya Bozkurt

Şeyma Aydınlık

Tolga Sütlü

Tuba Günel

Ümit Zeybek

Veysel Turan Yılmaz

Yasemin Delen Akçay

8

1st International Multidisciplanary Cancer Research Congress covers a wide rage of cancer field

topics including:

Biosimilars

Cancer immunotherapy

Cancer stem cells

CAR-NK Cells

Cell death (apoptosis/necrosis and

autophagy) in cancer

DNA damage

Epigenetics and cancer

Epithelial-mesenchymal transition

Exosomes in cancer

Kinases in cancer

KRAS in oncology

Lipids in cancer, lipidomics

Liquid biopsy: Future of oncology

Nanoparticles against cancer

Novel anticancer small molecules

Precision oncology

SPEAKER UNIVERSITY TITLE PAGE

Bülent Özpolat MD Anderson

Cancer Center, USA

Development of targeted

therapies in cancer – A historical

perspective – past present and

future

15

Hana Alguel Technical University

of Munich, Germany

From bench to bed: novel

concepts in mutant KRAS

driven cancers

16

Kıvanç Görgülü Technical University

of Munich, Germany

Autophagy and its role in

pancreatic cancer 17

Maryam

Moghaddam-Matin

Ferdowsi University,

Iran

Targeted therapy in colorectal

cancer 18

Burçin Güngör İstinye University,

Istanbul

Protein lipidation on cancer

metabolism 19

Sema Yılmaz Yeditepe University,

Istanbul

Molecular diagnosis in

childhood cancers, from

molecular side to bed side

20

Nihal Karakaş Medipol University,

Istanbul

Glioblastoma multiforme beyin

tümörünün erken tanı ve

tedavisine yönelik otoantikor

çalışmaları

22

Halil Kavaklı Koç University,

Istanbul

A molecule that destabilize

Cryptochrome 1 enhances

apoptosis in cancer cells

24

Ceyda Açılan Ayhan Koç University,

Istanbul

New therapeutic approaches

targeting MLL-AF9 leukemia

through epigenetic

reprogramming

25

9

Alexandre

Tavartkiladze

Tblisi State Medical

University, Georgia

Personalized vaccines and CAR-

T cell for cancer immunotherapy 27

Güneş Esendağlı Hacettepe

University, Ankara

Cancer cells adaptation to

immune responses 28

Konstantinos Dimas University of

Thessaly, Greece

Mouse models for

immunotherapeutic approaches 29

Süreyya Bozkurt İstinye University,

Istanbul

Diagnostic and prognostic

importance of cytogenetics in

hematologic cancers

30

Abdurrahman

Şengül

Bülent Ecevit

University,

Zonguldak

Antikanser Çalışmalarında

Platin Koordinasyon

Bileşiklerinin Rolü

31

Gülperi Öktem Ege University,

Izmir

Kanser kök hücrede güncel

tedavilere yaklaşım 32

Ata Özçimen Mersin University,

Mersin Aurora Kinases and Cancer 34

Şeyma Demirsoy Hacettepe

University, Ankara

A Parkinson-related gene in

melanoma: Exploringthe role of

the P5 typeATPase ATP13A2, a

Parkinson disease’sassociated

gene, in melanoma cell

proteostasis

37

Seçil Demirkol Canlı Hacettepe

University, Ankara

A novel transcriptomic based

gene panel for the

prognostication of pancreatic

cancer

38

Burcu Şengez

International

Biomedicine and

Genom Institute

(IBG), Izmir

The Transcription Factor Elf3 is

Essential for a

Successful Mesenchymal to

Epithelial Transition

39

Nazlıhan Aztopal Istinye University,

Istanbul

Kanser kök hücre ve epigenetik

yaklaşımlar 40

Didem Karakaş İstinye University,

Istanbul

Pankreatik Kanserde Tümör

Mikroçevresinin Hedeflenmesi 41

Şeyma Aydınlık Tübitak, Gebze

Hedefe yönelik tedavide kansere

özgü moleküler mekanizmaların

aydınlatılması

43

Ebru İnce Bostancı Dicle University,

Diyarbakır

Yeni antikanser ilaç keşfinde

genomik ve metabolomik:

fırsatlar ve zorluklar

44

Dilek Odacı

Demirkol

Ege University,

Izmir

Akıllı İlaç taşıyıcı sistemlerin

kanser hücrelerinin 45

10

hedeflenmesinde kullanımına

genel bakış

Dilek Ülker Çakır

Çanakkale Onsekiz

Mart University,

Çanakkale

Nanopartiküllerin biyokimyasal

ve hematolojik etkileri 46

Hasan İçen Dicle University,

Diyarbakır

Hayvanlarda görülen multiple

myelomalar 48

Mehmet Küçüköner Dicle University,

Diyarbakır

Kanserde çığır açan moleküler

hedef tedaviler 49

Ferda Arı Uludağ University,

Bursa

Kanser Tedavisinde Histon

Deasetilaz İnhibitorleri 51

Tuba Günel İstanbul University,

Istanbul

Over kanserinde liquid biyopsi

ile elde edilen moleküllerle

biyobelirteç çalışmaları

53

Oral Presentations

SPEAKER TITLE PAGE

Adnan Ayhanci

Stone Alkaline Water Induces Apoptosis of

Prostate Cancer Cells and Inhibits Tumor Cell

Induced Angiogenesis In Vitro

55

Asli Kurden Pekmezci MALT1 Paracaspase Inhibition Reduces

Hepatocellular Carcinoma Cell Survival 56

Ayca Üvez

In Vitro and In Vivo Investigation of Mangiferin

and Paclitaxel combination on Ehrlich Ascites

Carcinoma

57

Aydın Demiray Akciğer Kanseri ve KOAH’da Biyobelirteç Olarak

Aday miRNA Araştırması 59

Baran Erman Underlying Primary İmmunodeficiency in Patients

with Lymphoma 60

Berkcan Doğan

Determination of MiRNAs and Xeno-miRs

Targeting Most of the Genes Associated with

Colorectal Neoplasms and Central Obesity by "in

silico" Analysis

62

Burcu Salman Investigation of DHRS2 Gene Effects on Breast

Cancer Cell Line 63

Canan Vejselova Sezer

Bir Seramidaz İnhibitörü olan N-

Oleoylethanolamine Bileşiğinin A549 ve Beas-2B

Hücreleri Üzerindeki Sitotoksik ve Morfolojik

Değişimlerinin Araştırılması

65

Çevik Gürel

Doksorubisin İle İndüklenen Sıçan Nefrotoksisite

Modelinde Fluvastatinin Olası Koruyucu ve

Tedavi Edici Etkilerinin Histokimyasal ve

İmmünohistokimyasal Olarak Değerlendirilmesi

67

11

Cihangir Yandım Abnormal Expression Patterns of Repetitive DNA

Elements Uncovered in Breast Cancer 68

Damla Uludağ

Hedefe Yönelik Tedaviler İçin Çeşitli Kanser

Hücrelerinde IL13Rα2 Ekspresyonunun

Belirlenmesi

69

Dehan Çömez

Fluidic Shear Stress Regulates Biological

Behaviours of Hepatocellular Carcinoma Cells

Through c-Met Signalling

70

Derya Akçiçek

Investigation of the Anticancer Effect of

Haplophyllum Buxbaumii Plant on HCT-116

Colon Cancer Cells

71

Dilek Pirim

Identification of Shared Candidate Key Genes and

Pathways Associated with Cancer and Heart

Failure Pathogenesis Using Bioinformatic

Analyses

72

Ebru Temiz Investigation of Anti-cancer Properties of

Haplophyllum Ptilostylum Plant 73

Ece Gümüşoğlu The İmportance of Dysregulated miRNAs on

Ovarian Cysts and Tumors 74

Elif Merve Aydın

Investigation of Helicobacter Pylori Virulence

Factors and İmmune Response in Precancerous

Lesions

75

Esra Arslan Ateş

A Useful Tool for Distinguishing

Gene/Pseudogene Variants Detected via NGS:

Haplotype Analysis

76

Esra Bozgeyik

Identification of lncRNA Molecules İnvolved in

the Regulation of EGF-İnduced of Cellular

Proliferation

77

Feryal Akay

Morus Nigra (Karadut) Yaprağı Etanol

Ekstraktının Doğal bir DNA İleri Glikasyon Son

Ürünleri (DNA-AGE'ler) Bileşik Kaynağı Olarak

Değerlendirilmesi

78

Gamze Tan

The Antiproliferative and Apoptotic Effects of

Gold Nanoparticles Synthesized by Allium Cepa

Extract on Leukemia Cell Lines

80

Gülsün Bağcı The Role of c-Met Signaling in Organ Specific

Extravasation of HCC cells 81

Gürcan Günaydın The Effects of Cancer Associated Fibroblasts on

Tumor Associated Macrophages 82

Gülşah Torkay Cryptonin An Antimicrobial Peptide from Cicada

and its Effects on Melanoma Cell Lines 84

İbrahim Halil Kılıç

Güneydoğu Anadolu Bölgesinde Yetişen Patlıcan

Çeşitlerinin Kabuklarının Sitotoksik Etkilerinin

Saptanması

85

Hande Özkan

How Dose and Duration of Exposure Affect the

Rate of Acquired Resistance to Doxorubicin in

Gastric Cancer?

86

Hatice Mehtap Kutlu İnsan Meme Kanseri Hücrelerinde D-erythro-

MAPP’ın Antikanser Özelliklerinin Araştrılması 87

Hüseyin Özevren Experimental Traumatic Brain Injury Models 88

12

Işık Didem Karagöz In vitro Self-renewal Properties in A549 and

H1299 Non-Small Cell Lung Cancer Cells 89

Işıl Yıldırım

Immunohistochemical Determination of

Glutathione-S-Transferase Isoenzym Family

Expression in Invasive Lobular Breast Cancer

Tissue

90

İsmail Korkmaz Karadutun (Morus nigra) Anti Kanser Etkilerinin

Araştırılması 91

Kadir Eği The Investigation of Anticancer Effect of CEDAR

TAR Prepared by Traditional Methods 92

Lütfiye Kadıoğlu Dalkılıç

Mide Kanserinin Teşhisinde Kullanılabilecek

Biyobelirteç Adayı Genlerin Bİyoinformatik

Araçlar İle Belirlenmesi

93

Mehmet Tahir Hüsunet

L-Askorbik Asit’in (Vitamin C) İnsan

Promiyelösitik Kanser Hücre Hattında (HL-60)

MTT Testi ile Araştırılması

95

Melda Sarıman

Investigation of TMED9-ERAP1 Candidate Gene

Expressions Obtained from Multiple Myeloma

Transcriptome Data by RT-PCR

96

Melike Bayındır Bilgiç The Effects of The LIF Antagonist Molecule

(EC359) on the Chordoma Cells 97

Merve Nur Ataş Investigation of Apoptotic Effect of Betulinic

Acid in Renal Cancer Cells 98

Mümin Alper Erdoğan

Selective Cannabinoid-1/2 Receptor (CB1 and

CB2) Agonists Suppress Cell Proliferation and

Clonogenicity in Pancreatic and Breast Cancer

Cells

99

Murat Ferhat Ferhatoğlu

Usefulness of Simple Prognostic Markers of

Complete Blood Count to Predict Lymph Node

Metastasis in Colon Carcinoma

100

Necla Birgül İyison Characterization of BRI3 as a Novel Wnt/β-

catenin Pathway Target 101

Nur Ekimci Gürcan Sub-Culturing Tumorspheres Reduced the

Pluripotency of a Chordoma Cell Line 102

Onur Dirican Beyin Tümörü Dokularında CYP1A1, CYP1B1,

GST-M, GST-P ve MDR Proteinlerinin Rolü 103

Özen Leylek

Defining the Synergistic Sequential Application

Schedules for Chemotherapeutic–Molecular

Targeted Agent Dual Combinations in Gastric

Adenocarcinoma Cells

105

Özge Akbulut Targeting TACC3 as a Novel Therapeutic

Strategy for Breast Cancer Treatment 107

Özlem Demirci DNA Damaging Activities of Terbuthylazine 109

Pınar Kaygın hBD-1 ve hBD-3 Antimikrobiyal Peptidlerin

Ürotelyal Kanser Dokularındaki Ekspresyonları 110

Seda Baykal-Köse

Investigation of the Relationship Between Cellular

Adaptation and Therapeutic Resistance in Chronic

Myeloid Leukemia Cells under İmatinib Pressure

111

13

Selim Yalçın

Neutrophil-to-Lymphocyte Ratio and Platelet

Distribution Width Predicts Invasiveness of

Bladder Carcinoma

113

Semih Dalkılıç PİCOA VE TERFEZİA Türlerinin Anti-Kanser

Etkilerinin Araştırılması 115

Serap Sancar-Baş

The Effects of Mackia Amurensis

Leukoagglutinin on the Expressions of Some

Extracellular Matrix Proteins in Anaplastic

Thyroid Carcinoma Cell Line, 8505C

116

Sibel Bayıl Oğuzkan

An Investigation of the Antitumoral Activity of

Phycocyanin Derived from Spirulina (Arthrospira)

Platensis on Mice

117

Suna Bektaş

Effects of Maackia Amurensis Leukoaglutinin

Treatment on the Transcriptome of Human

Anaplastic Thyroid Cancer Cell Line 8505C

118

Süreyya Bozkurt Cytogenetic Anomalies in Lymphomas: A single

center study 119

Tevriz Dilan Demir

The Role of Nuclear P-glycoproteins in Nuclear

Sparing and Chemoresistance to Doxorubicin in

Gastric Adenocarcinoma Cell Models

120

Tuğba Güngör Synthesis, Antiinflammatory and Anticancer

Effects of Novel Amide Containing Compounds 122

Utku Özbey As a Potential Overall-Biomarker of Cancer Stem

Cells CD90: on Drug Resistance Perspective 124

Yeliz Yılmaz The Role of c-Met Neddylation in Hepatocellular

Carcinoma 125

Zelal Zuhal Kaya Keton Cisimlerinin in vitro İnsan Meme Kanseri

Hücrelerinin Canlılığını Azaltıcı Etkisi 126

Zeliha Emrence

Effect of Doxorubicin on Expression of RNA

m6A Methylation Enzymes in Leukemia Cell

Lines

127

Poster Presentations

Meltem Güleç Plants Used Against Cancer in Folk Medicine in

Turkey 129

Ayten Kılınçlı Targetıng Trem2 Reduces Prolıferatıon, Induces

Cell Death On Breast Cancer Cells 130

Bircan Çeken Toptancı

DNA Degradation by Water and Ethanol extracts

of Allium tuncelianum in the Presence of Copper

Ions: Implications for Anticancer Properties

131

Selahattin Can Özcan Role of NEK2A in PLK4 Induced Multipolar

Cell Divisions 132

Batuhan M. Kalkan Investigation of Nek2 Kinase Targets with Focus

on Centrosomal Unclustering 134

İpek Bulut

New Therapeutıc Approaches Targetıng Mll-Af9 Leukemıa Through Epıgenetıc Reprogrammıng

136

14

İsmail Koyuncu

Investigation of the free amino acid profile of

different breast cancer cell lines (MCF-7, MDA-

MB-231, and CRL-4010)

137

Elif Bozbulut

Hepatoselüler Karsinoma, Mide ve Kolorektal

Kanser Hastalarında Tedavi Öncesi ve Sonrası

Oksidatif Stresin Araştırılması

138

Gamze Tanriover

Cross-talk between tumor and stem cells:

determination of conditioned media cytokine

levels

139

Hatice DİNÇER

Yeni Sentezlenen Naftakinon Türevli

Bileşiklerin Üçlü Negatif Meme Kanseri

Hücreleri Üzerindeki Sitotoksik Etkilerinin

İncelenmesi

140

İlhan Yaylım

The Importance of FOXP3 C/A (rs:3761548) Genetic Variants and Protein Expressions in tumoral tissues of patients with Non-Small Cell Lung Cancer

141

Mehmet Emin Köse

Investigation of the Cell Cycle Specifity of Zn(II)

5,5-Diethylbarbiturate Complex on A-549 Human

Lung Cancer Cell Line

143

Ozyurt R Is Notch1 Associated with LeptinR, IL-6 and

TNF-α in the CRC Tumorigenesis? 144

Sayra Dilmac Effects of phosphorylation changes of E2F1 from

S235 on proliferation and migration 145

Selin Ulukaya The Role of p62 in Pancreatic Cancer

Development and Metastasis 146

Sedat Aydin

Expression levels of glutathione S transferase

isoenzymes in pleomorphic adenoma of the

parotid gland and normal gland

147

Sibel Gökşen New Orthotopic Mouse Colorectal Cancer Model 149

Invited speaker talk

15

Development of targeted therapies in cancer – A historical perspective – past present

and future

Bülent Özpolat

MD Anderson Cancer Center, USA


16

From Bench to Bed: Novel Concepts in Mutant KRAS Driven Cancers

Hana Algül

Comprehensive Cancer Center Munich, Technical University of Munich, Germany

e-mail: [email protected]

Mutations in the KRAS gene drive several common and deadly cancers. Unfortunately, most

mutant KRAS proteins cannot be targeted therapeutically. Among the mutant KRAS driven

cancers pancreatic ductal adenocarcinoma (PDAC) poses the most devastating malignancy.

Due to the late diagnosis, aggressive disease, and a lack of effective treatment options PDAC

is projected to become the second leading cause of cancer-related death by 2030. So far,

conventional oncology has failed to improve the outcome of patients with PDAC thus urging

for novel approaches. Fortunately, with the deeper understanding of the molecular basis of

PDAC new avenues and opportunities emerge at the horizon. Here, we will shed light on these

new opportunities and discuss new challenges to the implementation of an individualized

therapy approach in the context of precision medicine.


17

Levels of the Autophagy Related Gene 5 (Atg5) Determine Pancreatic Tumor Formation

and Metastasis in Mice

Kivanc Görgülü1*, Kalliope N. Diakopoulos1*, Marija Stevanovic1, Angeliki-Faidra

Karpathaki1, Jiaoyu Ai1, Derya Kabacaoglu1, Katrin J. Ciecielski1, Ezgi Kaya-Aksoy1,

Dietrich A. Ruess1, Sonja M. Wörmann1, Thomas Wartmann2, Yue Zhao2, Walter Halangk2,

Svetlana Voronina3, Alexey Tepikin3, Anna Artati4, Jerzy Adamski4 7, Michaela Aichler5,

Axel Walch5, Roland M. Schmid1, Martin Jastroch6, Götz Hartleben7, Stephan Herzig7, Bruno

Sainz Jr8, Christos S. Mantzoros9, Marina Lesina1 and Hana Algül1

1 II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich,

Germany

2 Klinik für Chirurgie Bereich Experimentelle Operative Medizin, Universitätsklinikum Magdeburg,

Magdeburg, Germany

3 Institute of Translational Medicine, University of Liverpool, Liverpool, UK

4 Institute of Experimental Genetics, Genome Analysis Centre, Helmholtz Zentrum München,

Neuherberg, Germany

5 Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany

6 Helmholtz Diabetes Center & German Diabetes Center (DZD), Helmholtz Zentrum München,

Neuherberg, Germany

7 Institute for Diabetes and Cancer, German Center for Diabetes Research (DZD), Neuherberg,

Germany

8 Department of Biochemistry, School of Medicine, Autónoma University of Madrid, Madrid, Spain

9 Division of Endocrinology, Diabetes, andMetabolism, Beth Israel Deaconess Medical Centre,

Harvard Medical School, Boston, Massachusetts

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease characterized by late-stage

diagnosis and metastasis. A multitude of underlying mechanisms has been suggested e.g.,

alterations in cell homeostasis and metabolism. Autophagy is a central regulator of homeostasis

and metabolism. Here we show for the first time that varying levels of the Autophagy related

gene 5 (Atg5) determine pancreatic carcinogenesis. Using an in vivo model of PDAC, we

generated mice with pancreas specific homozygous or heterozygous deletion of Atg5.

Homozygous deletion of Atg5 elevated tumor initiation but blocked progression; heterozygous

deletion of Atg5 increased tumor aggressiveness and metastasis. In vitro with in vivo analysis

reveal that Atg5-Heterozygosity not only influences cell homeostasis but also affects the tumor

microenvironment enhancing cell migration. Pro-tumorigenic inflammation, changes in

calcium homeostasis, and increased extracellular cathepsin activities collectively shape the

microenvironment enhancing metastasis. Thus, tumor stage and genetic background have to be

considered when targeting autophagy in PDAC.


18

Targeted Therapy in Colorectal Cancer

Maryam M. Matin1,2

1Department of Biology and Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad,

Iran 2Stem Cells and Regenerative Medicine Research Group, Academic Center for Education, Culture,

and Research (ACECR), Khorasan Razavi Branch, Mashhad, Iran

Colorectal cancer is the third most common cancer worldwide and its incidence has been

increasing in Iran in the past decade. Depending on the stage of colon cancer, various treatments

including surgery, chemotherapy, immunotherapy or targeted therapy might be considered in

the clinic. Despite many advances in these fields, patients still suffer from poor prognosis and

further studies are required on early detection and targeted therapy of this cancer. In the past

few years my lab has focused on different approaches to improve the efficacy of colorectal

cancer treatment. While use of natural products like ferutinin was beneficial, further research

indicated that safer and more efficient treatments can be achieved by targeted therapy. Some

examples include downregulation of certain long noncoding RNAs and also specific approaches

like "bacterial directed enzyme prodrug therapy" for which both in vitro and preclinical results

in mouse models with colorectal cancer will be discussed. However, despite all these promising

results, I think a combinatorial approach targeting multiple mechanisms would be more

appropriate in colorectal cancer therapy in the clinic.


19

Protein lipidation on cancer metabolism

Burçin Güngör

Department of Medical Biochemistry, Faculty of Medicine, Istinye University, İstanbul, Turkey

The importance of protein lipidation and lipid metabolism in cancer is well-recognized, but the

full spectrum of dysregulation of lipid post-translational modifications (PTMs) in cancer

remains largely unexplored. Till now, it has been known that a wide range of proteins, involved

in cancer, are modified by covalent linkage of fatty acids and/or isoprenoid groups. Attachment

of these hydrophobic groups plays a major role in regulating protein structure and function such

as correct subcellular localization of the protein. The saturated nature of the fatty acid promotes

insertion of palmitoylated proteins into liquid ordered domains of the membrane, known as

lipid rafts. Multiple signaling proteins are enriched in lipid rafts, and raft association promotes

intracellular signal transduction. Proteins actively functioning in malignant cell growth and

movement require lipidation. Therefore the action of lipid linkage and dissociation have

emerged as potential drug targets in a wide variety of disease states. Moreover, our

understanding of the dynamics of intracellular trafficking of lipid modified proteins has

dramatically expanded over the past few years. Ras proteins (K-, H, and N-Ras) are among the

best-known oncoproteins. H- and N-Ras are prenylated and then palmitoylated. Another group

of proteins possessing prominent lipidation features is Rho GTPases. Among these, Rac1 is one

of the key proteins for signal transduction for transcriptional regulation, ROS production, cell

motility, etc. Rac1 has double lipidation; prenylation and palmitoylation. Lipidation of Rac1

has been reported for protein localization to lipid rafts to regulate actin cytoskeleton thus cell

movement. Targeting Rac1 lipidation in cancer cells can be one of the important focus on the

therapeutic development for cancer cell invasion.


20

Molecular Diagnosis in Childhood Cancers

Sema Yilmaz

Health Science University Kartal Lutfi Kirdar Education and Research Hospital, Pediatric

Hematology/Oncology, Istanbul/Turkey

[email protected]

Cancer is a Genetic Disease (Theodor Boveri, 1914). Gene and chromosome abnormalities

observed in cancer[Gene mutations (oncogenes, tumor suppressor genes), Chromosome

structural, abnormalities (translocations, deletions, insertions) and Chromosome number

abnormalities (aneuploidy, polysomy)].

Molecular oncology (Personalized approach) provides, risk assessment, differential diagnosis,

prognosis prediction of treatment response pharmacokinetics, monitoring treatment response,

monitoring recurrence in patients without symptoms of cancer.

Diagnostic/Prognostic information

Molecular assays are currently available for testing on tumors including

• Fluorescent in situ hybridization (FISH)

• Polymerase chain reaction (PCR)

• Single nucleotide polymorphism (SNP)-based microarrays

• Next-generation sequencing (NGS) [Whole-genome sequencing (WGS), Whole

exome sequencing (WES) and RNA sequencing (RNAseq)

Next-generation sequencing (NGS), allows for massively parallel sequencing of genomic

fragments generating thousands to millions of short “reads” in a single run. Point mutations

including single nucleotide polymorphisms as well as small (generally, less than 20–30 base

pairs) insertions/deletions (indels) in large numbers of genes at once can be detected. NGS can

also identify unanticipated targetable mutations, copy number alterations, differentially

expressed genes, and gene fusions.

Pediatric cancers:

Pediatric cancers are significantly different from adult cancers because of mutation frequency

and type of altered genes, exhibit a lower mutational burden, relatively high prevalence of

specific structural variations (e.g., gene fusions and chromosomal rearrangements). Most of the


21

genetic alterations in pediatric leukemias and solid tumors involve well-known genes and

oncogenic pathways such as:

• Receptor tyrosine kinases,

• Phosphoinositide 3-kinase-AKT and related pathways, TP53, CDK12,

NOTCH1, ARID1 and

• Include amplification of other genes such as MYCN, MCL1, MDM2.

Precision cancer treatment

Precision cancer treatment is largely based on matching the patient’s tumor mutations with the

appropriate targeted therapy. The use of novel targeted therapy has greatly revolutionized

cancer treatment and is largely based on blocking actionable gene mutations or over-expressed

signaling transduction pathways.


22

Glioblastoma Multiforme Beyin Tümörünün Erken Tanı ve Tedavisine Yönelik

Otoantikor Çalışmaları

Nihal Karakaş

İstanbul Medipol Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji ABD, Rejeneratif ve Restoratif Tıp

Araştırmaları Merkezi (REMER)

Primer beyin tümörlerinin %60 kadarını gliomalar oluşturur ve gliomalar içerisinde ise Dünya

Sağlık Örgütü’nün sınıflandırmasına göre 4.evre (grade IV) olarak sınıflandırılan ileri grade’li

gliomalardan “Glioblastoma multiforme (GBM)” yetişkinlerde en sık görülen, agresif ve

malign bir beyin tümörüdür. GBM’in tedavisindeki bugünkü yöntemler, cerrahi tedavi ve bunu

takip eden radyoterapi ile kemoterapi uygulamalarıdır. Ancak hastalara GBM tanısı

konulduktan sonra yaşam süresi maalesef 1 yılı geçmemektedir. Dolayısıyla GBM ile

mücadelede, hastalığa özgü biyobelirteçlerin saptanması, erken tanı ve tedavi yöntemlerinin

geliştirilmesine öncülük edebilecektir.

Kanser oluşumu ile ilişkili olduğu düşünülerek vücut sıvılarında sirküle olan otoantikorların

araştırılması, özellikle kanserin erken tanısında yeni biyoişaretleyicilerin belirlenmesi açısından

etkin bir çalışma alanı oluşturmuştur. Akciğer ve meme kanseri gibi olgularda tespit edilen

otoantikorların, patolojik ve klinik olarak kanser tipine özgünlük gösterdiği tespit edilmiştir.

Buna karşılık, literatürde otoantikorların, beyin tümörleri ile ilişkisine yönelik, hastalığın

patolojisiyle ilgili olabilecek bilinen antijenlere özgü antikorlara ait tarama yapılmasına karşılık

olası yeni otoantikorlar ve ilişkili antijenlerin tespitine yönelik bir araştırma bildirilmemiştir.

Dolayısıyla, gliomalar içinde tedavisi en güç olan GBM’e özgü anti-GBM otoantikorların

araştırılması ve daha önce tanımlanmamış, yeni hedef antijenlerin keşfi GBM teşhis ve

tedavisinde bir dizi çalışmanın önünü açabilecek niteliktedir. Çalışmamızda, GBM ve non-

GBM glioma olarak gliomalar iki gruba ayrılmıştır ve hastaların tümör doku ve serumları

toplanmıştır. Kontrol olgu olarak ise tümör gelişimi oluşmamış epilepsi hastalarının örnekleri

kullanılmıştır. Elde edilen doku numunelerinde western blot analizleri yapılmış, primer antikor

yerine hasta ve kontrol serumları kullanılmıştır. Anlamlılık gösteren hedef proteinlerin tespit

edilebilmesi için kütle spektrometrisi analizleri ile aday proteinler tanımlanmıştır. Bu aday

proteinlere yönelik otoantikorların GBM serumlarında varlığı immünopresipitasyon ve

immünohistokimya yöntemleri uygulanarak çalışılmıştır.


23

Western blot analizleri ile serum örneklerinin, doku örneklerinden elde edilen proteinlerle

etkileştiği ve kontrol olgularına kıyas ile GBM olgularında anlamlı farklılıklar olduğu

saptanmıştır. Bu sonuçlara dayanarak 5 kontrol, 5 düşük grad ve 5 GBM olgularından elde

edilen proteinler kütle spekrometrisi yöntemi ile analiz edilmiştir. Biyoinformatik

değerlendirmeler sonucuda GBM olgularında düşük grad glioma ve kontrol olgularına kıyas ile

fazla eksprese edilen proteinler tespit edilmiştir. Bu aday proteinlerden birinin otoantijen

olduğu ve GBM hasta olgularında bu otoantijene güçlü oranda bağlanan özgün otoantikorların

varlığı immünopresipitasyon yöntemi ile belirlenmiştir. İmmünhistokimya çalışmaları

neticesinde GBM doku preparatlarında anlamlı derecede artmış otoantijen boyaması

gözlemlenmiştir. Bu bulgular kütle spektrometrisi ve immünopresipitasyon çalışmalarından

elde edilen bulgular ile uyumluluk göstermiştir. Sonuç olarak, bu çalışmamızda, GBM’ lerde

fazla ekspese edilen proteinlerden birini tanıyan bir otoantikor tespit edilmiştir. Ayrıca bu

otoantikorun, GBM için prognostik ve/veya diagnostik biyobelirteç adayı olduğu gösterilmiştir.

Böylece GBM’ye özgü bir otoantikor tanımlanmış ve bu yönüyle GBM hastalığının hasta

serumları kullanılarak erken dönem tanısına imkan sağlayabilecek bir buluş ortaya

konulmuştur.

Bu çalışma TÜBİTAK 216S nolu proje desteği ile gerçekleştirimiş ve patent başvuruları

başlatılmıştır.


24

Discovery of A Molecule That Destabilize Cryptochrome 1

Şeref Gül1, Kübra Akyel2, Tuğba Korkmaz3, Ibarhim Daniş4,5, Özgecan Şavlug İpek6,7, Fatih

Aygenli3, Ali Cihan Taskin8, Nuri Ozturk3, Narin Öztürk2, Durişehvar Özer Ünal4,5, Mustafa

Güzel6,9, Metin Turkay10, Alper Okyar2, Ibrahim Halil Kavakli1,11

1 Department of Chemical and Biological Engineering, Koc University, İstanbul

2 Faculty of Pharmacy Department of Pharmacology, Istanbul University, İstanbul, Turkey, 3 Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey, 4 İstanbul University Faculty of Pharmacy Department of Analytical Chemistry Beyazit-İstanbul,

5 İstanbul University Drug Research and Application Center (ILAM) Beyazıt İstanbul, 6 İstanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER),

Kavacik Campus, Kavacik-Beykoz/İstanbul, 7 Yildiz Technical University, Graduate School Of Natural And Applied Sciences, Department of

Chemistry, Besiktas/İstanbul, Turkey, 8 Animal Research Facility, Research Center For Translational Medicine, Koç University,

Rumelifeneri yolu, Sariyer, İstanbul, 9 İstanbul Medipol University, International School of Medicine, Department of Medical

Pharmacology, Kavacik Campus, Kavacik- Beykoz-İstanbul, 10 Department of Industrial Engineering, Koc University, İstanbul,

11 Department of Molecular Biology and Genetics, Koc University, İstanbul, Turkey.

We discovered a molecule that destabilizes Cryptochrome1 (CRY1) both in vitro and in vivo

using a structure-based drug design approach. The molecule, M47, enhanced the degradation

rate of CRY1 and modulated the period of U2-OS Bmal1-dLuc cells. In addition,

pharmacological studies with mice indicated that M47 had no toxic effect with half-life of ~6

h in blood. In addition, subcellular fractionation studies from mice liver indicated that M47

selectively decrease CRY1 level in the nucleus. Furthermore, M47 mediated CRY1 reduction

enhanced cisplatin-induced apoptosis in Ras-transformed p53-null fibroblast cells. Effect of

M47 on the oxaliplatin-induced apoptosis makes it a very promising agent to treat p53 mutant

dependent forms of cancer.


25

REWIRING THE EPIGENETIC NETWORKS AS A THERAPEUTIC APPROACH

IN MLL-REARRANGED LEUKEMIAS

Ipek Bulut1, Buse Cevatemre2, Adam Lee3, Arasu Ganesan3, Ceyda Acılan Ayhan2,4*

1Koc University, Graduate School of Science and Engineering, Istanbul, Turkey, 2Koc University, Research Center for Translational Medicine, Istanbul, Turkey,

3University of East Anglia, School of Pharmacy, Norwich, England, 4Koc University, School of Medicine, Istanbul, Turkey,

*To whom correspondence should be addressed

Defects in epigenetic pathways involving increased expression levels or abnormal patterns of

activity are one of the key drivers of cell proliferation in cancer. A particular example of these

defects is the MLL-AF9 fusion gene, seen in mixed-lineage leukemia (MLL). While the normal

function of the MLL gene regulates expression of developmental genes, chimeric MLL shows

abnormal activity playing an important role in the process of malignant transformation. The

main purpose of our studies is to reverse this transformation and to develop a personalized

medicine approach for the treatment of leukemia by targeting epigenetic processes dysregulated

in patients bearing the MLL-AF9 fusion protein.

Dual targeting in drug discovery is particularly important, as drugs with two targets reduce the

possibility of intrinsic or acquired drug resistance in tumors. While the normal function of MLL

gene has methyltransferase activity, the MLL-AF9 fusion protein recruits Lysine-Specific

Demethylase (LSD1) leading to activation of several genes that inhibit differentiation and cause

uncontrolled cell proliferation. Among the Histone De-Acetylases (HDACs), HDAC6 is

particularly unique, as it has targeted both in the nucleus and cytoplasm. Additionally, it is the

only HDAC, whose knockout mice is viable, making it an attractive target with potentially less

toxicity. Therefore, our project involves the use of LSD1 and HDAC6 inhibitors for dual

targeting.

The activity and the targeting efficiency of the inhibitors were verified by enzyme-based

profiling. The selectivity was measured through inhibition of other enzymes with similar

structure through in vitro enzyme inhibition and cellular thermal shift assays. Cell viability was

assessed using CTG assay, and the IC50 values were lower in cells containing MLL-AF9

compared to no MLL fusion. The epigenetic changes in response to these inhibitors were tested

via western blotting with epitope-specific antibodies. The differential expression of target genes

was evaluated using RT-qPCR, and a dramatic increase was observed CD11b and CD86


26

expression indicating that the drugs exhibited the expected changes. The synergy of these

molecules with the drugs used in the clinic was examined using both CTG and flow cytometry.

A significant synergy was observed with doxorubicin, which did not appear to be a result of

topoisomerase II inhibition. The mechanism of action for this synergistic effect is still under

examination.

In summary, the molecules under investigation showed promising activity in MLL-AF9

leukemic cells and will be characterized further as potential drugs in the clinic.


27

Treatment of refractory recurrent Gastrointestinal Stromal Tumors with adoptive

cellular immunotherapy (TILs) and Personalized Vaccine: a case report

A Tavartkiladze., R. Khutsishvili., P.Revazishvili., M.Maisuradze., L.Tavartkiladze

Institute for Personalized Medicine, Tbilisi State Medical University, Tbilisi, Georgia

We report the successful treatment of a patient with recurrent Gastrointestinal Stromal Tumors

with adoptive cellular immunotherapy and personalized vaccine. The patient is a young adult

with recurrent progressive disease refractory to aggressive multi-modality therapy including

repetitive surgical resection, radiation, and chemotherapy and all lines targeted therapy. He

received multiple courses of systemic intravenous administration of allogenous

phytohemagglutinin (PHA) activated T-lymphocytes in combination with a low dose of

interleukin-2 (IL-2) and personalized vaccine, which was adopted in laboratory from donors B-

lymphocytes and Patient’s Circulated Tumor Cells. The side-effects of this treatment were

limited and manageable. The patient achieved complete remission, as demonstrated by MRI

after 2 months from initiation immune therapy and confirmed by glucose-positron emission

tomography (PET) imaging 7 months after initiation of immune therapy. 14 months later, the

patient is still in remission with improving performance status. Adoptive cellular

immunotherapy utilizing allogenous phytohemagglutinin (PHA) activated T-lymphocytes and

personalized vaccine with low dose IL-2 appears to be a safe and effective therapy for a subset

of patients with primary, recurrent or progressive Gastrointestinal Stromal Tumors following

conventional therapy.

https://www.sciencedirect.com/topics/medicine-and-dentistry/immunotherapy

https://www.sciencedirect.com/topics/medicine-and-dentistry/low-drug-dose

https://www.sciencedirect.com/topics/medicine-and-dentistry/interleukin-2

https://www.sciencedirect.com/topics/medicine-and-dentistry/spontaneous-remission

https://www.sciencedirect.com/topics/medicine-and-dentistry/magnetic-resonance-imaging

https://www.sciencedirect.com/topics/medicine-and-dentistry/magnetic-resonance-imaging

https://www.sciencedirect.com/topics/medicine-and-dentistry/emission-tomography

https://www.sciencedirect.com/topics/medicine-and-dentistry/emission-tomography


28

Cancer Cells Adaptation to Immune Responses

Güneş Esendağlı

Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara-Turkey

The straightforward perspective that tumor cells are immunosuppressive has been contradicted

by the expression of potent costimulatory molecules on certain cancer cells. Indeed, the

stimulatory support offered by certain cancer cells such as myeloid leukemia or basal-like breast

cancer cells can provoke helper T cell responses. Unfavorably, this interaction can lead these

malignant cells to acquire an immune-suppressive capacity. Strong immune reactions are

required for the elimination of tumor cells. Tumor cells found in the circulation are considered

to be more susceptible to immune attack; the likelihood of these cells to come across with

immune cells is enhanced, and they are devoid of a protective (immunosuppressive)

microenvironment. On the other hand, anti-tumor immunity does not always correlate with

reduction in tumor growth and increase in patient survival. Moreover, immune-stimulatory

interventions have been shown to enhance the ability of immune cells to eradicate some, but

not all, tumor cells. Essentially, reduction in immune-provoking signals derived from the tumor

can diminish the effector phase of immune responses. The dogma in tumor immunology is that

the tumor cells express inhibitory molecules and anti-inflammatory cytokines to escape from

anti-tumor responses. However, it becomes more intriguing since the costimulatory molecules

can also be found on the tumor cells. Tumor cells hide from immune recognition and/or cope

with the immune attack. In other words, tumor cells which can successfully evade the anti-

tumor immunity may emerge as a consequence of adaptation to the selective pressure applied

by the immune system. Our results demonstrate the rapid adaptation capacity of leukemia,

breast cancer and lung cancer cells in response to anti-tumor immune responses. In our

experience, only a small subpopulation of cancer cells was able to provoke immunity, especially

through costimulation, adopts this de novo immunosuppressive character. Thus, during immune

evasion, tumor cells may benefit from being composed of heterogeneous subpopulations.


29

Mouse Models for Immunotherapeutic Approaches

Konstantinos Dimas

Department of Pharmacology, Faculty of Medicine, Health Science School, University of Thessaly,

Larissa, Greece

Any preclinical model system should mimic human cancer development. This includes models

that reproduce the genomic heterogeneity of human cancer, as well as develop a milieu that

incorporates the multitude of immune and stromal cell populations that make up the complex

tumor microenvironment. The gold standard for the development of standard cytotoxic cancer

therapies is to utilize xenograft models of human cancer cell lines engrafted into

immunocompromised mice to evaluate pharmacology, efficacy, and safety profiles of these

agents. However, the development of immunotherapy requires a model system with a

functionally intact immune system. Additionally, the immune system shows an inherent

heterogeneity and adaptability that to some extent explains the relative success of

immunotherapy, in that it is able to constantly adapt and evolve along with the tumor. However,

finding preclinical models that can recapitulate this adaptability in the face of tumor

heterogeneity remains a major impediment in the development of cancer immunotherapies. In

this brief talk, we discuss the current landscape of preclinical models available for preclinical

immunotherapeutic evaluation and the pros and cons associated with each model in this context.


30

Diagnostic and Prognostic Importance of Leukemia Cytogenetics

Süreyya Bozkurt

Department of Medical Biology, Faculty of Medicine, İstinye University, Istanbul Turkey

Cytogenetic Anomalies have a very important role in the prognosis and diagnosis of

hematologic cancers. Some cytogenetic abnormalities predict good prognosis while others

indicate poor prognosis. Cytogenetic is the gold standard for detecting to Ph chromosome and

monitoring cytogenetic response and is one of the most important prognostic markers in CML.

The emergence of additional chromosomal anomalies (ACA) besides the Philadelphia

chromosome in CML is thought to be a sign of disease progression. The frequencies of ACA s

in higher in CML-BC (Blastic crisis phase) patients than in CML-CP (Chronic Phase) patients

or AP patients. ACAs can be classified as major and minor route of abnormalities. The minor

route abnormalities are loss of Y chromosome, trisomy 21, hypodiploidy, hyperdiploidy, and

polyploidy. The treatment of the chronic phase Ph + CML is the oral administration of tyrosine

kinase inhibitors (TKI). Cytogenetic and molecular monitoring help selecting the most

convenient TKI drug and to optimize TKI treatment. The emergence of ACAs in CML

karyotype is an alarm for the development of AML or MDS. Cytogenetic findings play a central

role in diagnostic classification in AML as in all myeloid malignancies. In the development of

AML, some cytogenetic abnormalities function specifically as prognostic markers. Therefore

cytogenetic is another important diagnostic marker after induction therapy in AML. While del

(11q) is a marker of good prognosis, inv

(3) / t (3q) / del (3q) is a marker of poor prognosis in Myelodysplastic syndromes (MDS).

Despite highly advanced molecular techniques, conventional cytogenetic hematological

cancers remain the gold standard. Because cytogenetic anomalies shed light on new treatment

options as a guide in disease pathogenesis. It is also a guide in predicting prognosis and helps

in the selection of treatments to be applied.

References

Bennour A, Saad A, Sennana H. Chronic myeloid leukemia: Relevance of cytogenetic and molecular

assays. Crit Rev Oncol Hematol. 2016;97:263-74

François Guilhot. Cytogenetics in CML: more important than you think. Blood 2016 127:2661-2662

Ravandi F, Walter RB, Freeman SD. Evaluating measurable residual disease in acute myeloid

leukemia. Blood Adv. 2018 Jun 12;2(11):1356-1366

Hosono N. Genetic abnormalities and pathophysiology of MDS. Int J Clin Oncol. 2019;24(8):885-892

https://www.ncbi.nlm.nih.gov/pubmed/?term=Bennour%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26412717

https://www.ncbi.nlm.nih.gov/pubmed/?term=Saad%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26412717

https://www.ncbi.nlm.nih.gov/pubmed/?term=Sennana%20H%5BAuthor%5D&cauthor=true&cauthor_uid=26412717

https://www.ncbi.nlm.nih.gov/pubmed/26412717


https://www.ncbi.nlm.nih.gov/pubmed/?term=Ravandi%20F%5BAuthor%5D&cauthor=true&cauthor_uid=29895626

https://www.ncbi.nlm.nih.gov/pubmed/?term=Walter%20RB%5BAuthor%5D&cauthor=true&cauthor_uid=29895626

https://www.ncbi.nlm.nih.gov/pubmed/?term=Freeman%20SD%5BAuthor%5D&cauthor=true&cauthor_uid=29895626



https://www.ncbi.nlm.nih.gov/pubmed/?term=Hosono%20N%5BAuthor%5D&cauthor=true&cauthor_uid=31093808



31

ANTİKANSER ÇALIŞMALARINDA PLATİN KOORDİNASYON BİLEŞİKLERİNİN

ROLÜ

Abdurrahman Şengül

Zonguldak Bülent Ecevit Üniversitesi, Fen-Edebiyat Fakültesi, Kimya Bölümü 67100 İncivez,

Zonguldak

E-mail: [email protected]

Kanser, günümüzde dünyanın en önemli sağlık problemidir. Kanser, vücudumuzun herhangi

bir yerinde anormal hücrelerin kontrol edilemez bir şekilde büyümesi olarak tanımlanmaktadır.

WHO, kanserin genetik faktörlerin ve çevresel faktörlerin (UV, radyasyon, kimyasal

kanserojenler ve biyolojik kanserojenler) kişiyle etkileşimi sonucu meydana geldiğini rapor

etmiştir. Kanserin tipine, safhasına ve hastanın durumuna göre birçok farklı tedavi yöntemi

mevcuttur. Bu sunumda, 1965 yılında Barnett (Barney) Rosenberg’in Cisplatin’in (cis-

[Pt(NH3)2Cl2]) antitümör aktivitesini keşfetmesinden sonra kemoterapi alanında klinik

kullanımı onaylanmış olan platin(II)-koordinasyon bileşiklerinin özellikleri ve kanser

tedavisindeki rolleri vurgulanacaktır.

Cisplatin’in birçok kanser türüne karşı çok aktif olmasına rağmen ciddi yan etkilere sahip

olması ve bazı kanser tiplerinin Cisplatin’e direnç göstermesinden dolayı binlerce platin-esaslı

antitümör ilaç sentezlenmiştir. Bunlardan sadece birkaç tanesi (örneğin: Oxaliplatin) Cisplatin

direncini yenmesi ve daha az toksik özellik göstermesine rağmen günümüzde Cisplatin’den

daha etkili bir kemoterapi ajanı henüz keşfedilmemiştir. Günümüzde kanser hastalarının yarısı

Cisplatin ile tedavi edilmektedir. Yan etkileri daha az ve geniş spektruma sahip ilaç geliştirmek

için birinci nesil, ikinci nesil, üçüncü nesil ve yeni nesil antikanser platin ilaçları

sentezlenmiştir. Klasik kovalent bağlanma yapan ilaçların yerine daha sonra interkalasyon ve

groove bağlanma özelliği olan ilaçlar geliştirilmiştir. Günümüzde kinetik olarak daha kararlı

olan ve hücre içinde indirgenerek aktif hale geçen platin(IV) esaslı kompleksler büyük aşama

kaydetmiştir. Bunların yanında hidrofobik özellikleri olan dinükleer platin(II) bileşikleri de

umut verici sonuçlar göstermektedir. Bu çalışmada tüm bu bileşiklerin, in vitro ve in vivo

deneyleri sonucunda belirlenen antitümör aktiviteleri ve ilaçların moleküler yapıları ile olan

ilişkileri irdelenecektir.

Literatür ışığında, daha büyük antikanser özelliğe sahip fakat daha az yan etki gösteren

platin(II)-koordinasyon bileşikleri sentezlenmiş ve bunların DNA etkileşimleri ve sitotoksisite

çalışmaları grubumuz tarafından yapılmıştır.

mailto:[email protected]


32

Kanser Kök Hücresinde Güncel Tedavilere Yaklaşım

Gülperi Öktem

Ege Üniversitesi, Tıp Fakültesi Histoloji ve Embriyoloji ABD, İzmir

Ege Üniversitesi, Sağlık Bilimleri Enstitüsü Kök Hücre ABD, İzmir

Tümör kitlesindeki heterojen hücre popülasyonlarının çok küçük bir kısmını oluşturan Kanser

Kök Hücreleri (CSC) veya Tümör Başlangıç Hücreleri (TICs) malign progresyona sebep olan,

tedaviye yanıtı değiştiren hücrelerdir. Pek çok araştırma gurubu tarafından incelenen bu hücre

gurubu hedef tedavilerin de gözdesi haline gelmiştir. CSC’nin eradike edilmesininin kanseri

ortadan kaldıracağı yaygın olarak kabul görmektedir. Bu hücre gurubuna ait pek çok biobelirteç

araştırma gurupları tarafından identifiye edilmiş ve akademik makalelerde yayınlanmıştır.

Özellikle yüzey belirteçlerini hedefleyen spesifik monoklonal antikor tedavileri kanser tedavi

protokollerini değiştirmeye başlamıştır. Ancak görünen odur ki; günümüzde CSC’ni identifiye

etmek için kullanılan yüzey belirteçlerinin %73 ü insan embriyonik kök hücrelerinde (hESCs),

erişkin kök hücrelerinde ve somatik hücrelerde de bulunmaktadır. Bu da CSC’in epigenetik

veya genetik değişiklikler sonunda normal kök hücrelerden farklılaştığını göstermektedir. Bu

sebeple yüzey belirteçlerine göre kanser kök hücrelerinin izole edilmesi veya hedef tedavilerde

kullanılabilmesi için ancak spesifik bir beliteçin bulunması veya birden fazla yüzey belirtecinin

kullanılarak hedef tedavilerin yapılması uygun seçenek gibi görünmektedir. Bununla beraber,

kanser araştırmalarında değişen gündem son yıllarda egzozomların stratejik hedef olarak

karşımıza çıktığını göstermektedir. Hücresel sinyallemenin ana düzenleyicisi olarak, hem

normal hem de kanser kök hücreleri, tümör mikro-ortamını, büyümesini, ilerlemesini ve

varsayılan immünolojik fonksiyonunu değiştiren çeşitli otokrin ve parakrin sinayalleri orkestra

etmek için eksozomlar salgılanmaktadır. Bu endozomal yolak orijinli nano kesecikler

orijinlendiği hücrenin parmak izini taşıması yönü ile özellikle kanserin tanı ve tedavisinde

potansiyel belirteçler olma özelliği taşır, mRNA ve mikroRNA(miRNA)‘lar gibi taşıdıkları

kargo içerikleri ile kanser ilerleyişine, immün sistemden ve apoptozisten kaçış, anjiyogenez,

invazyon, metastaz gibi onkojenik hedefleri uyararak, amtitümör immün yanıtı değiştirerek

katkı sağlarlar. Hücreler arası iletişiminde eksozomların ve eksozomal miRNA vasıtası ile bilgi

aktarımı kanser gelişimindeki en önemli mekanizmalardan biridir. CSC’nin onkojenik fenotipi

teşvik etmek için tümör mikro ortamları ile nasıl etkileşime girdiği konusunda bilgilerimiz hala

eksiktir. Bu nedenle tanı ve tedavide tüm vücut sıvılarından elde edilebilen eksozomlar serum

ve plazmada korunmuş halde bulunan hücre tipine göre spesifik olan ve kökenlendiği tümör ile


33

ilgili biyobelirteçler içeren eksozomal miRNA’lar kullanılması çok daha büyük bir potansiyel

taşımaktadır.


34

AURORA KINASES AND CANCER

Ata Özçimen

University of Mersin, Department of Biology, Mersin-Turkey

Cancer is a disease caused by uncontrolled cell proliferation of bad tumors in the body. Human

cancer cells are often characterized by changes in the amount or organization of DNA resulting

from errors in mitosis, resulting in chromosome instability and aneuploidy. Auroras are the

serine/threonine kinase family, which are highly conserved throughout evolution in cell

division processes. As the main regulators of mitotic chromosome degradation, condensation

and orientation in the metaphase plate, spindle assembly and completion of cytokinesis play an

important role in cell division. They also play an important role in the proliferation of cancer

cells. Three mammalian subtypes were identified from these kinases as Aurora -A, -B and -C.

All the Aurora genes are characterized by 78–84% of identity between human and rodent

orthologs [3]. Phylogenetic trees show that the Aurora genes have originally evolved from a

single gene from urochordata ancestor (Tunicata). This gene was first called Increases in

Ploidy-1 (Ipl1). It has previously been identified in yeast genomes such as Saccharomyces

cerevisiae and Saccharomyces pombe, suggest that the functions of Aurora vary throughout

evolution. These only encode an Aurora–like homolog (Ipl1) [1,4]. The ancestral AURK gene

shares 41% of identity with the human AURKA gene. Aurora-A family is ubiquitous among

all vertebrates. In invertebrates and non-mammalian vertebrates, Ipl1 gene gave rise to AURKA

and AURKB/AURKC. The invertebrate Aurora-A and Aurora-B kinases have protein

families that are quite different from their vertebrate counterparts. The Aurora-B and Aurora-

C families in humans have emerged from a gene duplication event in mammals. These all three

were highly conserved in evolution processes [3]. Three mammalian Aurora kinases occur at

certain locations during mitosis. Aurora-A, which is the “polar kinases”, combines mainly

with separating centrosomes. The gene is encoded in human Chromosome 20q13 locus. It’s

overexpression and oncogenic transformation have inhibited the p53 activity [5]. Aurora-B,

which is the “equatorial kinase”, is a chromosomal transition protein. This gene is encoded in

human chromosome 17q13 locus. Aurora-B is a specific protein of Chromosomal Passenger

Complex (CPC), which manages kinase activity and localization. This complex is combined

with Inner Centromere Protein (INCENP), survivin (BIRC5), borealin (CDCA8) and non-

enzymatic sub-units [5,6]. Aurora-C, the least studied, appears to be localized in anaphase to


35

telophase centrosome, highly expressed in germ and testis. This gene is encoded in human

chromosome 19q13 locus.

Beside the increased expression in normal cell division and meiosis, all Aurora kinases have

differentially expressed in tumor cells. Aurora-A is highly amplified, overexpressed and

hyperactivated in several aneuploidy tumors such as gastrointestinal, nasopharyngeal, prostate

and breast cancers [3]. Aurora-B is also overexpressed in most cancer cells, which were

transformed from over proliferated non-cancerous cells. This gene is mainly active in stemness,

invasion and proliferation of tumors. Due to the actions of Aurora-B is a poor diagnostic factor

for several tumors such as hepatocellular carcinoma, non-small cell lung carcinoma and oral

squamous cell carcinoma [7]. Aurora-C is expressed in meiotically-active germ cells and in

human thyroid carcinomas, cervical and colorectal tumors. Aurora-C protein level is specially

correlated with the malignancies of colorectal carcinoma. However, Aurora-C overexpression

in tumors was not yet well described [8]. In the shed of light, molecular approach of the cancer

treatment is way to block proteins cause irregularity and their irregular important mediators in

cell division. One of the best approaches is suitable for Chromosomal Passenger Complex that

regulates kinetochore-microtubule associations in mitosis [2]. The therapeutic inhibition of

these kinases has probably become an attractive target for the development of potential anti-

cancer therapies due to their fundamental role during cell division. In fact, several small

molecule Aurora kinase inhibitors have already been developed, and some have shown

promising clinical efficacy in some human tumors in Phase-I and-II clinical trials [7].

As a result of the studies, one of the most advanced clinical compounds showing strong activity

against aurora kinases is Tozasertib (VX680), Alisertib (MLN 8237) and Danusertib

(formerly known as PHA-739358). Danusertib is named as Pan-Aurora Kinase inhibitor

because of its’ effective on all three Aurora kinases group. Further pharmaco-active studies of

pan-aurora kinase inhibitors might be potential anti-tumor compounds for cancer patients [7].

References

[1]. Brown, J. R., Koretke, K. K., Birkeland, M. L., Sanseau, P., Patrick, D. R. (2004). Evolutionary

relationships of Aurora kinases: Implications for model organism studies and the development of anti-

cancer drugs. BMC Evolutionary Biology, 10.1186/1471-2148-4-39. 10.

[2]. Bolanos-Garcia, V.M. (2005). Aurora kinases, The International Journal of Biochemistry & Cell

Biology 37. 1572–1577.

[3]. Willems, E., Dedobbeleer, M., Digregorio, M., Lombard, A., Lumapat, P. N., Rogister, B. (2018).

The functional diversity of Aurora kinases: a comprehensive review. Cell Div 13:7. 17.


36

[4]. Sasai, K., Katayama, H., Stenoien, D. L., Fujii, S., Honda, R., Kimura, M., Okano, Y., Tatsuka, M.,

Suzuki, F., Nigg, E. A., Earnshaw, W. C., Brinkley, W. R., Sen, S. (2004). Aurora-C kinase is a novel

chromosomal passenger protein that can complement Aurora-B kinase function in mitotic cells. Cell

Motility and the Cytoskeleton, 59(4), 249–263.

[5]. Seeling, J. M., Farmer, A. A., Mansfield, A., Cho, H., Choudhary, M. (2018). Differential

selective pressures experienced by the aurora kinase gene family. International Journal of Molecular

Sciences, 19(1), 1-16.

[6]. Adams, R. R., Carmena, M., Earnshaw, W. C. (2001). Chromosomal passengers and the (aurora)

ABCs of mitosis. TRENDS in Cell Biology 11(2). 49-54.

[7]. Kitzen, J.J.E.M., de Jonge, M.J.A., Verweij. J. (2010). Aurora kinase inhibitors. Critical Reviews

in Oncology/Hematology 73, 99–110.

[8]. Gavriilidis, P., Poutahidis, T., Giakoustidis, A., Makedou, K., Angelopoulou, K., Hardas, A.,

Andreani, P., Zacharioudaki, A., Saridis, G., Gargavanis, A., Louri, E., Antoniadis, N., Karampela, E.,

Psychalakis, N., Michalopoulos, A., Papalois, A., Iliadis, S., Mudan, S., Azoulay, D., Giakoustidis, D.

(2018). Targeting hepatocarcinogenesis model in C56BL6 mice with pan-aurora kinase inhibitor

Danusertib. Journal of Cancer, 9(5), 914-922.


37

A Parkinson-related Gene in Melanoma: Exploring The Role of The P5 Type ATPase

ATP13A2, A Parkinson Disease’s Associated Gene, in Melanoma Cell Proteostasis

Seyma Demirsoy 1,3, Peter Vangheluwe2 and Patrizia Agostinis1

1Laboratory of Cell Death Research & Therapy, Department of Cellular and Molecular Medicine, KU

Leuven, Belgium, 2Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU

Leuven, Belgium, 3Laboratory of Molecular Microbiology, Department of Medical Microbiology, Hacettepe University,

Ankara, Turkey

ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type

transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb

Syndrome (KRS), a form of autosomal Parkinson’s disease (PD). In spite of a growing interest

in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies

suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their

nature remains incompletely understood. To this end, we generated a set of melanoma and

neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N)

and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress

conditions, evoked by the proteasome inhibitör Bortezomib, endo-/lysosomal associated full-

length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the

intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic

degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-

proteins, a pattern also observed in patient-derived fibroblasts harboring ATP13A2 loss-of-

function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and

increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating

PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve,

compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation

of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity

–independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in

response to proteotoxic stress.


38

A Novel Transcriptomic Based Gene Panel for The Prognostication of Pancreatic

Cancer

Seçil Demirkol Canlı1, Ali Osmay Güre2

1Molecular Pathology Application and Research Center, Hacettepe University, Ankara, Turkey

2Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

PDAC is amongst the most lethal cancer types in the world with records of 5 year survival less

than 5%. A projection of cancer incidence and death rates to 2030 in US showed that pancreas

cancer will become the second cause of cancer-related death by 2030. A comprehensive study

of death rate trends in pancreatic cancer since 1970 indicated complex trends that are largely

unexplainable by known risk factors. For the evaluation of risk, currently the only FDA

approved biomarker for PDAC patients is CA19-9 with its limitations, and along with that

AJCC TNM staging and performance status are considered important prognosis indicators in

clinical practice. In order to identify a novel panel of prognostic genes, we utilized publically

available microarray and RNAseq data of PDAC tumors from GEO and TCGA. Expression of

20 genes was significantly associated with overall survival and recurrence free survival in four

datasets and a score generated based on the expression matrix of these genes have been

validated in two independent cohorts. The 20 gene score is dramatically elevated in metastatic

tissue, compared to primary tumor, and is higher in primary tumor compared to normal. Further

analyses of transcriptomic data from the prognostic sub-groups showed that the ‘low risk’

tumors have overall more immune cell infiltration, a higher CD8 T cell/Treg ratio. Analyses of

proteomic data from TCGA PAAD indicated higher levels of Cyclin B1, RAD51, EGFR and a

lower E-cadherin-Fibronectin ratio in ‘high risk’ group. In summary, our score defines both

prognostic and biological sub-groups among PDAC tumors, and has potential to have a

predictive role in terms of response to targeted therapy options.


39

The Transcription Factor Elf3 Is Essential for a Successful Mesenchymal to Epithelial

Transition

Burcu Şengez

Izmir Biomedicine and Genome Center, Izmir, Turkey

The epithelial to mesenchymal transition (EMT) and the mesenchymal to epithelial transition

(MET) are two critical biological processes that are involved in both physiological events such

as embryogenesis and development and also pathological events such as tumorigenesis. They

present with dramatic changes in cellular morphology and gene expression exhibiting acute

changes in E-cadherin expression. Despite the comprehensive understanding of EMT, the

regulation of MET is far from being understood.

To find novel regulators of MET, we hypothesized that such factors would correlate with Cdh1

expression. Bioinformatics examination of several expression profiles suggested Elf3 as a

strong candidate. Depletion of Elf3 at the onset of MET severely impaired the progression to

the epithelial state. This MET defect was explained, in part, by the absence of E-cadherin at the

plasma membrane. Moreover, during MET, ELF3 interacts with the Grhl3 promoter and

activates its expression.

Our findings present novel insights into the regulation of MET and reveal ELF3 as an

indispensable guardian of the epithelial state. A better understanding of MET will, eventually,

lead to better management of metastatic cancers.


40

Kanser Kök Hücre ve Epigenetik Yaklaşımlar

Nazlıhan Aztopal

İstinye Üniversitesi, Fen Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik Bölümü, İstanbul

Kanser tedavisi, son yıllarda yeni ilaçların da kullanıma girmesine rağmen, tatmin edici başarı

seviyesine ulaşmamıştır. Son yıllarda giderek artan kanıtlar, tedavi başarısızlığındaki en önemli

nedenlerden biri olarak tümör içerisinde kök hücrelerle benzer şekilde kendini yenileme ve

farklılaşma kabiliyetine sahip özel bir hücre alt grubunun, kanser kök hücrelerin (KKH),

varlığını işaret etmektedir. Tümörün çok küçük bir kısmını (%0,1-2) oluşturan bu hücre

grubunun; tümör oluşumu ve gelişimi, artmış invazif özellik, radyasyon ve kemorezistans ile

ilişkili oldukları düşünülmektedir. Bu nedenle, çoğunluğu oluşturan kanser hücrelerinin yanı

sıra KKH’lerini de hedefleyen tedavi modaliteleri önem kazanmaktadır.

Son yıllarda giderek artan kanıtlar, epigenetik değişikliklerin KKH özelliklerinin

düzenlenmesinde önemli bir role sahip olduğunu göstermektedir. KKH’lerinde, tümörün büyük

çoğunluğunu oluşturan ve kök hücre özelliği taşımayan kanser hücrelerinden farklı olarak;

hayatta kalma, kendini yenileme ve proliferasyon sürecinde kritik rol oynayan Wnt/ β-katenin

sinyalizasyonunun yeniden aktive olduğu ve tedavi direncine katkı sağladığı bilinmektedir. Her

ne kadar Wnt/ β-katenin sinyal yolağının aşırı aktivasyonu yolak bileşenlerinde genetik

mutasyonlar ile ilişkilendirilse de epigenetik mekanizmaların da bu düzenlemede oldukça

belirgin bir rol oynadığı ve terapötik olarak hedeflenebildiği gösterilmiştir. Özellikle, histon

asetilasyon programının değişimi farklılaşma süreci ile ilişkilendirilmekte ve bundan dolayı

farklılaşmayı arttırmak için histon deasetilazları (HDAC) hedefleyen ilaçlar kullanılmaktadır.

Epigenetik belirteçlerin terapötik birer hedef olarak önemi dikkate alındığında, KKH’lerini

hedefleyen ve bu hücreleri KKH özelliği taşımayan hücrelere dönüştürebilen bu

mekanizmaların anlaşılması ve aydınlatılması yeni tedavi seçenekleri/yaklaşımları sunabilir.

Dolayısıyla, tedaviye dirençten hatta tedavi sonrası nükslerin oluşumundan (kanser kök

hücreleri üzerinden) sorumlu tutulan Wnt/ β-katenin sinyal yolağını düzenleyen epigenetik

modifikasyonları daha iyi anlamak, terapötik bir hedef olarak rolünü tanımlamak ve yeni

epigenetik terapi yaklaşımları geliştirmek son yıllarda yaptığımız çalışmaların temelini

oluşturmaktadır.


41

Pankreatik Kanserde Tümör Mikroçevresinin Hedeflenmesi

Didem Karakaş

İstinye Üniversitesi, Fen-Edebiyat Fakültesi, Moleküler Biyoloji ve Genetik, İstanbul

Pankreatik kanser semptomlarının azlığı, erken deteksiyon ve efektif tedavi seçeneklerinin

bulunmaması nedeniyle en öldürücü kanser türleri arasında yer almaktadır. 5 yıllık sağ kalım

oranı %5’ten daha düşüktür ve hastaların %50’si ilk 6 ay içinde hayatını kaybetmektedir [1].

Günümüzde pankreatik kanserin tedavisi için sitotoksik ya da hedefli tedaviler gibi pek çok

yaklaşım denenmesine rağmen etkin bir sonuç alınamamaktadır. Son yıllarda yapılan

çalışmalarla, yalnızca karsinoma hücrelerinin değil, tümör mikroçevresinin de pankreatik

kanserin bu agresif doğasına katkı sağladığı ortaya konmuştur [2,3]. Bu nedenle sadece tümör

hücrelerini hedefleyen tedaviler başarısız olmaktadır ve etkin tedavi için karsinoma hücreleriyle

birlikte tümör mikroçevresinin de hedeflenmesi gerektiği düşünülmektedir.

Pankreatik kanser stroma bakımından en zengin kanserlerden biridir [4]. Tümör kütlesinin

yaklaşık %90’ı dezmoplastik stromadan oluşurken, tümör hücreleri tümörün sadece %10’lık

kısmını oluşturmaktadır [5]. Pankreatik kanseri eşsiz yapan bu yoğun stroma tedavi

başarısızlığındaki en büyük nedenlerden biri olarak görülmektedir. Çünkü son yıllarda yapılan

çalışmalar ile “tümör mikroçevresi” olarak da adlandırılan stromal hücrelerin tümör gelişimi,

invazyon, kemoresistans, apoptozisten ve immün sistemden kaçış ve metastaz süreçlerinde aktif

rol oynadığı bilinmektedir [6-8]. Bu nedenle günümüzde sadece tümör hücrelerini hedefleyen

tedavilerin başarısız olduğu ve tümör hücreleriyle birlikte stromal hücrelerin de hedeflenmesi

gerektiği bilinmektedir.

Bu kapsamda ilk olarak, pankreatik kanser ve mikroçevre hücreleri arasındaki etkileşim

mekanizmalarının aydınlatılması gerekmektedir. Pankreatik kanser hücreleri ile makrofajlar,

nöronlar ve pankreatik stellat hücreleri arasındaki interaksiyonların moleküler

mekanizmalarının incelenmesi ve bu mekanizmaların biyomarker olarak kullanılabilme

potansiyellerinin araştırılması ve bu interaksiyonları hedef alacak tedavi yaklaşımlarının

geliştirebilmesi son yıllardaki çalışmalarımızın temelini oluşturmaktadır.

Referanslar:

1. Michaud, D.S. 2017. “Pancreatic Cancer: Reference Module in Biomedical Sciences”, ABD.


42

2. Fujita, H. et al. 2010. “α-Smooth muscle actin expressing stroma promotes an aggressive tumor biology in pancreatic ductal adenocarcinoma”, Pancreas, 39, 1254–1262.

3. Miyamoto, H. et al. 2004. “Tumor–stroma interaction of human pancreatic cancer: acquired resistance

to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins”, Pancreas,

28, 38–44.

4. Feig, C. et al. 2012. “The pancreas cancer microenvironment”, Clin Cancer Res, 15,18(16),4266-76.

5. Li, J., Wientjes, M. G., Au, J.L. 2010. “Pancreatic cancer: pathobiology, treatment options, and drug delivery”, AAPS J., 2, 223–232.

6. Farrow, B., Albo, D., Berger, D.H. 2008. “The role of the tumor microenvironment in the progression of pancreatic cancer”, J. Surg. Res, 149,319–328.

7. Hanahan, D., Weinberg, R.A. 2000. “The hallmarks of cancer”, Cell, 100: 57–70.

8. Liotta, L.A., Kohn, E.C. 2001. “The microenvironment of the tumour-host interface”, Nature, 411,

375–379.


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HEDEFE YÖNELİK TEDAVİDE KANSERE ÖZGÜ MOLEKÜLER MEKANİZMALARIN

AYDINLATILMASI

Şeyma Aydınlık1, Egemen Dere1, Engin Ulukaya2

1Biyoloji Bölümü, Fen Edebiyat Fakültesi, Uludağ Üniversitesi, Bursa, Türkiye

2Tibbi Biyokimya Bölümü, Tıp Fakültesi, İstinye Üniversitesi, İstanbul, Türkiye

Metastatik kolon kanseri agresif kemoterapi rejimlerinin kullanılmasına rağmen, % 5'den az bir

hayatta kalma oranına sahiptir. Kolon kanserinde epidermal büyüme faktörü reseptörünün

(EGFR) prognostik ve prediktif önemi hala tartışmalıdır. Bu nedenle EGFR reseptörünü bloke

etmek amacıyla çalışmada tirozin kinaz inhibitörü canertinib kullanılmıştır. Bu amaçla Uludağ

üniversitesi kimya bölümü tarafından sentezlenenen Palladyum(Pd) (II) bileşiği

[PdCl(terpy)](sac).2H2O] ve bu bileşiğin canertinib ile kombinasyonunun kansere özgü

moleküler mekanizmalar üzerine etkileri araştırılmıştır. Ayrıca kemoterapötik ilaç 5-

Fluorourasil (5-FU) ve bu ajanın canertinib ile kombinasyonu ise pozitif kontrol olarak

kullanılmıştır.Dolayısıyla Palladyum (II) bileşiği ile canertinib tek başına ve kombinasyon

şeklinde, aynı şekilde 5- Fluorourasil tek başına ve canertinib ile kombinasyonunun insan kolon

kanseri hücre soyları (HCT-15, HT-29) üzerine sitotoksik etkileri SRB canlılık testi ile

belirlenmiştir. Hücrede gerçekleşen apoptozis, asidik vezikülasyon, mitokondriyal

depolarizasyon, flow sitometri, Anneksin-V-Cy3/SYTOX ikili boyama, akridin ve JC-1

boyama yöntemi belirlenmiştir. EGFR ilişkili yaşam yolaklarında, bu reseptörün inhibe

edilmesi ile gerçekleşen değişimler, epidermal mezenkimal dönüşüm (EMT) ve apoptoz/otofaji

ilişkili proteinlerdeki değişimler ise western blot yöntemi ile değerlendirildi. Ayrıca bu yaşam

yolağının inhibisyonunun kombinasyon tedavileri ile birlikte invazyon, yara iyileşmesi ve

koloni oluşturma yetenekleri üzerine etkileri belirlendi. Son olarak HUVEC endotel

hücrelerinde EGFR inhibisyonun kombinasyon tedavileri ile birlikte tüp oluşturma yeteneği in

vitro matrigel testi ile ve damar oluşumu üzerine etkileri ayrıca in vivo Chorio-allantoik

membran (CAM) testi ile değerlendirildi. Sonuç olarak, Pd (II) bileşiği ve canertinib

kombinasyonunun kolon kanseri hücre soylarında apoptozis ve sitotoksik etkilerinde artışa,

invazyon, yara iyileşmesi yeteneklerinde ve damar oluşumunda azalmaya neden olduğu

bulunmuştur.

*Bu çalışma Uludağ Üniversitesi Bilimsel Araştırmalar Projeler Birimi tarafından

desteklenmiştir (DDP(F)-2017).

Anahtar Kelimeler: Trozin kinaz inhibitörleri, Anjiyogenez, Otofaji, EMT, İnvazyon ve migrasyon


44

Yeni Antikanser İlaç Keşfinde Genomik ve Metabolomik: Fırsatlar ve Zorluklar

Ekrem Kum, Süleyman Özakın, Ebru İnce*

Dicle Üniversitesi Fen Fakültesi Biyoloji Bölümü 21280 Diyarbakır

*[email protected]

Elli yıldan uzun bir süredir, mikroorganizmalardan ve bitkilerden elde edilen küçük organik

moleküller çok sayıda kullanışlı kanser kemoterapötik ilacına kaynak teşkil etmiştir. Doğal

ürünlerin bu tip öncü bileşiklerinin tarama çalışmaları son yıllarda ivme kazanmış, karasal bitki

ve mikroorganizmalar kadar, deniz fauna ve florasını oluşturan bileşenlerinin de anti-kanser

aktiviteleri araştırılmaya başlanmıştır. Doğal ürünlerin biyosentezlerinin daha iyi anlaşılması

ile beraber, geleneksel ilaç veya ilaç öncülü tarama çalışmaları yerini genomik bilginin

kimyasal verilerle kombine edilmesi prensibe dayalı farklı stratejilere bırakmıştır. Anti-kanser

dahil olmak üzere farklı biyolojik aktivite ve emsalsiz kimyasal çeşitliliğe sahip mikrobiyal

doğal ürünlerin üreticisi olan Aktinomisetler kompleks bir yaşam döngüsüne ve diğer bakteriler

ile kıyaslandığında büyük genomlara sahiptirler. Yeni nesil dizileme teknolojileri sayesinde

genomlarında biyoaktif doğal ürün sentezinden sorumlu yüksek miktarda biyosentetik gen

kümesine sahip oldukları bulunmuştur. Bu makalede; mikrobiyal kaynaklı yeni biyoaktif

bileşik keşfinde genomik ve metabolomik tarama stratejileri, kullanılan biyoinformatik

programlar, avantaj veya zorluklarına değinilerek, son yıllarda bu teknoloji ile elde edilmiş yeni

anti-kanser ilaçların altı çizilmiştir.


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Kanser Hücrelerini Tanımak İçin Akıllı Biyofonksiyonel Yüzeylerin Tasarımı

Dilek Odacı Demirkol

Ege Üniversitesi Fen Fakültesi Biyokimya Bölümü 35100 Bornova-İzmir

Kanser, kendi dokularımızdan ortaya çıkar ve hastalıklı doku ve sağlıklı doku arasındaki

benzerlikler nedeniyle hem tespiti hem de tedavi yöntemlerinin seçimini zorlaştırır. Erken tanı

kanser hastasının prognozunun iyileştirilmesinde oldukça önemlidir. Şu anda, kanserin tespiti,

öncelikle görüntüleme tekniklerine veya hastalıktan şüphelenilen hücrelerin veya dokuların

morfolojik analizi ile yapılmaktadır. Nanoteknoloji alanındaki son araştırmalar kanser tanısı ve

tedavisi için şu anda mevcut uygulamaların dezavantajlarını bertaraf etmeye odaklanmıştır.

Kanserin erken teşhisi ve tedavisi amacıyla gerçekleştirilen nanomalzeme temelli araştırmaları

dört kategoriye ayırabiliriz [1]:

(1) Ekstraselüler kanser biyobelirteçlerinin belirlenmesi,

(2) Kanser hücrelerinin dedeksiyonu,

(3) in vivo dokularda kanserli hücrelerin tespiti ve

(4) Hedefe yönelik akıllı ilaç taşıyıcı sistemlerinin geliştirilmesidir.

Bazı nanoyapılı problar; avantaj sağlayan çeşitli benzersiz özellikleri ile kanserin erken teşhis

edilmesinde ve hedeflenmiş tedavisinde kullanılmaktadırlar [2]. Karbon yapılı

nanomalzemeler, polimerik yapılı malzemeler, metalik nanopartiküller, nanolifler ve veziküler

bu bağlamda kullanım potansiyeline sahip moleküllerdir [2,3]. Burada, nanomalzemeler ve

bunların biyolojik moleküllerle konjugasyonu sonrasında elde edilen yapıların kanserin teşhis

ve tedavisindeki kullanımı hakkında güncel yaklaşımlar tartışılacaktır.

Kaynaklar

[1]Fulya Ekiz Kanik, Alexandra Sneider, Prakash Rai, Dilek Odaci Demirkol, Suna Timur, Nanomaterials in Cell Imaging (Chapter 4). Innovations in Nanomaterials (Eds: Al-Nakib Chowdhury, Joe Shapter, and Abu Bin Imran) Nova Science Publishers New York, 2015.

[2]Bahar Güler, Bilal Demir, Emine Güler, Kadri Güleç, Ozan Yeşiltepe, Dilek Odacı Demirkol, Suna

Timur, Targeting and Imaging of cancer cells using nanomaterials (Chapter 8), Applications of

Nanobiomaterials (Ed: Alexandru Mihai Grumezescu), Elsevier, 2015.

[3]Emine Guler, Bilal Demir, Bahar Guler, Dilek Odaci Demirkol, Suna Timur, Biofuctionalized Nanomaterials for Targeting Cancer Cells, Therapeutic Nanostructures; Volume II: Drug Delivery (Ed: Alexandru Mihai Grumezescu), Elsevier, 2016.


46

Nanopartiküllerin biyokimyasal ve hematolojik etkileri

Dilek U. Çakır

Tıbbi Biyokimya Anabilim Dalı, Tıp Fakültesi, Çanakkale Onsekiz Mart Üniversitesi, Terzioğlu

Kampüsü

Günümüzde yine devrim niteliğinde olabilecek maddeyi atomik boyutlarda inceleyip işlemek

fikri ve uygulamaları, bilim ve teknolojiye yeni bir boyut kazandırmıştır. Yeni devrim ve

gelecek yüzyılın teknolojisi diye adlandırılan bu gelişmeler dönemi: nanobilim ve

nanoteknoloji dönemidir. Çözünmüş, hapsedilmiş veya adsorbe haldeki etkin maddeyi

kontrollü olarak açığa çıkaran, doğal ya da sentetik yapıdaki polimerlerle hazırlanan katı

kolloidal polimerik partiküler sistemlere nanopartiküller denilmektedir.

Hedeflendirilmiş nanopartikül taşıyıcıların kullanılması nano tıpta en önemli alanlardan biridir.

İlaç hedefleme; ilaç etken maddesinin hedef doku veya organda seçici ve kantitatif toplanma

yeteneği olarak tanımlanır. Nanoteknoloji temelli ilaç salınımı; ilacın geçirgenliğini, kontrollü

salınımını ve ilacın hedefe yönelik olması özelliklerini arttırmayı hedefler. Nanokapsülleri ya

da nanopartikülleri kullanan geleneksel ilaç salınım sistemi hedefe yönelik olmadığı için,

vücuttaki normal ve hassas hücreler üzerinde yan etki ve toksik etki oluşturabilir. Son

zamanlarda geliştirilen hedefli terapilerle nanopartiküllerin hedef bölgeye ulaşma şansı

artmıştır ve toksik etkileri ve ilaç dozu azalmıştır. Nanopartiküller, normal hücrelerde toksik

etki yaratmaktan kaçınarak, tümör hücrelerindeki ilaç konsantrasyonunu arttırmak için pasif ve

aktif hedefleme stratejilerini kullanır. Bu durum özellikle tümör kemoterapisinde, sadece

çevresindeki normal hücrelere dokunulmadan seçici şekilde tümör hücrelerine ilaç verilmesi

açısından önemlidir. Elde edilen mikro/nanopartiküllerin canlı vücudunda kullanılabilir

olabilmesi için fiziksel ve kimyasal özelliklerinin iyi bilinmesi ve hücreler ile allerjik, toksik,

karsinojenik reaksiyon vermemesi istenir. Nanoteknoloji alanındaki heyecan verici gelişmelere

rağmen, nano ölçekli materyallerin toksik etkilerinin ve sağlık açısından oluşturduğu riskin

değerlendirilmesi gerekir.Bu nedenle nanotıp alanındaki araştırma trendi, nano parçacıkların

toksik özelliklerinin daha iyi anlaşılması ve azaltılması üzerinedir.

İlaç taşıyıcı olarak kullanılan yavaş bozunan ya da bozunmayan nanopartiküllerde ilaç salımı

uzun sürer ve etki bölgesinde ilacın toplanmasına neden olur bu da sonuçta kronik inflamatuar

cevaba yol açar. Nanopartiküllerin yapısında bulundurduğu maddelerden gelen fizikokimyasal

özellikler nanopartiküllerin vücutta dağılımını, kan beyin bariyerini geçişi ve kan pıhtılaşma


47

yolaklarını tetikleyebilir. Altın ve polistiren içeren nanopartiküllerin hemoliz ve kan

pıhtılaşmasına neden olduğu gözlenmiştir. Karaciğer, dalak, böbrek, lenf nodları, kalp,

akciğerler ve kemik iliğinde nanoparçacıkların toksik etkisinin olduğu rapor edilmiştir.

Raporlarda nanoparçacıkların etkisi kısa dönem için gösterilmiştir ama uzun dönemdeki etkileri

ile ilgili çalışmalara da ihtiyaç vardır. Bu açıdan, nanomateryallerin ne kadar güvenli olduğu ve

ne derece toksik etkiye sahip oldukları henüz cevaplanmamış önemli sorulardır.

Nanoparçacıkların çevre, doğa ve insan sağlığına etkilerinin incelenmesi için sağlık otoriteleri

yeni çerçeve kurallar belirlemeye çalışmaktadır. Bu doğrultuda, klasik farmasötik kimya ve

biyoteknolojinin kullandığı ve biyomateryal ruhsatlandırılması için gerekli ve geçerli olan

testlerin yanında yeni bazı deneylerin de yapılması ve standart hale getirilmesi bir ihtiyaçtır.

Mevcut gelişmeleri göz önünde bulundurarak, hedefe odaklı kanser ve diğer hastalıkların

nanoparçacıklarla tedavi edilebilmesi için, nanomateryaller ve toksisite ile ilgili ileri düzeyde

çalışmaların yapılmasına ihtiyaç duyulmaktadır.


48

Hayvanlarda Multiple Myeloma (Plazmasitom, Plasma Cell Myelloma)

Hasan İçen

Veterinerlik Fakültesi, Dicle Üniversitesi, Diyarbakır

Multipl miyelom (MM), kemik iliğinde üretilen B lenfositlerinden köken alan plazma

hücrelerinin (plazma B hücreleri ya da plazmositler) proliferasyonu ile karakterize bir plazma

hücresi malignitesi olarak tanımlanan ve paraprotein olarak bilinen homojen bir

immünoglobulin salgılarlar. Çoğunlukla köpek, kedi ve insanlarda görülmesi ile birlikte fare,

at, inek, domuz ve tavşanlarda tespit edilmiştir. Tüm malign tümörlerin <%1'ini, malign

hematopoetik tümörlerin ise ~%8'ini, biyopsi ile teşhis edilen tüm primer ve sekonder kemik

tümörlerinin ise %3,6'sını oluştururlar.

Multipl miyelomun klinik belirtileri oldukça değişkendir ve çoklu organ yetmezliklerine yol

açabilir. Etkilenen hayvanlarda; uyuşukluk, halsizlik, topallık, kemik ağrısı, kanama (örn.,

Mukoza zarındaki peteşi, dişeti kanaması ve burun kanaması), poliüri / polidipsi ve nörolojik

bozukluklar gibi klinik belirtilerle beraber rejeneratif anemi, patolojik kemik kırıkları, böbrek

yetmezliği, immün yetmezlik sonucu sekonder enfeksiyonlar, pıhtılaşma bozuklukları, kronik

anemi, kalp yetmezliği gibi belirtilere de neden olabilir.

Hayvanlarda multipl miyelom teşhisi, kemik iliği aspirasyonunda veya biyopsisinde %20 den

fazla plazma hücrelerine rastlanılması, serum ya da idrarda Bence-Jones proteinlerinin tespit

edilmesi, ile radyografik bulgularla tanı konulabilir. Biyokimyasal olarak hiperproteinemi,

hiperviskozite ve hiperkalsemi görülebilir. Multipl miyelom için genelde palyatif tedavi ile

birlikte melfalan veya siklofosfamid ile prednizondan oluşur.


49

Kanserde Çığır Açan Moleküler Hedefler ve Tedavileri

Mehmet Küçüköner

Dicle Üniversitesi Tıbbi Onkoloji Bilimdalı, Diyarbakır

Kanser, çeşitli faktörlerin genetik materyal üzerinde yaptığı değişiklikler sonucu gelişen çok

basamaklı klonal bir hastalıktır. Kanser nedeni olan genetik değişikliklerin, onkolojik tanı

uygulamalarında önemle gözönünde bulundurulması gerekir. Kanser dokusunda bu tür

mutasyonların varlığı, tanı ve tedavi seçiminde yanıltıcı olabilir. Bu mutasyonların tek başına

varlığı kanser gelişiminin nedeni olarak değerlendirilmemelidir. Bu mutasyonlar, gelişen

kanser için yalnızca kolaylaştırıcı faktörler olarak bilinmektedir. Bu nedenle saptanan genetik

değişikliklerin tedavi hedefi olarak değerlendirilebilmesi için, o mutasyonun kanser dokusuna

özgü somatik mutasyon olduğunun ileri çalışmalarla gösterilmesi gerekir. Bu sebeplerle birçok

karsinom ve sarkomlar için moleküler testler kullanılmaktadır. Klinik uygulamalarda genel

olarak kullanılan moleküler yöntemler, dizi analizi, hibridizasyon veya PCR yöntemleridir.

Kanserde moleküler hedef tedavilerin başarılı örneklerinden biri akciğer kanseridir. Üzerinde

en çok çalışılan kanser tiplerinden biri olan akciğer kanserinde son yıllarda hem patogenezini

anlama hem de tedavi yaklaşımlarını belirlemede büyük gelişmeler yaşanmıştır. Pek çok

moleküler değişiklik driver mutasyonlar/moleküler değişiklik olarak tanımlanmıştır. Driver

mutasyonlar/moleküler değişiklikler kanserin başlangıcından ve gelişiminden sorumludur.

Özellikle hedefe yönelik tedavi sürecinde son derece etkili farmakolojik formları da bulunan

epidermal büyüme faktörü reseptörü (EGFR), anaplastik lenfoma kinaz (ALK) ve proto-

onkojen tirozin-protein kinaz 1 ROS (ROS1) gibi moleküler değişiklikler, akciğer kanseri

tedavisinde yeni bir çığır açmıştır. Büyüme faktör reseptörlerine karşı geliştirilen monoklonal

antikorlar ve hücre içi sinyal iletiminde rol alan tirozin kinaz reseptörlerini inhibe eden ilaçlar

kanser tedavisinde kullanılmaktadır. Son yıllarda T lenfosit ilişkili protein 4 (CTLA-4),

programlanmış ölüm-1 (PD-1) ve programlanmış ölüm ligandı 1’e (PD-L1) yönelik geliştirilen

antikorlar kanser tedavisinde ön plana çıkmışlardır. Ayrıca siklin bağımlı kinaz inhibitörleri,

Poli (ADP-Riboz) Polimeraz-1 (PARP) inhibitörleri bazı kanser türlerinde tedavi seçeneği

olmuştur.

Her geçen gün yeni bir hedefe yönelik tedavi ajanının ortaya atıldığı bu dönemde doğru hastaya

doğru tedavinin uygulanabilmesi için moleküler testler önem kazanmaktadır. Moleküler testler

sadece tedavi sürecinin belirlenmesinde değil, aynı zamanda tedavi direnç mekanizmalarının


50

anlaşılmasında ve çözümünde, hastalıkların prognozunun tahmininde ve daha da önemlisi

kanserin patogenezinin anlaşılmasında rol almaktadır.


51

KANSER TEDAVİSİNDE HİSTON DEASETİLAZ İNHİBİTÖRLERİ

Ferda Arı

Bursa Uludağ Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Moleküler Biyoloji Anabilim

Dalı 16059 Nilüfer/BURSA

Epigenetik terimi DNA dizisinde değişiklik oluşturmadan gen ekspresyonunda gelişen

kalıtımsal değişikliklerdir. Epigenetik mekanizmalar; DNA metilasyonu, histon

modifikasyonu, kromatinin yeniden modellenmesi olarak tanımlanmaktadır. Bu süreçler DNA

yapısına, DNA-bağlanma proteinlerinin yapısına, RNA ve protein degredasyonuna etki ederek

gen fonksiyonunun kontrolünü sağlamaktadır. Epigenetik mekanizmalarda ki işleyiş

bozuklukları özellikle kanser gibi çeşitli hastalıkların oluşumunda önemlidir.

Histon modifikasyonları histon proteinlerinin posttranslasyonel modifikasyonları olup; N-

terminal kuyruğunda gerçekleşir. Gen fonkiyonuna, kromatin yapısına, gen ekspresyonuna etki

edebilen histon modifikasyonları aynı zamanda DNA tamirinde ve replikasyonunda işlev

görmektedirler. Histon modifikasyonları çoğunlukla asetilasyon, metilasyon, fosforilasyon,

glikolizasyon şeklinde olmaktadır. Histonların asetilasyonunun, pozitif yüklerini nötralize

ettiği ve negatif yüklü DNA ile olan etkileşimlerini gevşettiği düşünülmektedir. Histonların

histon deasetilazlar (HDAC) tarafından deasetilasyonu, DNA ile olan etkileşimini sıkılaştırır

ve kapalı bir kromatin yapısına ve gen transkripsiyonunun engellenmesine neden olmaktadır.

Histon modifikasyonunun düzenlenmesinin yanı sıra HDAC'lar, transkripsiyon faktörleri,

şaperonlar ve sinyal molekülleri de dahil olmak üzere birçok histon proteininin post-

translasyonel asetilasyon durumunu düzenleyerek protein stabilitesinde, protein protein

etkileşimlerinde ve protein-DNA etkileşimlerinde değişikliğe neden olabilmektedirler.

HDAC’lar genetik olarak yüksek oranda korunmuş olsalar da kanserdeki rollerine odaklanan

mevcut veriler birçok tümör yapısında farklı ekspresyon düzeylerine sahip olduklarını

göstermiştir. HDAC’ların tümörijenezdeki birçok moleküler yolakta etkilerinden dolayı

hematolojik malignitelerde ve solid tümörlerde uzun süredir etkin hedef olarak

görülmektedirler. Bu amaçla çok sayıda HDAC inhibitörü (HDACi) doğal kaynaklardan

saflaştırılmış veya sentezlenmiştir. HDACi’leri hidroksamatlar, siklik peptidler, alifatik asitler

ve benzamitler olarak sınıflandırılmaktadır. HDACi, özellikle çeşitli kemoterapi ve/veya

radyoterapiyle kombinasyon halinde etkin bir anti-kanser tedavi seçeneği sunmaktadır. Ayrıca

birçok in-vitro çalışmada antitümör etkinliği farklı kanser modellerinde gösterilmiştir.

Vorinostat, romidepsin ve belinostat HDAC inhibitörleri T-hücreli lenfoma ve multipl miyelom


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için FDA tarafından onaylanmıştır. Solid malignitelerin tedavisi için ise Vorinostat, Valproik

asit, CHR-3996 ve Practinostat’ın devam eden veya tamamlanan klinik faz çalışmaları

bulunmaktadır. Bunlara ilaveten, son yıllarda bazı HDACi’lerinin kanser kök hücreleri ve

epitelyal-mezenkimal geçiş üzerine olan etkileri araştırılmakta ve umut verici sonuçlar

alınmaktadır.

Sonuç olarak, HDACi’leri kanserde epigenetik tedavide kullanılabilecek potansiyel moleküller

olup daha ileri klinik analizlerin yapılması gerekmektedir. Araştırmalar ilerledikçe şüphesiz ki

kanser tedavisinde HDACi’leri önemli bir yer alacaktır.


53

Over Kanserinde Likit Biyopsi ile Elde Edilen Moleküllerle Biyobelirteç Çalışmaları

Tuba Günel

İstanbul Üniversitesi Fen Fakültesi Moleküler Biyoloji ve Genetik Bölümü

Biyoteknoloji ve Genetik Mühendisliği Araştırma ve Uygulama Merkezi (BİYOGEM)

Tumor biomarkers are molecules that are produced by cancer cells or cells around them, which

can be measured in body fluids or in the blood during the diagnosis, screening or treatment of

cancer so-called “liquid biopsy”. Molecules that can be used as tumor biomarkers can be

counted as cytoplasmic proteins, enzymes, hormones, surface antigens, receptors, oncofetal

antigens (reemerging proteins in cancer that is normally lost after birth), oncogenes or their

products. An ideal tumor biomarker should be sensitive enough for early detection of small

tumors while retaining the specificity of the identified cancer type. Recent studies have

discovered a great number of non-invasive tumor diagnosis and screening has become an

important area of study

Contrary to tissue biopsy, through detection of circulating tumor cells (CTCs), tumor nucleic

acids (“circulating tumor DNA/RNA”), and exosomes, predictive and prognostic markers may

potentially be developed which is far less invasive. Hence early and multiple evaluations of the

disease can be made, including retrospective follow-up, identification of treatment effects and

investigation of clonal development. Isolation and characterization of CTCs, exosomes, and

circulating tumor DNA (ctDNA) will improve cancer diagnosis, treatment, and imaging. Liquid

biopsy can be performed “real-time” and at every stage of cancer. The asymptomatic patients,

it can be used to identify to improve early diagnosis and better intervention. Although, it has

some potential disadvantages such as; still is not certain to use in cancer diagnosis, difficulties

in analysis of data obtaining from high-throughput screening and lack of data verification

through clinical trials; it has significant potential for clinical cancer diagnosis in future

Ovarian cancer is one of the most important and fatal gynecological cancer types worldwide.

The biomarkers with high sensitivity and specificity obtained from the studies performed with

liquid biopsy will increase the five-year survival rate.

54

ORAL PRESENTATIONS

Oral Presentation

55

Stone Alkaline Water Induces Apoptosis of Prostate Cancer Cells and Inhibits Tumor

Cell Induced Angiogenesis In Vitro

Sila Appak-Baskoy1, lknur Kulcanay Sahin2, Ozgun Teksoy3, Mustafa Cengiz4, Asuman

Deveci Ozkan5, Gamze Guney Eskiler5, Namık Bilici6, Pinar Oztopcu Vatan3, Adnan

Ayhanci3

1Ryerson University, Faculty of Science, Toronto, Ontario, Canada

2Kırıkkale University, Vocational School of Health Services, Kırıkkale, Turkey

3Eskisehir Osmangazi University, Faculty of Science and Arts, Department of Biology, Eskisehir, Turkey

4Siirt University, Faculty of Education, Department of Elementary Education, Siirt, Turkey

5Sakarya University, Department of Medical Biology, Faculty of Medicine, Sakarya, Turkey

6Karabük University Faculty of Medicine Department of Medical Pharmacology, Karabuk, Turkey.

Prostate cancer is the second most common cancer in men. Treatment options to improve

overall survival rate of patients are limited since tumor cells acquire resistance to the

chemotherapeutic drugs. Thus, new alternative therapeutic strategies are urgently needed to

improve the overall life quality and expectancy of the patients. Stone alkaline water (SAW) is

a high alkaline mineral water that is obtained by breaking certain stone and it may have

anticancer effects due to its rich mineral content. Among the most noteworthy minerals in SAW

are sodium (Na) and magnesium (Mg). In this study, we aimed to determine anticancer effects

of SAW on PC-3 and DU-145 prostate adenocarcinoma cell lines. Our findings indicated that

SAW treatment significantly decreased cell viability and induced apoptotic cell death through

accumulation of cells at G0/G1 phase. Additionally, SAW treatment was found to be more

effective in PC-3 cells that have more metastatic activity than DU-145 cells that have a

moderate metastatic activity. Furthermore, treatment of the prostate cancer cells with SAW

inhibited tumor cell induced HUVEC tube formation and migration stating that SAW inhibits

tumor cell derived angiogenesis in vitro.

Key Words: Prostate cancer, Alkaline Water, Apoptosis, Angiogenesis

Oral Presentation

56

MALT1 PARACASPASE INHIBITION REDUCES HEPATOCELLULAR

CARCINOMA CELL SURVIVAL

Asli Kurden Pekmezci1,2, Ozlem Silan Coskun1,2, Ece Cakiroglu1,2, Dilara Demirci2, Serif

Senturk1,2

1 Dokuz Eylul University Izmir International Biomedicine and Genome Institute,

2 Izmir Biomedicine and Genome Center

Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths around the world

and is associated with several etiological factors including infections with hepatitis B and C

viruses, heavy alcohol consumption and chronic aflatoxin B1 exposure. Treatment strategies

for HCC are currently limited. Hepatocarcinogenesis process involves alterations in several

molecular pathways including activation of NF-κB pathway. MALT1 (Mucosa-Associated

Lymphoid Tissue Lymphoma Translocation Protein 1) is a cytosolic signaling molecule which

acts both as a scaffold protein and as a protease for NF-κB pathway activation. To date, the

contribution of MALT1 to HCC cell survival has not been studied. In the present study, we

investigated the effects of MALT1 inhibition on HCC cell survival. For this purpose, we

depleted the MALT1 levels in HCC cell lines via RNAi system and small molecule inhibitor

and we analyzed the cell survival with different methods. Based on the preliminary results,

shRNA-mediated gene silencing or pharmacological inhibition with MI-2 reduced cell growth

in vitro, and this decrease in cell growth is associated with inhibition of cell proliferation as

well as reduced expression of well-known NF-kB downstream targets, an indirect indication of

NF-κB pathway inactivation. Conclusively, our results suggest that MALT1 plays an important

role in HCC cell growth. Further studies focusing on delineating the molecular mechanisms

underlying the growth regulating effects of MALT1 are currently underway. By all means, we

think that pharmacological inhibition of MALT1 in HCC is a promising avenue to be explored

further.

Key Words: Hepatocellular carcinoma, NF-κB, MALT1, RNAi

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57

In vitro and In vivo Investigation of Mangiferin and Paclitaxel combination on Ehrlich

Ascites Carcinoma.

Ayca Uvez1, Engin Ulukaya2, Elif Ilkay Armutak1

1Istanbul University-Cerrahpasa, Faculty of Veterinary Medicine, Department of Histology and

Embryology, Istanbul-Turkey

2Istinye University, Faculty of Medicine, Department of Clinical Biochemistry, Istanbul-Turkey

Aims: Recent years, combinatory usage of various natural compounds in cancer therapy is

aimed to increase activate of chemotherapeutic agents Mangiferin is a polyphenol that has been

reported may have an important role in cancer. The aim of this study was to investigate in vitro

and in vivo effects of Mangiferin and Paclitaxel alone and in their combinations on cell

proliferation, apoptosis, autophagy and survival pathway In addition, the effect on angiogenesis

was evaluated by HUVEC tube formation method and in vivo CAM model.

Methods: The effect of Mangiferin (6.25-300 µM) and Paclitaxel (0.25-15.9 µM) alone and in

combination on (Ehrlich Ascites Carcinoma) EAC and HUVEC cells were assessed by ATP

cytotoxicity test. In combination with Mangiferin and Paclitaxel, apoptosis, autophagy, and

survival markers were determined by western blot. The effect of Mangiferin (12.5-50 µM) and

Paclitaxel (1.99-15.9 µM) alone and in combination on HUVEC cells were evaluated at 0-16 h

by tube formation test. Mangiferin (12.5-5 mg/ml) and Paclitaxel (0.3-0.0375 mg/ml µM) alone

and in combination were treated in CAM assay. Seventy eight female Balb/c mice with EAC

solid tumors were administered 12.5 mg/kg Paclitaxel by subcutaneously and 25-50-100 mg/kg

Mangiferin by gavage. Tumor volumes determined by caliper then proliferation, apoptosis,

autophagy, and survival markers were evaluated by immunohistochemistry.

Results: The ATP test results showed that the cytotoxic effect increased synergistically in a

dose and cell type dependent in the combination group. According to evaluation of western blot

results, the increase of LC3B with the decrease of p62 along with the increase of FAS with

cleavage of PARP1 was shown that apoptosis occurred through the external pathway in the

presence of autophagy. Furthermore, the survival parameters were suppressed. The in vitro

results were parallel to in vivo. The most effective dose on tumor size, tumor weight, apoptosis,

autophagy, and survival were determined as Mangiferin 50 mg/kg alone and combination. In

vitro tube formation test was observed that Mangiferin 50 µM dose was effective alone. On the

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in vivo CAM assay, mangiferin was shown more effective antiangiogenic effect in combination

groups.

Conclusions: According to results, Mangiferin and Paclitaxel combinations selectively

increased apoptosis in the presence of autophagy, decreased proliferation and survival

parameters, suppressed tumor size and observed strong antiangiogenic effect. In vivo, low doses

of Mangiferin combination with Paclitaxel were shown stronger synergistic effect than alone.

Mangiferin could be a promising potential for the development of new strategies in the

treatment of cancer.

Keywords: Mangiferin, Breast Cancer, Paclitaxel, Apoptosis, Autophagy.

The present work was supported by the Research Fund of Istanbul University-Cerrahpasa.

Project No. TDK-2017-26107.

Oral Presentation

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Akciğer Kanseri ve KOAH’da Biyobelirteç olarak aday miRNA Araştırması

Aydın Demiray

Pamukkale Üniversitesi Tıbbi Genetik Anabilim Dalı, Denizli, Türkiye ([email protected])

Genel bilgiler: Akciğer kanserleri, kanser nedenli ölüm sebepleri arasında ilk sırada, insidansı

üçüncü sırada yer almaktadır (1). Akciğer kanserlerinin %85’i Küçük Hücre Dışı Akciğer

Kanserleri (KHDAK) oluşturmaktadır (1). Kronik Obstrüktif Akciğer Hastalığı (KOAH),

genellikle zararlı partikül veya gazlara ciddi maruziyetin neden olduğu havayolu ve/veya

alveoler anormalliklere bağlı kalıcı hava akımı kısıtlanması ve solunumsal semptomlarla

karakterize, yaygın, önlenebilir ve tedavi edilebilir bir hastalıktır (2). Yapılan çalışmalarda

KOAH akciğer kanseri gelişimi açısından artmış risk ile ilişkilendirilmiştir. Akciğer kanseri

hastalarının % 40-70'ini KOAH hastaları oluşturmaktadır (3). Bu durumun ana etmeni sigara

kullanımı öngörülmektedir. Biz bu çalışmamızda sigara kullanan 30 KHDAK ve 30 KOAH

hastalarında serum düzeyinde prediktif bir faktör olarak miRNA araştırması yaptık.

Materyal-Metod: Pamukkale Üniversitesine başvuran 30 KHDAK ve KOAH hasta

serumlarından miRNA izolasyonu elde edilerek cDNA (ABM) çevirimi gerçekleştirilmiş. Bu

cDNA’lar literatürde belirlenen 20 miRNA (ABM) ile panel oluşturularak incelenmiştir.

Sonuçlar SPSS 17. Paket programında analiz edilmiştir.

Sonuç: Elde edilen miRNA profili normal akciğer hücre hattı MRC-5 normalizasyonu

sağlanmıştır. Analiz sonucunda KHDAK kanseri hastalarında hsa-miR-26a ve hsa-miR-93

KOAH hastalarına oranla daha yüksek ifade edildiği ve hsa-miR-20b ifadesi de her iki grupta

yüksek olarak saptanmıştır.

Tartışma: KOAH hastalarının sigara içimine bağlı olarak KHDAK oluşumu rapor edilmiştir

(3). Buna bağlı olarak sigara içen KOAH hastalarında KHDAK oluşumu prediktif miRNA

taraması yapılmıştır. Pinelo ve arkadaşlarının ve Leidinger ve arkadaşlarının yapmış oldukları

çalışmalar ile sonuçlarımız örtüşmektedir (4,5). Bizim çalışmamızda KHDAK hastalarında hsa-

miR-26a ve hsa-miR-93 yüksek ifade edilirken her iki grupta hsa-miR-20b yüksek ifade

edilmiştir. Ayrıca hsa-miR-26a ve hsa-miR-93 ise PI3K/AKT yolağında aktifleyen

miRNA’lardır. hsa-miR-26a PTPN13 hedefleyerek src aktivasyonu sonucu EGFR alt yolağını

aktiflediği hsa-miR-93 ise PTEN baskılanması sonucu PI3K/AKT yolağını aktiflediği rapor

edilmiştir. hsa-miR-26a ve hsa-miR-93 ikisi de KHDAK’lerinde onkomiR olarak rapor


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edilmiştir (6,7). Ayrıca hsa-miR-20b literatürde akciğer kanserlerinde BTG3 geni üzerinden

p53 baskılanmasını ortadan kaldırdığı rapor edilmiştir (8). hsa-miR-20b her iki grupta artmış

olmasının moleküler yolakların aydınlatılması KOAH’ın KHDAK dönüşümünü

aydınlatabileceği öngörülmektedir.

Referans

(1) Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A. Cancer Treatment and Survivorship Statistics, 2012. CA-Cancer J Clin 2012; 62: 220-241.

(2) Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2017. Available from: http://goldcopd.org.

(3) Wu AH, Fontham ET, Reynolds P, Greenberg RS, Buffler P, Liff J, et al., Previous lung disease and risk of lung cancer among lifetime nonsmoking women in the United States. American journal of epidemiology,1995:141(11);1023-1032.

(4) Petra Leidinger, Andreas Keller, Anne Borries, Hanno Huwer, Mareike Rohling, Junko Huebers, Hans-Peter Lenhof, Eckart Meese Specific peripheral miRNA profiles for distinguishing lung cancer from COPD. Lung Cancer 2011:74;41-47

(5) Sonia Molina-Pinelo, M. Dolores Pastor, Rocı´o Suarez, Beatriz Romero-Romero, Miriam Gonza´lez et al. MicroRNA clusters: dysregulation in lung adenocarcinoma and COPD. Eur Respir J 2014; 43: 1740–1749

(6) Shudi Xu, Tao Wang, Zhiwei Yang, Ying Li, Weijie Li et al. miR-26a desensitizes non-small cell lung cancer cells to tyrosine kinase inhibitors by targeting PTPN13. Oncotarget 2016:29(7);45687-45701

(7) Wei Yang, Jinquan Bai, Dayong Liu, Shuwei Wang, Nan Zhao et al. MiR-93-5p up-regulation is involved in non-small cell lung cancer cells proliferation and migration and poor prognosis. Gene 2018:647;13-20

(8) LIJUN PENG, SHAOBIN LI, YUCHAN LI, MINGHUI WAN, XISHENG FANG et al. Regulation of BTG3 by microRNA-20b-5p in non-small cell lung cancer. ONCOLOGY LETTERS 2019:18;137-144

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Underlying primary immunodeficiency in patients with lymphoma

Baran Erman

Department of Molecular Biology and Genetics, Istinye University, Istanbul, Turkey

[email protected]

Primary immunodeficiencies (PIDs) are rare and heterogeneous congenital diseases of immune

system. The most common clinical symptoms in PID patients are high susceptibility to

infections, inflammation, allergy, autoimmunity and malignancies. The vast majority of

primary immunodeficiency diseases shows monogenic inheritance and almost 400 genetic

defects have been identified so far.2 The overall prevalence of PIDs is approximately 1:10.000.

However, the high consanguinity and fertility rates lead to a higher prevalence in some countries

like Turkey.

After severe infections, malignancy is the most prevalent cause of death in both children and

adults with PIDs. The possible explanation is impaired immunosurveillance mechanisms leads

cancer development in the patients with PID. PIDs more often associated with cancer include

common variable immunodeficiency (CVID), Wiskott–Aldrich syndrome, ataxia-

telangiectasia, and severe combined immunodeficiency. Hematologic cancers, especially

lymphoma, are the most common type of malignancies in PID patients.

We identified PID mutations in two patents following with lymphoma by next generation

sequencing (NGS). In last decade, NGS technologies have been facilitated rapid genetic

diagnosis in complex congenital diseases like PIDs. We applied whole exome sequencing and

determined two distinct PIDs due to mutations in CARD9 and CD70 genes in the patients. The

first patient is a 8-year-old boy received hematopoietic stem cell transplantation from HLA-

matched mother due to EBV-associated lymphoma. NGS analysis revealed homozygous

missense, c.332C>T mutation in CD70 gene. Other two siblings of the patient had the same

mutation, as well. In the other patient, a 14-year-old boy, disseminated lymphadenopathies were

detected. He had also weight loss and lymphoma was considered first. However, we found

homozygous, nonsense c.883C>T mutation in CARD9 gene.

In conclusion, underlying PID should be considered in the patients presented with lymphoma.

Moreover, NGS based approaches may be the first option for a rapid and accurate genetic

diagnosis in PID patients.

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Determination of MiRNAs and Xeno-miRs Targeting Most of the Genes Associated

with Colorectal Neoplasms and Central Obesity by "in silico" Analysis

Berkcan Dogan1,2

1Uludag University, Faculty of Medicine, Department of Medical Genetics, Bursa, Turkey, 2Istanbul University, Institute of Graduate Studies in Science, Department of Biology and Genetics,

Istanbul, Turkey

[email protected]

Scope: Recent studies have found a close connection between obesity and the development of

colorectal neoplasia. It is known that central obesity is associated with an increased risk of

colorectal cancer and that these diseases have common genes associated with pathogenesis.

Hsa-miR-335-5p, targeting the highest number of identified mutual genes, regulated by DNA

methylation that has been reported to possess both tumor suppressor and tumor promoter

activities. Also, upregulation of hsa-miR-335-5p correlated with obesity in adipose tissue in

mice. The purpose of this study specifies common genes for colorectal neoplasms and central

obesity (CN&CO) and microRNAs (miRNAs) and Xeno-miRs targeting these genes that may

involve in the pathogenesis of CN&CO diseases’ via “in silico” analysis.

Methods and Results: Biomolecules that were reported to be associated with both conditions

have been searched by using Ingenuity Pathways Knowledge Base (IPKB) in Ingenuity

Pathway Analysis (IPA) software (QIAGEN Inc., Germany). Nineteen common genes (ACE,

ACE2, ADRA1A, ADRA1B, ADRA1D, CA1, CA12, CA14, CA2, CA5A, CA7, CYP2E1, ESR1,

GCG, GH1, GHR, NISCH, REN and SLC5A2) are involved in the pathogenesis of CN&CO by

IPKB. Using miRNet (www.mirnet.ca), six genes (ACE, CA12, CA14, CA5A, CYP2E1, REN)

targeted by hsa-miR-335-5p in nineteen genes have been found. Hsa-miR-335-5p is upregulated

in CN&CO and suppresses its target genes as reported by IPA software and it is correlated with

other experimental studies. Subsequently, analysis conducted with Dietary MicroRNA

Database showed that Xeno-miR bta-miR-335 was found only in Bos taurus, and this matched

%100 sequence similarity.

Conclusion: Hsa-miR-335-5p targets the highest number of colorectal cancer- and obesity-

related genes and shows same sequence of bta-miR-335 in bovine. MiR-335-5p can have a

therapeutic value for cancer- and obesity-related disorders. Consequently, personalized dietary

interventions can improve obesity-colorectal cancer management if these results can be

validated by using human cell lines and model organisms experimentally.

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Investigation of DHRS2 Gene Effects on Breast Cancer Cell Line

Burcu Salman1, Sema Sırma Ekmekci1, Suzan Çınar2, Neslihan Abacı1

1Department of Genetics, Aziz Sancar Institute of Experimental Medicine, İstanbul University, Turkey

2Department of Immunology, Aziz Sancar Institute of Experimental Medicine, İstanbul University,

Turkey

Breast cancer is the most common malignancy in women and takes the second place between

all cancers. Even though recent research increased life quality of patients and therapy

succession, our knowledge on molecular mechanisms and the tumor behavior on this cancer is

limited. Interactions between p53 and Mdm2 molecules are widely researched but the

regulatory mechanisms of these proteins are still not fully known. Mdm2 and p53 proteins affect

cell metabolism and regulate each other. The dehydrogenase/reductase gene we selected to

study, DHRS2, regulates p53 via suppressing Mdm2. We aimed to investigate the relationship

between p53 and DHRS2. To better understand this regulation, DHRS2 gene expression on

MCF7 cell line was suppressed via RNAi. On the control group and DHSR2 siRNA applied

experiment group, apoptosis percentage was measured on the flow cytometry. In addition, cell

proliferation and viability were evaluated with MTT assay and cell staining with trypan blue.

Last of all migration test were performed using scratch assay method which is very commonly

used nowadays. 24- and 48-hours groups were compared. Apoptotic cell count was lower on

DHRS2 suppressed cells according to the control group. Again, results on DHRS2 gene

suppressed cells showed us decelerate wound recovery and decreased cell viability and

proliferation. Decreased apoptotic cell count on experiment group supports increased protein

amount of Mdm2 and the decreased p53 because of active Mdm2. Our findings on the cell

migration could use as a lead on this research area. This thesis research shows the specialty of

being the first research on our country that specifically investigates DHRS2 gene on breast

cancer.

This work was supported by Scientific Research Projects Coordination Unit of Istanbul

University (grant number 30936).

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Bir seramidaz inhibitörü olan N-Oleoylethanolamine bileşiğinin A549 ve Beas-2B

hücreleri üzerindeki sitotoksik ve morfolojik değişimlerinin araştırılması

Emre Çömlekçi1, Hüseyin İzgördü1, Canan Vejselova Sezer1*, O. Tansel Korkmaz2, Hatice

Mehtap Kutlu1

1Eskişehir Teknik Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Eskişehir, Türkiye 2Osmangazi Üniversitesi, Tıp Fakültesi, Fizyoloji Anabilimdalı, Eskişehir, Türkiye

*Sorumlu yazar: [email protected]

Sfingolipid metabolizmasında bulunan enzimler hücrenin sağ kalım ve ölümünde önemli roller

oynamaktadır ve bu metabolizmanın merkez molekülü olan seramidin kanserli hücreleri

apoptoza teşvik ettiği ortaya konmuştur. Yeni nesil kanser çalışmalarında sfingolipidler önemli

bir yer tutmaktadır. Seramid ve sfingozin katabolizmasından sorumlu bir enzim olan

seramidazların inhibitörü olan N-Oleoylethanolamine (NOE) seramidin sfingosine

dönüşümünü engellemektedir ve bununla birlikte farklı kanser hücrelerinde apoptozu

tetiklediği bildirilmiştir. Yaptığımız çalışmada, N-Oleoylethanolamine (NOE) bileşiğinin

akciğer kanseri hücreleri ve sağlıklı akciğer hücreleri üzerindeki sitotoksisitesinin ölçülmesi ve

hücreler üzerindeki morfolojik değişiklerin saptanması amaçlanmıştır. Sitotoksisiteyi tespit

etmek için MTT testi kullanılmıştır. A549 ve Beas-2B hücreleri, 96 kuyucuklu plakalarda 24

saat boyunca farklı konsantrasyonlarda muamele edilmiştir. İnkübe edilen hücreler, 570 nm

dalga boyunda bir plaka okuyucu kullanılarak spektrofotometrik olarak ölçülmüştür. Elde

edilen absorbanslardan canlılık yüzdeleri hesaplanmış ve IC50 değeri belirlenmiştir. A549

hücrelerine farklı konsantrasyonlarda N-Oleoylethanolamine (NOE) uygulanması ile 24 saat

içinde hücre ölümünde önemli bir artışa neden olduğu görülmüştür. Bu çalışmada NOE ile

tedavi edilen A549 ve Beas-2B hücreleri, morfolojik değişikliklerin tespit edilmesi amacıyla

konfokal mikroskop altında incelenmiştir. A549 ve Beas-2B hücreleri, sitotoksisite analizi ile

hesaplanan IC50 değeri ile uygulanan NOE konsantrasyonları ile inkübe edilmiştir. İşlem gören

hücreler Alexa fluor-488 phalloidin ve akridin oranj ile tespit edilerek boyanmış ve daha sonra

konfokal mikroskop altında gözlemlenmiştir. Konfokal mikroskopi ile NOE'nin insan akciğer

kanseri hücreleri üzerindeki apoptotik etkileri gösterilmiştir. Çalışmamızdan elde edilen

sonuçlara göre, N-Oleoylethanolamine (NOE) bileşiğinin insan akciğer kanseri tedavisinde bir

anti kanser ajan olma potansiyeline sahip olduğu gösterilmiştir ve daha ileri deneylerin

gerçekleştirilmesi tarafımızdan önerilmektedir.

Anahtar Kelimeler: N-Oleoylethanolamine, NOE, akciğer kanseri, apoptoz.

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Doksorubisin İle İndüklenen Sıçan Nefrotoksisite Modelinde Fluvastatinin Olası

Koruyucu ve Tedavi Edici Etkilerinin Histokimyasal ve İmmünohistokimyasal Olarak

Değerlendirilmesi

Fatih Oltulu1, Çevik Gürel1,2

1Ege Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilin Dalı, İzmir, Türkiye 2Harran Üniversitesi Tıp Fakültesi, Histoloji ve Embriyoloji Anabilim Dalı, Şanlıurfa, Türkiye

Amaç: Doksorubisin, solid tümörlerin ve hematolojik malignitelerin tedavisinde sıklıkla

kullanılan fakat başta kalp ve böbrek olmak üzere birçok organda ortaya çıkan akut ve kronik

yan etkiler nedeniyle kullanımı önemli ölçüde kısıtlanan antrasiklin türevi bir antibiyotiktir. Bu

çalışmada amaç, doksorubisin ile indüklenen nefrotoksisite modelinde fluvastatinin olası

koruyucu ve tedavi edici etkilerinin ve bu etkilerin altında yatan moleküler mekanizmalarının

araştırılmasıdır.

Gereç ve Yöntemler: Bu çalışma için 35 adet yetişkin erkek Sprague Dawley türü sıçan

kullanılmıştır. Sıçanlar rastgele; Kontrol (n=7), Fluvastatin (n=7), Doksorubisin (n=7),

Profilaksi (n=7) ve Tedavi (n=7) gruplarına ayrılmıştır. Kontrol grubuna deneyin 1.,4. ve 7.

günlerinde serum fizyolojik uygulaması yapılmış başka herhangi bir uygulama veya girişim

yapılmamıştır. Fluvastatin grubundaki hayvanlara ise 7 gün boyunca, gavaj ile 6 mg/kg/gün

fluvastatin uygulaması yapılmıştır. Doksorubisin, Proflaksi ve Tedavi gruplarında, deneyin 1.,

4. ve 7. günlerinde, 7,5 mg/kg/gün doksorubisin uygulaması yapılarak nefrotoksisite modeli

indüklenmiştir. Proflaksi grubuna, 1., 4. ve 7. günlerde doksorubisin uygulamasından bir saat

önce olacak şekilde, deneyin ilk gününden başlanarak 7 gün boyunca gavaj ile 6 mg/kg/gün

fluvastatin uygulanmıştır. Tedavi grubundaki hayvanlara ise 8. günden itibaren 7 gün boyunca

gavaj ile 6 mg/kg/gün fluvastatin uygulanmıştır. Deney protokolünün sonunda, sakrifiye edilen

sıçanlardan alınan örnekler histokimyasal, immünohistokimyasal, biyokimyasal ve Real-Time

PCR analizleri ile değerlendirilmiştir.

Bulgular: Histopatolojik ve biyokimyasal değerlendirmede doksorubisin grubunda

vakualizasyon, kollajen miktarında artış, Bowman kapsülünde kalınlaşma ve Bowman

boşluğunda daralma, intersitisiyel ödem, infiltrasyon, tübüler dilatasyon, tübül epitel

hücrelerinde dökülmeler, MDA seviyesinde artış ve SOD aktivitesinde azalma gibi patolojik

değişiklikler saptanmıştır. Bunanla birlikte, proflaksi ve tedavi gruplarında, doksorubisin

grubunda gözlemlenen patolojik bulguların çoğunun bulunmadığı görülmüştür. Yapılan

immünohistokimyasal ve Real-Time PCR analizlerinde elde edilen sonuçlar ise doksorubisin

uygulamasının böbreklerde mTOR mRNA ve protein ekspresyonlarını baskıladığını buna

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karşın LC3 mRNA ve protein ekspresyonunda ise anlamlı bir artışa neden olduğunu

gözlemlenmiştir. Öte yandan, proflaksi ve tedavi gruplarında doksorubisin grubuna oranla,

mTOR ekspresyonlarında artış ve LC3 ekspresyon seviyelerinde ise anlamlı bir azalma olduğu

tespit edilmiştir.

Sonuç ve Tartışma: Yapılan analizlerden elde edilen sonuçlar, doksorubisin kaynaklı

nefrotoksisitede fluvastatinin, antioksidan sistemin aktivasyonu ile lipit peroksidasyonunun

inhibisyonu ve otofaji yolağının regülasyonu yoluyla bu kemoterapötik ajanın kullanımından

kaynaklanan patolojileri önleyebileceğini göstermektedir. Ek olarak, fluvastatinin profilaktik

olarak kullanımının, tedavi amaçlı kullanımına kıyasla daha olumlu sonuçlar verdiği

gözlemlenmiştir.

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Abnormal Expression Patterns of Repetitive DNA Elements Uncovered in Breast Cancer

Cihangir Yandım1, 2,, Gökhan Karakülah2, 3*

1İzmir University of Economics, Faculty of Engineering, Department of Genetics and Bioengineering, 35330, Balçova, İzmir, Turkey

2İzmir Biomedicine and Genome Center (IBG), Dokuz Eylül University Health Campus,

35340, İnciraltı, İzmir, Turkey 3İzmir International Biomedicine and Genome Institute (iBG-İzmir), Dokuz Eylül University,

35340, İnciraltı, İzmir, Turkey

e-mails: [email protected] ; [email protected]

*corresponding

Repetitive DNA elements, which constitute more than half of the human genome with more

than a thousand types of distinct motifs 1,2, are normally hardly expressed in healthy cells with

a stable genome3. Notably, a common feature of breast cancer is genomic instability, which is

associated with heterogeneity and resistance to treatment 4. The contribution of repeats to this

pathogenic phenomenon largely remained unexplored mostly due to difficulties in mapping the

repeats using sequencing data. Only a handful of repeats (i.e. HERVK, HSATI and II) were

previously reported in breast cancer literature 5-7, and a genome-wide analysis was needed to

get a global picture. Hence, we analysed an RNA sequencing dataset comprising the

transcriptomes of ER+/HER2- breast tumours obtained from 22 patients along with their

healthy breast tissues 8. We used ultimate bioinformatics tools that allow proper repeat

quantification 9,10 and identified 56 repeat motifs being significantly dysregulated in tumours;

many of them being novel for cancer literature. Among these, D20S16 and REP522 satellites

globally correlated well with breast cancer driver genes such as AKT, mTOR, KRAS, BRCA1

and BRCA2. We also studied how the repeat motifs could interfere with the expressions of

proximal genes through regression analysis, and determined transposons such as L2b, L2c,

MER5B or LTR5_Hs to be potentially involved in the dysregulation of breast cancer genes.

Our results not only open a new avenue of research for breast cancer but also provide a guiding

bioinformatics pipeline on how repetitive DNA, which is often overlooked, could be studied in

the context of cancer genomics. The repeats identified in this study may serve as biomarkers

for assessing genomic instability or prognosis in breast cancer patients, and warrant further

research in terms of understanding complex events that take place in the axis of genomic

distortion underlying this devastating disease.

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References

1 de Koning, A. P., Gu, W., Castoe, T. A., Batzer, M. A. & Pollock, D. D. Repetitive elements

may comprise over two-thirds of the human genome. PLoS Genet 7, e1002384,

doi:10.1371/journal.pgen.1002384 (2011).

2 Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature

409, 860-921, doi:10.1038/35057062 (2001).

3 Saksouk, N., Simboeck, E. & Dejardin, J. Constitutive heterochromatin formation and

transcription in mammals. Epigenetics & chromatin 8, 3, doi:10.1186/1756-8935-8-3

(2015).

4 Kwei, K. A., Kung, Y., Salari, K., Holcomb, I. N. & Pollack, J. R. Genomic instability in

breast cancer: pathogenesis and clinical implications. Molecular oncology 4, 255-266,

doi:10.1016/j.molonc.2010.04.001 (2010).

5 Ichida, K. et al. Overexpression of satellite alpha transcripts leads to chromosomal

instability via segregation errors at specific chromosomes. International journal of

oncology, doi:10.3892/ijo.2018.4321 (2018).

6 Johanning, G. L. et al. Expression of human endogenous retrovirus-K is strongly associated

with the basal-like breast cancer phenotype. Scientific reports 7, 41960,

doi:10.1038/srep41960 (2017).

7 Zhu, Q. et al. Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer. Molecular

cell 70, 842-853 e847, doi:10.1016/j.molcel.2018.04.023 (2018).

8 Solovyov, A. et al. Global Cancer Transcriptome Quantifies Repeat Element

Polarization between Immunotherapy Responsive and T Cell Suppressive Classes.

Cell reports 23, 512-521, doi:10.1016/j.celrep.2018.03.042 (2018).

9 Papin, C. et al. Combinatorial DNA methylation codes at repetitive elements. Genome

research 27, 934-946, doi:10.1101/gr.213983.116 (2017).

10 Yandim, C. & Karakulah, G. Expression dynamics of repetitive DNA in early

human embryonic development. BMC genomics 20, 439, doi:10.1186/s12864-019-

5803-1 (2019).

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Hedefe Yönelik Tedaviler İçin Çeşitli Kanser Hücrelerinde IL13Rα2 Ekspresyonunun

Belirlenmesi

Damla Uludağ1, Ozan Topcu1, Nihal Karakaş1,2

1İstanbul Medipol University, Regenerative and Restorative Medicine Research Center (REMER), Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810

2Istanbul Medipol University, School of Medicine, Department of Medical Biology, Kavacik Campus, Kavacik-Beykoz/ISTANBUL 34810

Çeşitli kanser türlerinde uygulanan standart tedaviler, hastalığın prognozunun olumlu yönde

seyri için yetersiz kalmakla birlikte yalnızca kanser hücrelerini hedeflememektedir. Bu durum

uygulanan tedavilerin sağlıklı hücreler üzerine olumsuz etkilerinin varlığı ile sonuçlanmaktadır.

Hedefe yönelik tedavilerde ise esas amaç kanser hücrelerinin selektif olarak hedeflenerek

ortadan kaldırılmasıdır. Bu kapsamda hedefe yönelik reseptörlerden biri, kanser hücrelerine

özgü olan interlökin-13 (IL-13) reseptörüdür. IL-13, normal hücrelerde ya hiç ya da az düzeyde

eksprese edilen fakat çeşitli kanser hücre hatlarında yoğun olarak eksprese edilen IL13Rα2

reseptörlerine yüksek afinite ile bağlanır. Böylece IL-13 ve yüksek afinite gösterdiği IL13Rα2

reseptörü hedefe yönelik terapötiklerin oluşturulmasında kanser seçiciliği yönüyle oldukça

önemlidir. Bununla birlikte, kanser hastalığı ve prognozuna dair klinik öncesi araştırmalarda

tümördeki gerilemenin takibi için kullanılan yöntemler de sınırlıdır. Diğer taraftan

görüntülenebilir tümör çalışmaları, hedefe yönelik tedavilerin etkinliğini en hassas şekilde

ortaya koymaktadır.

Bu doğrultuda çalışmamızda; yeşil floresan proteini (GFP) ile biyolüminesan görüntüleme

ajanlarından Firefly lusiferazı (Fluc) birlikte içeren lentiviral vektörler kullanılarak, bu ajanları

eksprese eden kanser hücre hatları oluşturulmuştur. Ardından terapötik hedefleme için IL13Rα2

yüzey reseptörünün çeşitli kanser hücrelerindeki ekspresyon seviyeleri belirlenmiştir. Terapötik

hedefleme sonucunda hücre canlılığının in vitro analizi, bu hücrelerde canlılığın tespit

edilmesini sağlayan luminesan sinyal ölçümü için Fluc’a özgü D-luciferin kullanılarak

gerçekleştirilmiştir. Hücre canlılık analizi sonrasında kontrol hücrelerine kıyasla IL13Rα2

eksprese eden hücrelerde hedefe yönelik terapötik kullanımının hücre ölümüne yol açtığı tespit

edilmiştir.

Bu proje, TUBİTAK 117S421 nolu 1001 projesi kapsamında desteklenmektedir.

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Fluidic Shear Stress Regulates Biological Behaviours of Hepatocellular Carcinoma

Cells Through c-Met Signalling

Dehan Comez1,3*, Gulsun Bagci1,2*, Hande Topel1,2*, Ezgi Bagirsakci1,2, Yeliz Yilmaz1,3,

Aysim Gunes1, Nese Atabey1

• Equal contribution 1Izmir Biomedicine and Genome Center, Balcova, Izmir, Turkey

2Department of Medical Biology and Genetics, Health Sciences Institute, Dokuz Eylul University,

Balcova, Izmir, Turkey 3Izmir International Biomedicine and Genome Institute, Balcova,Izmir, Turkey

Tumour metastasis is the most common reason of the cancer related death. Metastasis process

requires cancer cells to detach primer tumour and intravasation to blood circulation and survive

the shear stress in blood stream, homing and proliferation in the secondary organ. Fluidic shear

stress (FSS) in blood circulation effects tumour cell’s survival, apoptosis, invasion, metastasis

and proliferation. Several clinic-pathological studies underline that in HCC intravascular

invasion is poor prognosis factor and it is significantly important in metastasis. When tumour

cells intravasate to sinusoidal capillaries, shear stress stimulates apoptosis. Frequency of

intrahepatic metastasis is high in HCC and it occurs as invasion dependent manner indicating

survival of tumour cells under FSS is crucial step for intrahepatic and extrahepatic metastasis.

Nevertheless, mechanisms behind process hasn’t been illuminated adequately yet. Our previous

studies showed that HGF/c-Met pathway plays crucial role in cell survival, invasion, metastasis

during hepatocarcinogenesis. In this study we aimed to examine the role of HGF/c-Met pathway

on survival and proliferation of HCC cells under FSS. In order to do this, we used peristaltic

pump to apply FSS to HCC cells which have low (HuH-7 and HepG2) and high (SNU-449) c-

Met levels. c-Met expression and activation levels were determined by RT-qPCR and Western

blotting, cell survival and proliferation analysis by trypan blue and (3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide) (MTT) in HCC cells. The effect of FSS on colony

formation, epithelial-mesenchymal transition (EMT) and invasion were determined by 3D

microfluidic system. Our data showed that, FSS treatment increased the expression and

activation of c-Met stimulating EMT phenotype, cell survival, proliferation and invasion in

HCC cells and c-Met inhibitor (SU11274) reversed these effects suggesting HGF/c-Met

pathway has critical role on the survival of HCC cells under FSS and induces aggressive

phenotype and c-Met inhibitors could be used to inhibit survival of circulating tumour cells’

metastasis in HCC.

Keywords: HCC metastasis, fluidic shear stress, circulating tumour cell, cancer

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Investigation of the Anticancer Effect of Haplophyllum Buxbaumii Plant on HCT-116

Colon Cancer Cells

Ataman Gönel, Derya Akçiçek, İsmail Koyuncu, Nihayet Bayraktar, Ebru Temız

Department of Medical Biochemistry, Harran University, Sanliurfa, Turkey

Colon cancer is the second leading cause of cancer-related deaths in the world. Mostly, the main

treatment option is postoperative chemotherapy. Although the cytotoxic effects of

chemotherapeutic drugs are to ensure the complete destruction of cancer cells, healthy cells are

damaged through these drugs. The discovery of phytotherapeutic agents in which the toxic

effect for healthy cells is minimized can be promising for cancer patients. The aim of this work

was to investigate the anticancer feature of Haplophyllum buxbaumii plant, which is grown

endemically in Şanlıurfa.

In our study, MTT method was used to investigate the cytotoxic effect of HCT-116 colon cancer

cell line and healthy (HUVEC) cells from extracts obtained from Haplophyllum buxbaumii

plant. Flow cytometric annexin V analysis, cell cycle analysis (BD Cycletest ™ Plus DNA

Reagent), intracellular ROS effect analysis (flow cytometry analysis) and apoptotic

morphological change analysis (fluorescence Acridine orange / ethidium bromide staining)

were performed to detect the apoptotic activity of the extract material. At the end of the study,

the IC50 value of the cytotoxicity found by MTT method in HCT-116 cells was determined at

14.17 μg / ml. For the normal cells, IC50 dose was found to be 70 µg/ml. It was concluded that

the cytotoxic effect was caused by apoptosis in HCT-116 cells and there was no apoptotic effect

in HUVEC cell.

This study showed that Haplophyllum buxbaumii plant extract may have the potential of

chemotherapeutic effect for the HCT-116 colon cancer cells.

Keywords: HCT-116, HUVEC, Haplophyllum Buxbaumii, Cytotoxicity, Annexin V

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Identification of shared candidate key genes and pathways associated with cancer

and heart failure pathogenesis using bioinformatic analyses

Dilek Pirim

Uludag University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics,

Bursa, Turkey

Background: Emerging evidence suggests that heart failure (HF) may contribute to the

oncogenic process and tumor development. High incidence of cancer in patients with HF

highlights the link between cancer and HF which needs to be elucidated to alleviate the

worldwide burden of the two diseases. The objective of our study is to identify the potential

common key genes and molecular pathways associated with cancer and HF.

Methods: Microarray data sets (GSE9128, GSE103512) were downloaded from Gene

Expression Omnibus and integrated analyses were performed by using bioinformatic tools.

Differentially expressed genes (DEGs) were analyzed in GEO2R using Limma R package.

Functional and pathway enrichment analyses of DEGs were performed by NetworkAnalyst

online analytics tool. STRING database was used for construction of protein-protein

interaction network. Network was visualized by Cytoscape software; hub genes were

determined using plug in Cytohubba. The expression levels of hub genes were evaluated in

independent datasets through C/VD and ENCORI databases for validation purposes.

Results: A total of 54 genes were identified to be dysregulated in HF and 4 types of cancers

(Breast, colorectal, non small cell lung and Pancreatic cancer). Top most enriched Kyoto

Encyclopedia of Genes Genomes (KEGG) categories was related immune system and most

significant Gene Ontology (GO) term related biological process was the response to abiotic

stimuli. Network analyses revealed top ten hub genes with the highest scores in Maximal

Clique Centrality ranking as VEGFA, JUN, DUSP1, FOS, CXCL8, EGR1, MYC, CXCL2,

NR4A2, FOSL2 and five hub genes including VEGFA, MYC, CXCL8, CXCL2, FOSL2 were

found to be significantly different expressed in independent datasets related both HF and

cancer.

Conclusion: This study shed light on the shared critical pathways and potential genes

regarding the common molecular mechanism of the pathogenesis of HF and cancer. Our

results pinpoint candidate molecular targets for further investigation and management of

cancer.

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Investigation of Anti-cancer Properties of Haplophyllum Ptilostylum Plant

Ebru Temız, İsmail Koyuncu Ataman Gönel, M.Orhan Tunçel


Characterizing by uncontrolled cell growth, cancer is one of the most common diseases. Since

the current methods damage to the immune system of the body during the treatment process, a

new treatment method, which has less side effect and high specificity, have been searched.

Phytotherapy, which has become widespread as one of the cancer treatment methods in recent

years, has many advantages. The present study aims to investigate the antiproliferative effects

of Haplophyllum Ptilostylum extract on liver cancer. The cytotoxic effects of the extract

obtained from Haplophyllum Ptilostylum on liver cancer cell line (SNU-423) and control cell

line (HUVEC) were examined by MTT method. In addition, flow cytometric; annexin V

analysis, cell cycle analysis, mitochondrial membrane potential analysis and apoptotic

morphological change analysis (fluorescence Acridine orange/ethidium bromide staining) were

performed to detect the apoptotic activity of the extracted material. At the end of the study, the

cytotoxic effect (IC50) was measured as 5μg/ml in liver cancer cells and 102μg/ml in control

cells. The apoptotic activities in SNU-423 and HUVEC cells were measured as %25.6 and

%7.9, respectively; the effects of the extract on the cell cycle were found as follows: Sub-G1:

%2,3 and G0/G1: %55,0 in SNU-423; and Sub-G1: %0 and G0/G1: %58,2 in HUVEC. The

mitochondria membrane potentials in SNU-432 and HUVEC were recorded as %33.2 and %

15.7, respectively. According to the obtained results, Haplophyllum Ptilostylum plant creates

an apoptotic activity by disrupting the mitochondria membrane potential in the liver cancer cell.

Based on these grounds, we suggest that Haplophyllum Ptilostylum plant can be tested as a

pharmacologic agent in different cancer types.

Keywords: Haplophyllum Ptilostylum, SNU-423, Liver Cancer, Cell Death, Cytotoxicity

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The importance of dysregulated miRNAs on ovarian cysts and tumors

Ece Gumusoglu1, Tuba Gunel1, Mohammad Kazem Hosseini1, Nogayhan Seymen2, Taylan Senol3, Uğur Sezerman2, Samet Topuz4, Kılıç Aydınlı5

1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey.

2Acıbadem University, School of Medicine, Department of Basic Sciences, Biostatistics, Istanbul Turkey.

3Bağcılar Training and Research Hospital, Department of Obstetrics and Gynecology, İstanbul,

Turkey. 4Istanbul University, Department of Obstetrics and Gynecology, Istanbul Medical Faculty, Istanbul,

Turkey. 5Medicus Healthcare Centre, Istanbul, Turkey

Ovarian cancer is the fifth most important cause of cancer-related deaths among women and

the most lethal gynecologic malignancy. Epithelial ovarian cancer (EOC) is asymptomatic and

few screening tests are available. In our study, we aimed to find the importance of dysregulated

miRNAs in ovarian cyst and their expression profile difference between ovarian cancer cases.

Another goal of our study is to identify novel circulating miRNAs to be used as therapeutic

prediction of EOC. In this study, we studied three different samples: serums of EOC patients,

healthy individuals (HI) and benign ovarian cysts (BOC). Their miRNA expressions have been

compared by microarray. Microarray data were analyzed according to miRNA expressions the

relation between miRNAs target genes and EOC were examined by bioinformatic tools. 75 and

66 significantly dysregulated miRNAs were identified by microarray in BOC vs. EOC and BOC

vs. HI comparison, respectively. Then, we focused on common miRNAs that found in both

comparison and finally 46 important miRNAs were detected which can represent the only

common sample group, BOC. After these findings, we also considered miRNA profiling in

EOC and HI, and surprisingly any common miRNAs were found with these 46 miRNAs. Thus,

we analyzed them depending on their potential importance on BOC pathogenesis. After

bioinformatic analysis, our findings indicated that there are several biological processes and

pathways which can be considered to be related BOC development.

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Investigation of Helicobacter Pylori virulence factors and immune response in

precancerous lesions

Elif Merve Aydın1†‡, Tevriz Dilan Demir2‡, Ayça Sayı Yazgan3* Arzu Tiftikçi 4*

1Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University,

Istanbul, Turkey

2Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University, Istanbul, Turkey

3Molecular Biology- Genetics and Biotechnology Research Center, Istanbul Technical University, Istanbul, Turkey

4Internal Medicine Gastroenterology Department, Acibadem University, Istanbul, Turkey

†Presenting Author, ‡Equal contribution to the study

The developmental stages of gastric cancer could be classified as chronic gastritis, atrophic

gastritis, intestinal metaplasia and dysplasia. It is not possible to determine whether patients

with gastric pathology will develop gastric cancer, as the genetic and immunological aspects of

gastric pathogenesis are not fully known. At the same time, the relation between the

characteristic of the virulence factors of H. pylori and the impact on the host's immune response

is not fully understood. Therefore, we aimed to investigate the ten H. pylori virulence factors

and adaptive immune response in patients with gastric pathology.

This study included in total 32 patients; 20 with active chronic gastritis, 3 with inactive chronic

gastritis, and 9 with intestinal metaplasia. 15 subjects who had no signs of inflammation and

did not carry infection were included to this study as control. Three tissue samples were taken

from the antrum of the stomach during the endoscopy. DNA and RNA was extracted from two

of the received tissue samples and third biopsy specimen was used to determine the

pathogenesis of gastric tissue and H.pylori presence using histological staining. DNA samples

were used to detect ten H. pylori virulence factor genes; ureA, ureB, hpaA, cagA, napA, dupA,

oipA, vacA m1/m2, vacA s1/s2, babA using conventional PCR. qRT-PCR was used to

investigate the expression of regulatory T cell specific transcription factor, FOXP-3; Th-1

specific cytokine IFN-γ, and immune checkpoint ligand PD-L1 at mRNA level. The most

common virulence factors in patients with active chronic gastritis were shown to be napA, hpaA

and cagA. The expression level of IFN -ɣ was increased and FOXP-3 was decreased in gastric

precancerous lesions. Conversely, PD-L1 expression level was higher in precancerous lesions.

Overall, we investigated virulence factors of Helicobacter pylori and immune response of the

host in gastric precancerous lesions.

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A Useful Tool for Distinguishing Gene/Pseudogene Variants Detected via NGS:

Haplotype Analysis

Esra Arslan Ateş1,2, Ahmet İlter Güney2, Tuba Günel1 1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul.

2Marmara University Pendik Training and Research Hospital, Department of Medical Genetics,

Istanbul.

e-mail: [email protected]

Introduction: Lynch Syndrome is an inherited cancer prone disease mostly associated with

colon, endometrium, ovarian and gastrointestinal tract cancers. Mutations affecting missmatch

repair genes (MLH1, PMS2, MSH2, MSH6) are responsible from Lynch Syndrome. PMS2 has

a psedudogene called PMS2CL showing a high sequence similarity to PMS2 gene. Here we

report a novel mutation in PMS2 gene associated with Lynch Syndrome which was proven to

be on the active gene using haplotype analysis.

Case Report: A 69 year-old woman referred to us because of endometrium cancer. She had a

history of colon cancer diagnosed at the age of 66. Endometrial tumor showed high

microsatellite instability and MLH1 and PMS2 expression loss were detected in

immunohistochemical analysis. There were no other cancer history in her family. BRAF V600E

mutation and MLH1 promoter hypermethyation were not detected in tumor tissue. On the basis

of immunohystochemical findings MLH1 and PMS2 genes were screened for germline

mutations from peripheral blood lymphocytes via next generation sequencing. We detected a

novel 47bp insertion (c.1362_1407dup; p.Pro470Valfs*3) in exon 11 of PMS2 gene with a

variant fraction of 26%. The false positive result due to pseudogene was excluded by haplotype

analysis using the SNPs that allow us to distinguish PMS2 and PMS2CL.

Conclusion: PMS2 gene is a MMR system component which has an important role in DNA

replication and homologous recombination, located on chromosome 7p and consist of 15 exons.

There is a pseudogene, PMS2CL, which shows 98% sequence similarity to exons 9 and 11–15

of PMS2 and it is important to distinguish the variations between gene and pseudogene

sequences. NGS data enable us to assess the haplotypes in an amplicon using the distinct SNPs

on gene and psedudogene.


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77

Identification of lncRNA molecules involved in the regulation of EGF-induced of cellular

proliferation

Esra Bozgeyik

Tekirdag Namik Kemal University, Faculty of Medicine, Department of Medical Biology, Tekirdag, Turkey

Lung cancer is one of the leading causes of cancer-related deaths worldwide. According to 2019

cancer statistics, it is estimated that over 230000 new lung cancer cases will occur and 143000

people will die due to lung cancer. Moreover, lncRNAs (long non-coding RNA) are functional

RNA molecules longer than 200 nucleotides that do not have protein coding capacity and play

important roles in the vital cellular processes such as proliferation, growth, division,

differentiation, survival, and migration of cells. Due to their wide range of biological functions,

deregulation of these transcripts highly contributes to molecular pathogenesis of cancers

including lung cancer. EGF is key factor that is involved in the regulation of cell proliferation.

Since EGF is a very chief protein for the maintenance of normal physiological processes,

deregulation of EGF-induced signaling significantly contributes to the several hallmark

capabilities of human cancers. Thus, determination of lncRNA molecules involved in the

regulation of EGF-induced promotion of cellular proliferation is of great interest to illuminate

the molecular pathology of human cancers. Accordingly, in this study we aimed to identify

differentially expressed lncRNAs involved in the regulation of EGF-induced stimulation of

cellular proliferation. As a result, expression levels of MALAT1, ANRIL and MCM3AP-AS1

lncRNAs was found to be increased whereas expression levels of LOC105372161 and MEG3

were found to be decreased. However, only changes in the expression of LOC105372161 were

found to be statistically significant, suggesting that LOC105372161 is involved in the regulation

of EGF-induced cell proliferation and BCL2 might be the putative target of LOC105372161.

Keywords: EGF, LOC105372161, lncRNA, lung cancer.

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Morus nigra (Karadut) yaprağı etanol ekstraktının doğal bir DNA ileri glikasyon son

ürünleri (DNA-AGE'ler) bileşik kaynağı olarak değerlendirilmesi

Feryal Akay, Bircan Çeken Toptancı, Nesrin İnceören, Göksel Kızıl, Murat Kızıl

University of Dicle, Faculty of Science, Chemistry Department, Diyarbakır, Turkey

Gelişmekte olan ülkelerde nişasta bazlı gıdaların tüketiminin artması ile metabolik hastalıklarda

hızlı bir artış meydana gelmiştir. Glikoz kanda insulin tarafından tanınırken fruktoz tanınmaz

ve lipogenezise katılarak hücre içi lipit birikimini arttırır. İndirgen şekerler; biyolojik makro

moleküllerde bazı reaktif ürünler ve AGE’leri (İleri Glikasyon Son ürünleri) oluşumuna neden

olur. Diyabetin sürekliliği dokularda protein birikimine ve Maillard reaksiyonuna sebep

olabilir. İleri glikasyon son ürün reseptörü (RAGE) ekspresyonuna neden olabilir ve karşılıklı

olarak AGE formasyonuda hiperglisemiyi hızlandırır. Bu ürünler çeşitli mekanizmalarla bir dizi

hastalığın patojenezinde birikerek doku hasarına neden olabilir.

AGEs’ler hücrelerin çekirdek ve sitoplazmalarında birikerek DNA hasarı oluşturur. Böylece

kanserli hücre oluşabilir. AGEs reaktif oksijen türlerini arttırarak antioksidan sistemlere hasar

verebilirler.

AGEs ciltteki elastikliği zayıflatarak yaşlanmayı da aktif hale getirir. Eğer deriye yakınsa deri

kanserine sebep olur. UV ile aktifleşerek proteinleri modifiye edip yine DNA hasarına neden

olur. Derideki glikolizlenmiş proteinler foto-aktif olarak, foto-yaşlanma ve foto-kanserde eşit

derecede işlev görürler.

Bazı doğal gıda bileşenleri AGE'lerin oluşumuna ve birikmesine karşı koruyucu bir etki

gösterebilir. Oksidasyon birçok AGE'nin oluşumunda rol oynayan çok önemli bir adım

olduğundan, başlangıçta antioksidan, radikal temizleme veya metal şelatlama özelliklerine

sahip polifenoller AGE inhibitörleri olarak araştırılmaktadır.

Çalışmanın amacı farklı ekstraksiyon teknikleri kullanılarak elde edilen Morus nigra (Karadut)

yaprağı etanol ekstraktlarının; toplam fenolik, flavonoid, DPPH ve hidroksi radikalini

söndürme aktivitelerini araştırlaması ve bu parametrelerde en yüksek aktivite gösteren ekstrakt

ve M. nigra yaprağının etken maddelerinden Quersetinin; DNA-fruktoz ve DNA-aspartam

sisteminde in vitro antiglikasyon kapasitesini Floresans spektrometresi, UV spektrometresi,

fruktozamin (NBT) testi ve agarose jel elektroforez tekniklerini kullanarak araştırmaktır.

Sonuç olarak, fruktozun ve aspartamın DNA da oksidatif hasara neden olduğu ve M. nigra

yaprak ekstraktının ve quersetinin, antioksidant aktivitesinin olduğu, hidroksi radikali kaynaklı

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DNA oksidasyonunu inhibe ettiği tespit edildi, ancak fruktoz kaynaklı DNA-AGE oluşumunu

etkin bir şekilde inhibe ettmediği tespit edildi. M. nigra yapraklarında ana etken madde olarak

bulunan Quersetinin fruktoz kaynaklı DNA kesimini konsantrasyona bağlı olarak önlediği

belirlendi, ancak M. nigra yaprağı etanol ekstraktı DNA kesimini önlemeyip, DNA kesimini

artırdığı tespit edildi.

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80

The antiproliferative and apoptotic effects of gold nanoparticles synthesized by Allium

cepa extract on leukemia cell lines

Gamze Tan1*, Kamile Öztürk1, Mehtap Tarhan1

1Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey

*[email protected]; [email protected]

In biomedicine, the use of metallic nanoparticles prepared by using plant extracts is increasing.

It is preferred because the extract of the plants contains many different metabolites, which are

especially useful in the reduction of metal ions. They are also low-cost, easily available, and

environmentally friendly alternatives to chemical reducing agents. In addition, plants have been

used in the treatment of many diseases and obtaining medicines in modern medicine. Allium

cepa (Onion) with a large number of functional compounds has been used to protect human

health, thanks to their therapeutically useful properties, such as antibacterial, anti-inflammatory

and antioxidant activities in particular. In this study, it is aimed to show the synthesis of gold

nanoparticles using the aqueous extract of onion (O-AuNPs), and evaluate their antiproliferative

and apoptotic effects on four leukemia cell lines.

The chemical structure and physical properties of bio-fabricated AuNPs were confirmed with

TEM, zeta potential, and ICP-OES. Furthermore, cellular responses of O-AuNPs was

investigated on K562, HL-60, CEM and Raji leukemia cell lines by using MTT test for 48 and

72 hours. Pro-apoptotic Bax, anti-apoptotic Bcl-2 and Caspase-3 gene expression levels were

determined by using real time quantitative RT-PCR method.

According to TEM images, O-AuNPs had spherical in shape with approximately 13.5 nm in

size. The zeta potential result showed that surface charge of the particles was about -14.4 mV.

O-AuNPs decreased cell viability in all cell lines in both dose and time dependent manner.

Moreover, O-AuNPs induced apoptosis via changing the expression Bax, Bcl-2 and caspase 3

gene in all cell line.

In conclusion, our results suggest that O-AuNPs have antiproliferative and apoptotic effects on

leukemia cell line through by up-regulating Bax/Bcl-2 gene expression ration and increased

caspase-3 levels.

Keywords: Allium cepa, antiproliferative effect, leukemia, apoptosis, gold nanoparticles

Oral Presentation

81

The Role of c-Met Signaling in Organ Specific Extravasation of HCC cells

Gulsun Bagci1,2*, Dehan Comez1,2 *,Hande Topel1,3*, Ezgi Bagirsakci1,2, Yeliz Yilmaz1,3, Aysim Gunes1, Gizem Batı Ayaz4, Devrim Pesen Okvur4, Nese Atabey1

*Equal contribution 1Izmir Biomedicine and Genome Center, Balcova, Izmir, Turkey

2Department of Molecular Biology and Genetics, Izmir International Biomedicine and Genome

Institute, Balcova,Izmir, Turkey 3Department of Medical Biology and Genetics, Health Sciences Institute, Dokuz Eylul University,

Balcova, Izmir, Turkey 4Izmir Institute of Technology, Department of Molecular Biology and Genetics, Izmir, Turkey

HCC (Hepatocellular Carcinoma) is the fifth most common cancer worldwide and the third

cause of cancer deaths. Due to difficulties in diagnosis, many patients are diagnosed at late

stages. HCC is associated with high mortality rate due to high recurrence and metastasis risk in

patients after surgical resection. Metastasis consists of multiple steps as dissemination of cancer

cells from primary tumor, intravasation into blood stream, survival in circulation, extravasation

to secondary organs and colonization. c-Met signaling pathway mediates embryogenesis and

organ regeneration, its aberrant activation can promote invasion, motility, proliferation,

angiogenesis and evading apoptosis in several cancers including HCC. Recent data suggest that

activation of c-Met signaling might be an important factor in the regulation of tumor metastasis.

However, role of c-Met signaling in the organ specific extravasation is not defined in literature

yet. Lab-on-a-chip is integrated 3D cell culturing to study for tumor-microenvironment

interactions, drug screening, studying metastatic patterns of cancer cells to distant organs. In

this study, a Lab-on-chip device was used to mimic the extravasation capacities of SNU-449,

SNU-398, SNU-398-Met HCC cells to investigate role of c-Met on the organ specific

extravasation. Homing cells were THLE-2, BEAS-2B and HS-27A to represent liver epithelial,

lung epithelial and bone marrow/stroma cells respectively. HUVEC cells were used to mimic

endothelial barrier. HCC cells were exposed to 0.5 dyn/cm2 FSS for 4 hours to simulate FSS in

hepatic sinusoid. As a result, activation of c-Met signaling is significantly increased organ

specific extravasation of HCC cells and c-Met inhibitor SU11274 decreased extravasation rate

of HCC cells approximately 70% in total. Our data support that c-Met signaling could play role

in organ specific extravasation especially in intrahepatic metastasis and bone metastasis and

SU11274 could be used as anti-cancer drug to prevent metastasis especially to prevent HCC

and can be used for therapeutic purposes.

Keywords: HCC, c-Met, metastasis, extravasation

Oral Presentation

82

The Effects of Cancer Associated Fibroblasts on Tumor Associated Macrophages

Gurcan Gunaydin

Department of Basic Oncology, Cancer Institute, Hacettepe University, Ankara, Turkey

[email protected]

Introduction: Monocytes polarize into either M1 or M2 macrophages. Most macrophages in

tumor microenvironment display M2-like-features. Monocytes/macrophages demonstrated

plasticity and investigating the mechanisms underlying this plasticity will pave the way for

more effective and successful diagnostic/therapeutic approaches cancer treatment. Therefore,

the current study aims to shed light on the effects of cancer-associated-fibroblasts(CAFs) on

macrophage-differentiation in breast-cancer patients.

Materials/Methods: An-enzymatic-protocol utilizing Collagenase-I/Hyaluronidase was used

to obtain CAFs and normal-fibroblasts(NFs) from patients undergoing total-

mastectomy/reduction-mammoplasty. Surface-expressions of markers (e.g. α-smooth-muscle-

actin[α-SMA],vimentin), which differentiate CAFs/NFs, were analyzed with

immunocytochemistry. CAF/NF cultures were used to acquire conditionedmediums(CMs).

Peripheral-blood-mononuclear-cells were isolated from healthy-volunteers’ peripheralblood

via Histopaque-1077. CD14+ monocytes, CD4+ T-cells were isolated from PBMCs via

magneticseparation. Surface-expressions of CD206,CD163 were analyzed with flow-

cytometry. Functional-effects of CAF/NF-educated-monocytes on CD4+ T-lymphocytes were

demonstrated via CFSE proliferation assays with CD4+ T-cells (activated-with-CD3/CD28-

magnetic-beads). Transwell-migration-assays were utilized to demonstrate the alterations in

monocyte-recruitment due to CAFs/NFs. E-cadherin,vimentin protein expressions were

determined by Western-blot. In addition, alterations in MDA-MB-231 breastcancer- cell-

invasion due to CAF/NF-educated-monocytes were investigated with Transwell-inserts.

Results: Although CAFs expressed α-SMA;NFs did not. CAFs were able to potently recruit

monocytes. This recruitment may be mediated through Monocyte-chemotactic-protein-1(MCP-

1) or stromal-cellderived- factor-1(SDF-1) cytokines, since monocyte migration was

significantly reduced as a result of MCP- 1 or SDF-1 inhibition through MCP-1 or CXCR4(a-

chemokine-receptor-specific for SDF-1) blockingantibodies. CD163, CD206 (associated-with

M2-macrophages) expressions were higher in CAF-educatedcells than in NF-educatedcells. T

cell-mediated immune-responses were shown to be suppressed by CAFeducated- monocytes.

While CAF-educated-monocytes augmented breast-cancer-cell-invasion, NFeducated-

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83

monocytes did not. Moreover, CAF-educated-monocytes increased vimentin

expression,decreased E-cadherin-expression in breast-cancer- cells. Last but not least, CAFs

differentiated M1-macrophages to M2-like-macrophages, as CD163-expression significantly

increased in M1-macrophages due to CAFs.

Discussion: CAFs polarized monocytes to M2-like pro-tumoral macrophages phenotypically

and functionally; whereas NFs did not. CAFs were shown to be very potent in recruiting

monocytes. MCP-1 and SDF-1, which are secreted from stromal-cells, may provide evidence

to be pivotal monocytechemotactic- cytokines.

Keywords: breast cancer, CAFs, monocytes, macrophages, oncoimmunology, tumor

microenvironment

Authors declare no conflict of interest.

Oral Presentation

84

Cryptonin: An Antimicrobial Peptide from Cicada and its Effects on Melanoma Cell

Lines

Gülşah Torkay, Serap Sancar-Baş

İstanbul University, Science Faculty, Biology Department, Molecular Biology Section, Vezneciler,

34134, Fatih, ISTANBUL

Introduction: Antimicrobial peptides are important part of the innate immune response and

synthesized by various organisms ranging from bacteria to mammals. These peptides

generally have 12-50 amino acids and most of them are positively charged. Accumulating

data have shown that antimicrobial peptides not only have effects on microorganisms but

also may have toxic effects on tumor cells. So, we can say that some of these peptides are

not only “antimicrobial” anymore. Since they have relatively high positive charge, they can

interact with tumor cell membranes which have anionic character like bacterial membranes.

Aim: In this study, we aimed to investigate the effects of cryptonin which is an antimicrobial

peptide defined in the hemolymph of Cicada, Cryptotympana dubia (Park et al., 2007) and

the anticancer activity has not studied before. Cryptonin has an alpha-helix structure in

aqueous solution, 24 amino acids in length, 50% hydrophobicity and +8 net charge. For these

reasons, cryptonin can bind to membrane bilayers to form pores and cause tumor cells to

death.

Methods: The anti-proliferative effects of cryptonin was studied with MTT test on metastatic

and non-metastatic melanoma cell lines and possible lytic effects were studied with lactate

dehydrogenase and HMGB1 release. The morphology of cell death type was studied with

acridine orange/ethidium bromide staining. Besides, reactive oxygen levels were measured.

Results: We detected that cryptonin decreased the cell viability in the melanoma cell lines in

time and dose dependent manner via damaging to cell membranes. This lytic effect leads to

releasing of lactate dehydrogenase and HMGB1 protein, which are the marks of necrotic cell

death. The ethidium bromide stained nucleus number is also increased in higher peptide

concentrations.

Discussion: Taken together, the antimicrobial peptide cryptonin may have therapeutic

potential for treatment of melanoma, especially in metastatic cell lines leading immediate

cell death.

Key-words: Cryptonin, Antimicrobial peptide, Melanoma, Necrotic cell death

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85

Güneydoğu Anadolu Bölgesinde Yetişen Patlican Çeşitlerinin Kabuklarının Sitotoksik

Etkilerinin Saptanması

İbrahim Halil Kılıç, Işık Didem Karagöz, Başak Simitçioğlu, Sami Serhat Tonus, Bekir

Sıddık Kurt

Biyoloji Bölümü, Gaziantep Üniversitesi, Gaziantep

Günümüzde bazı bitki özütleri gıda sanayisinde kullanılmak üzere marketlerde görülmeye

başlamıştır. Bazı bileşiklerin antioksidan kapasitesinin, sentetik olanlardan daha yüksek olduğu

yine dünya nüfusunun çoğunluğu için bitkiler, ilaç hammaddesi olarak yaygın olarak

kullanmaktadırlar. Özellikle gelişmekte olan ülkelerde nüfusun % 80’i sağlık gereksinimlerini

ilk etapta geleneksel tıbbi bitkilerden sağlamaktadır. Tıbbi ilaç dışında bitkiler endüstri

sektöründe de oldukça önemli yere sahiptir. Gelişen ilaç sektörüne rağmen birçok hastalığa

çözüm bulunmamakla birlikte yeni etkin bileşik keşfi için araştırmalar hızla devam etmektedir.

Ülkemizin önemli sebzelerinden olan patlıcanın özellikle Güneydoğu Anadoluda bölgesinde

patlıcanın farklı çeşitleri üretilmekte ve tüketilmekte olup özellikle dolmalık çeşidi (kuru

dolmalık) tüm ülkemize bu bölgeden gönderilmektedir. Gaziantep’te yetişen Dolmalık patlıcan,

Kemer patlıcan, Nizip patlıcan ve Birecik patlıcanı olmak üzere dört çeşit patlıcan kabukları

soyulup kurutulmuş ve su,metanol,hekzan ve diklormetan özütleri elde edilmiştir. Özüt

verimleri hesaplanmıştır.

Sitotoksisitenin değerlendirilmesi amacıyla kulanılan enzimatik test yöntemlerinden biri olan

MTT testi ile belirlenmiştir. H1299 ve HUVEC hücre hatları kullanılmıştır. Özellikle Dolmalık

ve Kemer patlıcan çeşitlerinin Metanaol özütleri H1299 kanser hücreleri üzerine toksik etki

gösterirken sağlıklı HUVEC hücre hatları üzerine sitotoksik etkisi göstermemiştir. Seçici

sitotoksik etki saptanmış olması etken bileşiklerin saflaştırılmasına temel veriler

oluşturmaktadır.

Anahtar kelimeler: Dolmalık patlıcan, Nizip patlıcanı, Sitotoksite

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86

How dose and duration of exposure affect the rate of acquired resistance to doxorubicin

in gastric cancer?

Hande Özkan1,2†, Gülnihal Özcan2,3*

1 Koc University Graduate School of Health Sciences, Istanbul, Turkey 2 Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey

3 Koc University School of Medicine, Istanbul, Turkey

†Presenting Author *Corresponding author, e-mail: [email protected]

Chemotherapy is the mainstay for the treatment of numerous cancers. However, patients mostly

develop chemoresistance due to the presence of naturally resistant cancer cells within the tumor

mass or acquired resistance after chemotherapy. Therefore, there is an urgent need to develop

new therapeutic strategies that can overcome chemoresistance. Today’s chemotherapy

protocols are based on the administration of chemotherapeutics at maximum tolerated doses

with intermittent chemotherapy sessions. Although these protocols decrease the size of the

tumor at the short-term, remaining cancer cells adapt themselves to the presence of

chemotherapeutics, escape from their anti-cancer action and develop chemoresistant clones

which cause the regrowth of the tumor at later stages. Therefore, an increase in long-term

survival could not be achieved with this strategy. Recently it was suggested that doses below

maximum tolerated dose applied over a longer period of time may decrease the rate of acquired

resistance to chemotherapeutics and increase overall survival, despite a lower anti-cancer

efficacy at the short-term. To test this hypothesis in gastric cancer, we exposed gastric

adenocarcinoma cells to doxorubicin, one of the most potent and most commonly used

chemotherapeutics in cancer, with a high dose-intermittent protocol or low dose-continuous

protocol. Our preliminary studies suggest that, high doses of doxorubicin applied over a short-

term may induce a higher increase in resistance index compared to the application of lower

doses over a longer period of time. We also observed that a specific group of multi-nucleated

giant cells were selected by high dose-intermittent application and these cells replenished the

original cell population during the drug-free interval. On the other hand, the topology of the

initial cell population was mostly preserved with low dose-continuous exposure. These results

suggest that, low dose-continuous administration of doxorubicin might be tested as a strategy

to decrease rate of acquired resistance in gastric cancer.

Funding/Support: “The authors gratefully acknowledge use of the services and facilities of the Koç University Research Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is solely the responsibility of the authors and does not

necessarily represent the official views of the Presidency of Strategy and Budget.”


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87

İNSAN MEME KANSERİ HÜCRELERİNDE D-erythro-MAPP’ın ANTİKANSER

ÖZELLİKLERİNİN ARAŞTIRILMASI

Hüseyin İzgördü1, Canan Vejselova Sezer1, Emre Çömlekçi1, Hatice Mehtap Kutlu1*

1Eskişehir Teknik Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Eskişehir

*Sorumlu yazar:[email protected]

Giriş ve Amaç: Kanser günümüzde öldürücü hastalıklar arasında ilk sıralarda yer almaktadır.

Kanser, tedavi sürecinde hastalara büyük zorluklar yaşatan ve tedavisi çok pahalı olan bir

hastalıktır. Meme kanseri erkeklerde ve kadınlarda görülen bir kanser çeşidir. Meme kanseri,

hem gelişmiş hem de gelişmekte olan ülkelerde tüm kanserler içinde üçüncü sırada yer

almaktadır. Kanser tanısı konduktan sonra hastaların hayatta kalmaları ortalama olarak 5 yıl ile

sınırlıdır. Bu sebeple ileriye dönük daha etkin tedavi biçimlerinin geliştirilmesine ihtiyaç

duyulmaktadır. Seramid, sfingozin ve sfingozin-1-fosfat; hücre proliferasyonunu ve apoptozu

kontrol etmekte görev alan sfingolipid metabolitleridir. Seramid ve sfingozin hücre ölümüne,

yaşlanmasına ve hücre döngüsünün durdurulmasına aracılık etmektedirler. Bu çalışmanın

amacı bir seramidaz inhibitörü olan D-erythro-MAPP’ın insan meme kanseri (MCF-7) hücreleri

üzerindeki potansiyel antiproliferatif, proapoptotik ve sitotoksik etkilerinin araştırılmasıdır.

Gereç ve Yöntemler: Bu çalışmada D-erythro-MAPP’ın insan meme kanseri hücreleri

üzerindeki sitotoksisitesi MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide) kolorimetrik testi ile incelenmiştir. MTT deney sonuçlarından D-erythro-MAPP’ın

insan meme kanseri hücrelerin yarısını öldüren IC50 konsantrasyonu belirlenmiştir. D-erythro-

MAPP’ın neden olduğu hücre ince yapısal ve morfolojik değişiklikler geçirimli elektron

mikroskobu (TEM) ve konfokal mikroskopi yöntemi ile araştırılmıştır.

Sonuçlar: MTT sonuçlarından elde edilen verilere göre D-erythro-MAPP’ın MCF-7

hücrelerinin canlılığını 24 saatlik bir uygulama sonucunda doza bağımlı olarak düşürmüştür.

D-erythro-MAPP’ın MTT sonucuna göre IC50 değeri 4,4µM olarak bulunmuştur. Konfokal

mikroskopi ve TEM bulguları ise çekirdekte yapısal anomali, membranlarda tomurcuklanma,

kromatin yoğunlaşması, çekirdek ve hücre iskeletinde parçalanmalar gibi proapoptotik etkiler

ile bağlantılı olarak değerlendirilmiştir. Sonuç olarak D-erythro-MAPP’ın bu çalışma

sonucunda ciddi bir antikanser ajanı olma potansiyele sahip olduğu ortaya konulmuştur ve daha

ileri ve detaylı kanser araştırmalarına ihtiyaç vardır.

Anahtar Kelimeler: D-erythro-MAPP, MCF-7, TEM, Konfokal

Bu çalışma TÜBİTAK 118Z943 no’lu 1002 projesi ile desteklenmiştir.

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88

Experimental traumatic brain injury models

Hüseyin Özevren

Departments of Neurosurgery, Dicle University School of Medicine, Diyarbakir, Turkey.

Head trauma is still a serious cause of morbidity and mortality. It has physical, psychological,

social and economic effects on societies. In spite of many diagnostic and therapeutic

approaches, the point reached is unfortunately still not at the desired level. Therefore, new

treatment approaches that can shed light on the treatment are very important for both the

problem and the head trauma patient. Experimental studies for head trauma are the most

appropriate experimental methods to investigate neuroprotective methods

The experimental model of traumatic brain injury was first made in the mid-20th century.

Denny-Brown and Russell divided experimental brain injury into acceleration and compression

in 1941; have used the term acceleration for blunt closed head trauma and the term compression

for penetrating injury of the skull. Over the years, several improvements have been made.

In the 1980s, there was a huge explosion in traumatic brain injury experimental research. In

parallel with the development of instruments used in cell and molecular biology, 3 new models

have been developed. These models; fluid percussion damage, controlled cortical effect, weight

reduction from high

It was accepted that the main animal used in experimental studies was rodents. They are used

because of their similarity to the human brain, easy to grow and cost-effective.

Therefore, new treatment approaches that can shed light on the treatment are very important for

both the problem and the head trauma patient. Experimental studies on head trauma are the most

appropriate experimental methods to investigate neuroprotective methods.

Keywords: Experimental, Neuroprotective. Traumatic brain injury

Oral Presentation

89

In vitro Self-renewal Properties in A549 and H1299 Non-Small Cell Lung Cancer Cells

Hiba H. A. Khair and Isik Didem Karagoz,

Department of Biology, Faculty of Art and Science, Gaziantep University, Gaziantep, Turkey

Lung cancer remains at the top of the list of cancers with the highest incidence and mortality

rates worldwide. The cancer stem cell paradigm proposes that a small subpopulation of cancer

cells called cancer stem cells (CSCs) can self-renew, recapitulate tumor heterogeneity by

differentiating into different lineages of cells that constitute the bulk of the tumor and

substantially lead to tumor recurrence. Accumulating evidence suggests that a small population

of stem-like cells are responsible for the initiation, progression, and metastasis of lung cancer.

This study aims to assess cancer stem-like properties in A549 and H1299 non-small cell lung

cancer cells. The spheroid formation assay was used to assess self-renewal ability indicated by

tumorspheres formation and to calculate sphere-forming efficiency in vitro. The limiting

dilution assay was used to calculate the minimum number of cells needed to be cultured to see

at least one spheroid in vitro. Cells derived from both cell lines could form tumor spheroids.

The sphere-forming efficiencies of A549 and H1299 cells were 4.14 ± 1.06% and 5.81 ± 1.28%,

respectively. However, H1299 cells produced more and larger tumorspheres when compared to

A549 cells. The mean diameter of tumor spheroids in H1299 and A549 cells were 95.25 ± 41.03

and 80.70 ± 31.72 μm, respectively (p=0.00003). In limiting dilution assay, A549 and H1299

single cells produced spheroids at 23.5 ± 43.7% and 62.5 ± 49.4%, respectively. The frequency

of sphere-forming cells (stem-like cells) was found to be 6.06-fold higher for H1299 cells than

those for A549 cells. These results suggest that the self-renewal ability of H1299 cells is higher

and more pronounced compared to A549 cells. As a conclusion, both A549 and H1299 cells

comprise stem-like cells with self-renewal ability.

Key words: Lung cancer, Cancer stem-like properties, Self-renewal.

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90

IMMUNOHISTOCHEMICAL DETERMINATION OF GLUTATHIONE-S-

TRANSFERASE ISOENZYM FAMILY EXPRESSION IN INVASIVE LOBULAR

BREAST CANCER TISSUE

Işıl Yıldırım1, Bilge Aydın Türk2, Pınar Kaygın3, Serpil Oğuztzüzün3

1Beykent University, Vocational School, Pharmacy Services Program, Biochemistry Area, 34500

İstanbul, Turkey,

2Adıyaman University, Faculty of Medicine, Department of Pathology, 02100, Adıyaman, Turkey

3Kırıkkale University, Faculty of Science, Department of Biology, 71450, Kırıkkale, Turkey

Corresponding author email: [email protected]; [email protected]

Aim: GST isozymes are present in a variety of normal and malignant human tissues including

breast carcinomas. The aim of the present study was to evaluate the association between GSTM,

GSTA1, GSTP1 of invasive lobular breast cancers tissues.

Method: Breast biopsy specimens from at least 14 invasive breast lobular carcinoma women

whose diagnosis and treatment protocols were determined between 09.07.2007 - 01.12.2018

were used at Adiyaman University Medical Pathology Department by Ethics Committee with

resolution of 2019 / 1-28. The protein expression of GSTM, GSTA1, GST1 in breast carcinoma

cells, determined by immunohistochemistry. GST was correlated with clinical like age and

several pathological parameters of prognostic significance including tumor size, nodal status,

estrogen receptor protein positivity, or progesterone receptor protein positivity.

Results: When pathologic diagnosis and histological gland conditions were considered for

GSTM1 protein, invasive lobular carcinoma was found protein staining grade +1 for right breast

tissue, in normal tissue and in tumor tissue was 16.6 %, 50 %, respectively. In both invasive

lobular carcinoma breast right and left, +3 staining was 33.3 %. GSTA1 protein was not

expressed for both histological glands. GSTPi protein expression invasive lobular carcinoma

for both right and left was found 66.66 % and 33.3 % for staining +1 degree. Since P > 0.05

right and left breast carcinoma, GSTM1, GSTA1 and GSTPi were not statistically different in

the degree of staining. Mean age for invasive lobular breast carcinoma right gland 59.85±17.61,

while mean age for invasive lobular carcinoma left breast 49±5.38. Tumor seize for invasive

lobular carcinoma right gland varies between 0.1 cm and 2.5 cm according to biopsy material,

but tumor size of left glands was found 0.2 cm and 5.4 cm. Estrogen receptor 62 % ± 0.36 for

invasive lobular carcinoma left glands, but for left glands was 68%±0.45.


Oral Presentation

91

KARADUTUN (Morus nigra) ANTİ KANSER ETKİLERİNİN ARAŞTIRILMASI

Semih Dalkılıç, Sevda Kırbağ, Lütfiye Kadıoğlu Dalkılıç, İsmail Korkmaz, Şule İnci

Fırat Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Moleküler Biyoloji ve Genetik Programı, ELAZIĞ

Kanser tüm dünyada oldukça yaygın olarak görülen bir hastalıktır. Dünya Sağlık Örgütü’nün

verilerine göre kanserin görülme sıklığı her yıl daha da artmaktadır. Yapılan tahminlere göre

2030 yılına gelindiğinde 22 milyon yeni kanser vakasının ortaya çıkması beklenmektedir.

Günümüzde özellikle tıp alanında, diagnostik ve tedavi yöntemlerinde elde edilen ilerlemeler

sayesinde kanser tedavisinde önemli başarılar elde edilmiştir. Ancak tüm bu gelişmelere

rağmen özellikle bazı kanser türlerinde elde edilen başarı tatmin edici değildir. Yeni ilaç hedefi

moleküllerin tanımlanması ve daha etkili ilaçların geliştirilmesi için yoğun çalışmalar

yapılmaktadır.

Bunlara ilave olarak bazı doğal maddelerin kanser tedavisinde etkili olabileceği ileri

sürülmektedir. Bu tür uygulamalar neredeyse insanlık tarihi kadar eskidir ve günümüzde bile

uygulanmaktadır ve alternatif tıp veya tamamlayıcı tıp olarak adlandırılmaktadır.

Halk arasında da özellikle doğal yoldan elde edilen kırmızı meyveler şifalı olarak

görülmektedir. Bu çalışmada amacımız kırmızı meyvelerden biri olan karadutun (Morus nigra)

anti kanser özelliğinin incelenmesidir.

Doğal yollardan elde edilen karadut (Morus nigra) meyvesinin, anti kanser etkisi, meme kanseri

(MDA MB 231) ve prostat kanseri (PC3) hücre hatları kullanılarak, MTT (3- (4,5-dimetiltiazol-

2-il) -2,5-difeniltetrazolyum bromür) Assay ile test edildi. Karadut (Morus nigra) meyvesinin

ekstresi kanser hücre hatları üzerine 1/10, 1/25, 1/50, 1/75 ve 1/100 oranlarında 72 saat süreyle

uygulandı.

Karadutun (Morus nigra) farklı konsantrasyonlardaki ekstresinin, prostat ve meme kanser

hücrelerinin canlılığını farklı oranlarda etkilediği gözlenmiştir.

Anahtar Kelimeler: Karadut, Ekstrakt, Meme kanseri, Prostat kanseri, MTT

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The Investigation Of Anticancer Effect Of Cedar Tar Prepared By Traditional Methods

İsmail Koyuncu, Kadir Eği, Ebru Temiz, Ataman Gönel


Cancer is one of the leading causes of morbidity and mortality worldwide and it ranks first after

cardiovascular diseases. Colon adenocarcinoma is the most common cancer of gastrointestinal

system, although it is seen in different populations in different populations. Various methods

such as chemotherapy, radiotherapy, surgical methods, immunotherapy, gene therapy and

alternative therapy are applied together or separately in cancer treatment. Chemotherapy is the

most commonly used method. The biggest handicap of chemotherapy is the destruction of

normal cells while killing cancerous cells. Therefore, there has now been a trend towards plant-

based drugs to provide a more effective treatment for cancer with minimal toxicity. In this study;

the Toros Mountains in Turkey (Tarsus) is grown and used for centuries among the people,

from the cedar trees have been viewed etnofarmosötik feature anticancer activity of tar obtained

by conventional methods. In this study, the cytotoxic effect of cedar tar HCT-116 (colon cancer

cell line) and HUVEC (human normal cell line) cells was determined using MTT Assay method.

At the end of 24 hours, the cytotoxic effect (IC50) on colon cancer was calculated to be 30

µg/ml, whereas the effect on normal cells was approximately twice as high.

Apoptotic effect was examined by Annexin-V, Cell Cycle, JC-1, ROS method. Cedar tar HCT-

116 cells showed apoptotic activity by disrupting mitochondral membrane potential.

Morphological images were also determined by Acridine orange / Ethidium bromide staining

methods. The effect of cedar tar on cell cycle was observed in 70% of G0-G1 phase. In addition,

apoptotic effect correlated with ROS increase.

In this study, as a result of the data obtained by examining some chemical and biological activity

properties of tar, it will be determined whether there will be a new herbal medicine candidate

for cancer treatment.

Keywords: Tar, Cytotoxicity, Cell Death, Colon Cancer, HCT-116

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93

MİDE KANSERİNİN TEŞHİSİNDE KULLANILABİLECEK BİYOBELİRTEÇ

ADAYI GENLERİN BİYOİNFORMATİK ARAÇLAR İLE BELİRLENMESİ

Semih Dalkılıç, Lütfiye Kadıoğlu Dalkılıç

Fırat Üniversitesi Fen Fakültesi Biyoloji Bölümü Moleküler Biyoloji ve Genetik Programı

Geçtiğimiz yüzyıl süresince insidansında bir azalma olsa da mide kanseri, kanserle ilişkili

ölümlerde ikinci sırada yer almaktadır. Özellikle doğu Asya’da en yaygın kanser türüdür (1).

Yapılan bu çalışmada National Center for Biotechnology Information (NCBI) Gene Expression

Omnibus (GEO) veri tabanında bulunan GSE54129 kodlu veri seti kullanılmıştır(2). Bu veri

seti içerisinde 111 adet Mide kanseri tümör dokusu örneği, 21 adet normal mide dokusu

kullanılarak genom boyu gen ifade analizi yapılmıştır.İfade analizi Affymetrix Human

GeneChip 133 Plus 2.0 (HG-U133 Plus 2.0) mikrodizinlerine hibridize edilmiştir. İşlenmemiş

gen ekspresyon verisi (CEL dosyaları) bu veritabanından alınarak biyoinformatik analizler

yapılmıştır. Tüm analizler Applied Biosystem’e ait olan Transcriptome Analysis Console 4.0

(TAC) ile yapılmıştır.

Analiz sırasında öncelikle verinin kalite kontrolü ve önişlem (preprocessing) analizleri

yapılmıştır. Önişlemler, görüntü analizi (Image Analysis), arkaplan düzeltmesi (Background

Correction), normalizasyon (Normalization) ve özetleme (Summarization) adımlarından

oluşmaktadır. Bu işlemlerden sonra bu veri üzerine varyans filtreleme uygulanmıştır. Varyans

filtreleme işleminden sonra ise kanser ve kontrol grubu arasında karşılaştırma yapılarak bu iki

grup arasında ifade düzeyi farklılık gösteren genler (DEG) belirlenmiştir. Tüm bu analizler

RMA (Robust Multi-Array Average) metodu ile gerçekleştirilmiştir. Yalancı pozitif sonuçların

benzerliğini düşürmek için ham p değerleri üzerinden Benjamini-Hochberg düzeltmesi

yapılmıştır. Daha sonra p<0.05 için kat değişimi (log2 fold change, FC > 5) olacak şekilde bir

cut-off değeri belirlenmiştir.

Elde edilen farklı ifade edilen gen listesi daha sonra DAVID (The Database for Annotation,

Visualization and Integrated Discovery v6.8) veritabanına yüklenerek gen zenginleştirme

(GeneSet Enrichment) ve fonksiyonel kümeleme analizleri (Functional Annotation Clustering)

yapılmıştır. Bu veritabanına entegre olan KEGG (Kyoto Encyclopedia of Genes and Genomes)

veritabanı üzerinden de yolak analizleri yapılmıştır.

Elde edilen sonuçlara göre mide kanserinde neoplastik transformasyon sırasında bir çok genin

ifade düzeyi sağlıklı kontrol bireylerle kıyaslandığı zaman anlamlı düzeyde değişmektedir. Elde

Oral Presentation

94

edilen bu sonuçlara göre bu genler mide kanserinin teşhis edilmesinde veya moleküler

tiplendirmesinde kullanılabilecek biyobelirteç adayı olabilecek genlerdir.

Anahtar Kelimeler: Mide kanseri, Gen ifadesi, Biyoinformatik, Biyobelirteç

Oral Presentation

95

L-Askorbik Asit’in (Vitamin C) İnsan Promiyelösitik Kanser Hücre Hattında (HL-60)

MTT Testi ile Araştırılması

Mehmet Tahir Hüsunet1, Mehmet Bertan Yılmaz2, Hasan Basri İla1

1Çukurova Üniversitesi Fen Edebiyat Fakültesi Biyoloji Bölümü Adana/Türkiye;

[email protected]

2Çukurova Üniversitesi Tıp Fakültesi Tıbbi Biyoloji Bölümü Adana/Türkiye

Amaç: L-Askorbik Asit (LAA) Proliferatif Etkisinin İnsan Promiyelösitik Kanser Hücre

Hattında (HL-60) MTT Testi ile Araştırılması

Giriş: L-Askorbik Asit insanlar tarafından sentezlenmeyen, takviye diyet ile dışardan

alınan ve antioksidan özellik gösteren bir maddedir. LAA, birçok meyve ve sebzenin

yapısında bulunmaktadır. LAA içeren gıdalar aynı zamanda antioksidan özellikler de

gösterebilmektedir. Çalışmamızda LAA’nın HL-60 hücre hattı üzerindeki proliferatif

etkisine bakıldı. Bu amaçla LAA konsantrasyonları 0,5 mg/mL, 1 mg/mL ve 2 mg/mL

olarak belirlendi ve MTT testi yapıldı. Buna göre; LAA’nın, HL-60 insan promiyelositik

lösemi hücre hattında 570 nm’deki absorbans sonuçları incelendiğinde doza bağlı bir

proliferasyon artışı görüldü. 0,5 mg/mL ve 1 mg/mL dozları kontrol düzeyinde artışa neden

olurken en yüksek doz (2 mg/mL) hücre canlılığını kontrole göre çok önemli düzeyde

(p<0,001) artığı görüldü.

Materyal-Metod: HL-60 hücre hattının %5 CO2, 37 oC sıcaklık ve nemli ortamda hücre

kültürü yapılmıştır. RPMI-1640 besi ortamı kullanılarak 24 saatlik kültür yapılmıştır.

Kültürün 20. saatinde MTT maddesi verilerek kültür 24. saatte sonlandırılmıştır. Daha sonra

spektrofotometrede 570 nm absorbans değerleri alınarak sonuçlar değerlendirilmiştir.

Sonuç: En yüksek dozun (2 mg/mL) HL-60 insan promiyelositik lösemi hücre hattını ciddi

oranda bölünmeye teşvik ettiği görüldü (p<0,001). Bu durumun daha iyi anlaşılabilmesi için

ileri düzey moleküler çalışmalara ihtiyaç duyulmaktadır. Moleküler çalışmaların MTT testi

ile paralel sonuçlar vermesi durumunda kanser tedavisi gören insanlara antioksidan

içermeyen diyetler tavsiye edilebilecektir.

Oral Presentation

96

Investigation of TMED9-ERAP1 Candidate Gene Expressions Obtained from Multiple

Myeloma Transcriptome Data by RT-PCR

Melda Sarıman1,2, Büşra Karaçam1, Mesut Ayer3, Sema Sırma Ekmekci1, İlknur Suer4, Kıvanç Çefle4, Şükrü Palanduz4, Şükrü Öztürk4, Neslihan Abacı1

1Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine,

Istanbul, Turkey 2Molecular Cancer Research Center, Istinye University, Istanbul, Turkey

3Clinic of Hematology, İstanbul Haseki Training and Research Hospital, İstanbul, Turkey 4Department of Internal Medicine, Division of Medical Genetics, İstanbul University İstanbul School

of Medicine, İstanbul, Turkey

Multiple Myeloma (MM) in the plasma cell dyscrasias group is characterized by transformation

of B cells into malignant plasma cells originating from bone marrow. Monoclonal Gammopathy

of Undetermined Significance (MGUS) is an asymptomatic premalignant plasma cell disorder

to be characterized by the presence of serum M-protein. The expression levels of genes were

investigated in our previous study by RNA-Sequencing from cell pools of MM patients and

healthy bone marrow controls. MM group was compared with healthy bone marrow donor

group over RPKM values and candidate genes with the highest expression in MM group were

determined by filtering with control group. These genes were analyzed by molecular properties

of the genes determined by using bioinformatics tools such as Gene Set Enrichment Analysis

(GSEA), MSGDB (Broad Institute), Panther, and String databases, and biological pathways

within the cell, and the pathways and protein-protein interactions were analyzed in silico.

Candidate genes obtained from transcriptome data are very valuable and specific, and their

direct or indirect relationships of these genes have been revealed and this study was aimed to

contribute to the elucidation of MM pathogenesis.

In our study, bone marrow materials from 30 newly diagnosed, untreated MM patients and from

11 MGUS and 9 healthy bone marrow donor groups were used. Expression levels of the

candidate genes (TMED9 and ERAP1) that we identified previously were examined by qRT-

PCR method. The results of MM group, the expression of TMED9 and ERAP1 genes were

found to be higher than that of MGUS and healthy bone marrow donor group.

According to these results, we think that ERAP1 and TMED9 genes in MM will directly or

indirectly contribute to the elucidation of the pathogenesis of the disease.

Keywords: Multiple Myeloma, Bioinformatic, qRT-PCR, TMED9, ERAP1

This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University

(grant number 30846).

Oral Presentation

97

The Effects Of The Lıf Antagonıst Molecule (Ec359) On The Chordoma Cells

Melike Bayindir Bilgic, Nur Ekimci Gurcan, Utku Ozbey, Aysegul Kuskucu, Omer Faruk

Bayrak Yeditepe University, Institute of Biotechnology, Istanbul

Introduction: Chordoma is a type of malignant bone cancer observed on the skull base, axial

skeleton and around the rump-bone, which is thought to originate from ectopic-notochord

residues in the embryonic period (1). Chordomas are resistant to conventional chemotherapy

and radiotherapies as well as slow growth. Total block resection is usually used as the first

choice in the treatment of chordoma. In addition, radiotherapy is among the treatment methods

used (2, 3). Resistance to chemotherapy, recurrence and metastasis capacity make it difficult to

treat chordoma. Therefore, new treatment foci and strategies targeted for development are of

great importance for chordoma. Our study group has previously demonstrated that Leukemia

Inhibitory Factor (LIF) molecule increases chordoma aggressiveness (4). EC359 is a LIF

antagonist molecule. The EC359, LIF antagonist has been shown to inhibit a wide variety of

tumor types, including tumors resistant to standard therapies, including NSCLC, breast, ovary,

colon and pancreas. In this study, the efficacy of EC359 on chordoma cells was investigated.

Materials and Methods: The EC359 molecule was dissolved with DMSO. The EC359

solution prepared at different concentration values was treated with 6 different chordoma cell

lines (U-CH1, U-CH2, Um-CHOR1, Mug-CHOR1, JHC7, YU-CHOR1). After 8 days of study,

viability was determined by 8th day MTS assay.

Results: During the eight-day experimental period, viability of chordoma cells treated with

EC359 decreased compared to the control group. This significant decrease in viability of EC359

treatment in chemotherapy / radiotherapy resistant chordoma was found to be worth further

research. Therefore, Westernblot and qPCR investigations of the samples will also be carried

out to clarify the mechanism in downstream pathways.

(1) Chugh R, Tawbi H, Lucas DR, Biermann JS, Schuetze SM, Baker LH. Chordoma: The

Nonsarcoma Primary Bone Tumor. The Oncologist. 2007 Jan 11;12(11):1344–50.

(2) Bailey CS, Fisher CG, Boyd MC, Dvorak MFS. En bloc marginal excision of a multilevel cervical

chordoma: Case report. Journal of Neurosurgery: Spine. 2006 May 1;4(5):409–14.

(3) Carrabba G, Dehdashti AR, Gentili F. Surgery for clival lesions: open resection versus the expanded

endoscopic endonasal approach. Neurosurgical Focus. 2008 Dec 1;25(6):E7.

(4) Gulluoglu, S., Sahin, M., Tuysuz, E. C., Yaltirik, C. K., Kuskucu, A., Ozkan, F., ... & Bayrak, O. F.

(2017). Leukemia inhibitory factor promotes aggressiveness of chordoma. Oncology Research

Featuring Preclinical and Clinical Cancer Therapeutics, 25(7), 1177-1188.

Oral Presentation

98

Investigation of Apoptotic Effect of Betulinic Acid in Renal Cancer Cells

Merve Nur Ataş1, Barış Ertuğrul1, Elif Sinem İplik2, Bedia Çakmakoğlu1, Arzu Ergen1

1Istanbul University, Aziz Sancar Institute of Experimental Medical, Department of Molecular

Medicine, Istanbul, Turkey 2Istanbul Yeni Yuzyil University, Faculty of Pharmacy, Istanbul, Turkey


Kidney cancer or renal cancer is the fourteenth most frequent cancer in the world. With its

increasing incidence in the last few years, the renal cell carcinoma is the most common kidney

cancer type. Apart from the environmental factors, increasing activities of expressions of genes

and/or their pathways make cells become cancerous. AKT-1 and mTOR promote cell growth,

proliferation and survival, participate in cellular homeostasis and have ability to suppress

apoptosis in various ways.

Betulinic acid which is a lupane-type pentacyclic triterpenoid is derived from various plants.

Betulinic acid exhibits wide range biological activities such as anti-inflammatory, anti-viral,

anti-bacterial and particularly anti-cancer activities. It is known that betulinic acid promotes

apoptosis in cancer cells along with no toxic effect in normal cells. This effect makes betulinic

acid a potential anti-cancer drug. In our study, clear cell renal carcinoma cell line CAKI-2 and

healthy cell line MRC-5 are used to research the effect of betulinic acid on apoptosis and gene

expression levels. In order to identify the toxic effect of betulinic acid, WST-1 and to detect

apoptosis Annexin-V were conducted. Expression analysis of AKT-1 and mTOR genes were

conducted with Real-Time PCR to measure the effect of betulinic acid in gene expressions

levels.

As a result of this study, the apoptotic activity of betulinic acid on kidney cancer cell line was

detected with Annexin-V. In gene expression analysis, there was statistically significant

decrease in AKT-1 expression level while mTOR expression level was increased. Betulinic acid

with its apoptotic effect on renal cancer cell line and non-toxic effect on normal cell line is a

potential anti-cancer drug promising for future studies.

Keywords: Betulinic acid, renal cancer, apoptosis

Acknowledgment: The present work was supported by the Research Fund of Istanbul University.

Project No: 31565

Oral Presentation

99

Selective cannabinoid-1/2 receptor (CB1 and CB2) agonists suppress cell proliferation and clonogenicity in pancreatic and breast cancer cells

Nergiz Hacer Turgut1, Mumin Alper Erdogan1

1Izmir Katip Celebi University, Faculty of Pharmacy, Department of Pharmacology, Izmir,TURKEY 2Izmir Katip Celebi University, Faculty of Medicine, Department of Physiology, Izmir,TURKEY

Introduction/Aim: Cancer ranks first among the causes of morbidity and mortality all over the

world and is expected to continue to be the main cause of death in the coming years. Therefore,

novel molecular targets and therapeutics strategies are urgently needed. In many cases, some

reports show that levels of endocannabinoids and their receptors are increased in cancer, a

situation that frequently correlates with tumor aggressiveness. Recent studies have suggested

that cannabinoid-1/2 receptors contribute to the tumor growth in a variety of cancers including

pancreas, colon, prostate and breast cancer. Understanding how cannabinoids are able to

regulate essential cellular processes involved in tumorigenesis, such as cell proliferation and

cell death are crucial for improving existing and developing new therapeutic approaches for

cancer patients. In this study, we investigated the impact of CB1-2 receptor agonists on cell

proliferation and clonogenicity in pancreatic(PANC1) and breast(MDA-MB-231) cancer cells.

Material/Methods: The impact of CB1/2 receptors on cell proliferation and clonogenicity was

investigated using selective CB2-agonist-L-759633, CB1-agonist-ACEA and CB1-agonist-

ACP A in our cancer cells, utilizing MTS and colony formation assay, respectively.

Mechanisms of these cell behaviours are also under investigating by Western blot.

Results: CB1/2 receptor agonists led to a significant decrease in proliferation and colony

formation in all cells with the doses of 1, 10, 50, 100, 250µM for 72 h and 14 days, respectively.

Based on our findings, these agonists led to the inhibition of both cell viability and clonogenic

growth as a dose dependent manner. Our first western blot data is also suggesting that CB1/2

agonists may inhibit proliferation and tumorigenesis through the upregulation of apoptotic

proteins in these cells.

Conclusion: Our data suggests that CB1/2 agonists have the therapeutic potential through the

inhibition of survival of these cancer cells and also might be linked with further cellular

mechanisms for the prevention.

Keywords: Cannabinoids, Cancer, CB1/2 Receptor, Agonist, Proliferation, Clonogenicity, Pancreatic

Cancer, Breast Cancer, Neuroblastoma

Oral Presentation

100

Usefulness of simple prognostic markers of complete blood count to Predict lymph node

metastasis in colon carcinoma

Murat Ferhat Ferhatoğlu

Istanbul Okan University, Faculty of Medicine, Department of General Surgery

Background: According to estimates from the World Health Organization (WHO) in 2015,

cancer is the first or second leading cause of death before age 70 years in 91 of 172 countries.

Cancer incidence and mortality are rapidly growing worldwide. With rapid population growth

and aging worldwide, the rising prominence of cancer as a leading cause of death partly reflects

marked declines in mortality rates of stroke and coronary heart disease, relative to cancer, in

many countries. According to GLOBOCAN 2018 data, colon cancer is the fourth most

commonly diagnosed type of cancer and the sixth most common cause of cancer deaths. Lymph

node metastasis is common in patients with colorectal cancer. And, it is a known fact that lymph

node metastasis in patients with colon cancer is an indicator of a poor prognosis. Complete

blood count is a laboratory test frequently used in clinical practice and comprises white blood

cell, red blood cell and platelet counts, and their morphological indices. In this study, we tried

to asses the association between the parameters of complete blood count and lymph node

metastasis in colon cancer.

Materials and Methods: This is a retrospective cohort study conducted in a state hospital from

January 2010 to December 2015. One hundred and five colon carcinoma patients following an

elective colon surgery were enrolled in the study. Pathology results and preoperative complete

blood count parameters were collected.

Results: There were significant positive correlations between leukocyte, lymphocyte,

neutrophil measurements and metastatic lymph node positive cases (0.001, 0.02, 0.014;

respectively).

Conclusion: We found a positive correlation between leukocyte, lymphocyte, neutrophil

measurements and metastatic lymph node positive cases.

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101

Characterization of BRI3 as a Novel Wnt/β-catenin Pathway Target

İzzet Akiva, Necla Birgül İyison Department of Molecular Biology and Genetics, Boğaziçi University North Campus Kpark Building,

Bebek, Istanbul, Turkey

Background: The Wnt/β-catenin signaling is an evolutionary conserved pathway which has

important functions in vertebrate early development, axis formation, cellular proliferation and

morphogenesis. The activation of this pathway leads to translocation of the transcriptional

activator β -catenin into the nucleus where it activates T-cell factor/Lymphoid enhancer factor

(Tcf/Lef) family of transcription factors, which regulate expression of developmental and cell

cycle-related genes. Apart from its roles in various cellular processes, Wnt/β-catenin signaling

pathway is also one of the most important intracellular pathways for cancer progression. A

significant number of identified target molecules of this pathway, are known to have

tumorigenic characters.

Objective: Previous studies confirmed BRI3 gene to be one of the transcriptional target genes

of this pathway, but it has not been associated with cancer up to now and its function remained

largely unknown. Further characterization of this gene in order to elucidate its biological roles

and eventual implications in cancer, is one of the main objectives of this study.

Methods: Overexpression studies in Huh7 cells, Luciferase Reporter Assay, Western Blotting,

Quantitative Real-Time PCR Analysis, Cell Proliferation and Cell Migration Assay, Xenograft

Assay in SCID mice, RNA Isolation from tumor tissues and RNA-Sequencing.

Results: Functional characterization of this novel Wnt/β-catenin pathway target has been

carried out by using both in vitro and in vivo techniques. BRI3 is found to be upregulated in

response to TNF-α treatment and overexpression of BRI3 results in an increase in NFkB

promoter activity. Cell proliferation and migration assays show that, Huh7 cells stably

expressing BRI3 gene have greater proliferative and invasive capabilities compared to control.

Furthermore, in vivo xenograft experiments show that stable overexpression of this gene in

Huh7 cell lines results in significantly larger tumor sizes in SCID mice. In order to determine

the possible interacting pathways in this tumorigenesis process, RNA-Seq analysis was carried

out from BRI3 expressing tumors and control tumors from SCID mice.

Acknowledgements: This study is supported by funding from TUBITAK - 108T183 and BAP (Boğaziçi

University Research Projects) 08B101.

Keywords: Wnt/β-catenin pathway, BRI3, Xenograft, RNA-Sequencing.

Oral Presentation

102

Sub-Culturing Tumorspheres Reduced the Pluripotency of a Chordoma Cell Line

Nur Ekımcı Gurcan1,2, Melike Bayındır Bılgıc1,2, Utku Ozbey1,2, Aysegul Kuskucu2, Omer

Faruk Bayrak2

1 Yeditepe University, Institute of Natural and Applied Sciences, Department of Biotechnology

2 Yeditepe University, School of Medicine, Department of Medical Genetics

Cancer is a result of accumulated mutations altering pathways that control cellular growth,

division and apoptosis. Cancer stem cells (CSCs) are a minor subpopulation within tumors that

have self-renewal and differentiation potential which provides the cancer enhanced resistance

capacity to cellular stress, therapeutic agents and radiation. Studies suggest that invasion and

metastasis capabilities of cancer cells are driven by increased epithelial mesenchymal transition

(EMT) status and associated with CSC phenotype. CH22 is a Chordoma cell line, which is a

rare, malignant bone cancer with poor prognosis. Chordoma cells are highly resistant to

chemotherapeutic drugs. Tumorsphere formation model is a known CSC enrichment method

based on culturing and eliminating non-stem like cancer cells on an ultra-low attachment

surface with specific culture medium conditions. Surviving cancer cell subpopulations are the

ones that exhibit higher stem-like characteristics. However, tumorsphere formation protocols

differ whether they suggest a repetitive sub-culturing and sphere formation or termination of

the experiment with the initial spheres.

In this study, parental CH22 cells, first generation tumorspheres achieved from CH22 cells and

sub-cultured tumorspheres are compared based on pluripotency, EMT and MET related genes

expression changes via qPCR.

Results revealed that sphere formation increases pluripotency markers OCT4, SOX2, Nanog,

KLF4 and c-Myc comparing parental CH22 cell line, but sub-cultured sphere formation

decreases that elevated expression levels. MET markers E-Cad, Cytokeratin 19 and Muc1 are

not elevated in both sphere groups as expected. Surprisingly, some of the EMT related genes

N-Cad, Twist, Snail and Slug are not altered significantly with sphere formation assays.

However, ZEB2 and fibronectin increased with sphere formation and sub-culturing spheres lead

an additional increase in these genes.

This study suggests that sub-culturing tumorspheres may reduce stem-like properties, instead

of CSC enrichment.

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103

Beyin Tümörü Dokularında CYP1A1, CYP1B1, GST-M, GST-P ve MDR Proteinlerinin

Rolü

Onur Dirican1, Pınar Kaygın1, Serpil Oğuztüzün1, Gülçin Şimşek3, Yusuf İzci4, Cahit Kural4, Tülay Çoban5, Sezen Yılmaz Sarıaltın5, Oğuz Kul2, Işıl Yıldırım6

1Kırıkkale Üniversitesi, Fen Fakültesi, Biyoloji Anabilim Dalı, Kırıkkale,Türkiye.

2 Kırıkkale Üniversitesi, Veteriner Fakültesi, Patoloji Anabilim Dalı, Kırıkkale,Türkiye.

3 Sağlık Bilimleri Üniversitesi, Keçiören Eğitim ve Araştırma Hastanesi, Tıbbi Patoloji Anabilim Dalı,

Ankara,Türkiye.

4Sağlık Bilimleri Üniversitesi, Gülhane Eğitim ve Araştırma Hastanesi, Beyin Cerrahisi Anabilim

Dalı, Ankara, Türkiye.

5 Ankara Üniversitesi, Eczacılık Fakültesi, Farmasötik Toksikoloji Anabili Dalı, Ankara, Türkiye.

6 Beykent Üniversitesi, Eczane Hizmetleri Meslek Yüksekokulu,Eczane Hizmetleri Bölümü, İstanbul,

Türkiye.

Beyin tümörü dokularında, immunohistokimyasal yöntemle yapılan bu çalışmada; CYP1A1,

CYP1B1, GST-M, GST-P ve MDR protein ekspresyonları açısından değerlendirilmiştir.

Dokuların elde edildiği hastaların yaş, cinsiyet, alkol kullanımı, sigara kullanımı, tümör

lokalizasyonu, metastatik durumları, kemoterapik veya radyoterapik tedavi alma durumları ve

sağ kalım oranları göz önünde bulundurularak, karşılaştırmalı olarak ortaya konulması

amaçlanmıştır. Çalışma 2017-2019 yılları arasında tümör teşhisi ile beyin cerrahisi bölümü ve

kliniğine başvuran 141 hastadan cerrahi müdahale ile elde edilen ve incelenmesi için patoloji

bölümüne iletilen taze dokuların, immunohistokimyasal metod ile ekspresyon durumları

skorlanarak (0=ekspresyon yok, 1=zayıf ekspresyon, 2=güçlü ekspresyon) değerlendirilmeye

alınmıştır. Hastalardan alınan verilerde, ilgili parametrelere bağlı olarak; 83'ü (%58,9) erkek,

58'i (%41,1) kadındı. Tüm yaşlar için yaş ortalaması 49.44 (6-83) idi; kadınlarda ortalama yaş

50.25 (6-77) ve erkeklerde ortalama yaş 48.91 (11-83) idi. Hastaların 44'ü (%31,2) sigara

içiyordu ve 15'i (%10,6) alkol kullanıyordu. Hastaların 52'sine (%36,9) radyoterapi, 30'una

(%21,3) kemoterapi uygulandı. Çalışmanın sonunda 94 (%66.7) hasta yaşıyordu.

İmmunohistokimyasal metod sonrası kullanılan protein belirleyicilere bağlı olarak boyama

yoğunluğu istatistiksel olarak değerlendirildiğinde; CYP1A1 için; tümör (%6,7) ve normal

(%5,4) dokuları arasında ekspresyon paternleri açısından benzer bulundu (p=0.627). CYP1B1

için ekspresyon oranı tümörlü dokularda 4 kat gibi yüksek bir oranda gözlenirken, tümör

(%41,6) ve normal (%9,4) dokuları arasında oldukça önemli ekspresyon seviyesi farkı görüldü

(p<0.0001). Tümör dokularındaki GST-M ve GST-P ekspresyonu, normal dokularda ki

ekspresyonundan neredeyse 3 kat daha yüksek olduğu belirlendi (GST-M ve GST-P

ekspresyonları için; p<0.0001). Tümör dokularında, normal dokularla karşılaştırıldığında daha

yüksek MDR ekspresyonu saptandı. Tümör dokularının 65'i (%43.6) zayıf ve 22'si (%14.8)

güçlü, normal dokuların 9'u (% 6.0) zayıf ve 1'i (%0.7) güçlü MDR ekspresyonuna sahip olduğu

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gözlenmiştir (p<0.0001). Yapılan bu çalışma da, beyin tümörlü dokularda, CYP1A1’e oranla

oldukça yoğun eksprese olduğu tespit edilen CYP1B1, GST-M, GST-P ve MDR proteinlerinin

dokudaki tespiti, yapılacak benzer çalışmalarda, metabolik sinyal yolaklarının anlaşılması ve

prognostik faktörler açısından müdahalesinin sorgulanması gerektiği düşünülmektedir.

Anahtar Kelimeler : Beyin tümörü, immunohistokimya, CYP, GST, MDR.

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Defining the synergistic sequential application schedules for chemotherapeutic–

molecular targeted agent dual combinations in gastric adenocarcinoma cells

Özen Leylek1,2,†, Gülnihal Özcan2,3,*

1Koc University Graduate School of Health Sciences, Istanbul, Turkey 2Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey

3Koc University School of Medicine, Istanbul, Turkey

†Presenting Author, *Corresponding author, e-mail:[email protected]

Gastric cancer is the fifth most common cancer and the third leading cause of deaths from

cancer. It is diagnosed mostly at the metastatic stage where there is no curative strategy. Though

chemotherapeutics and molecular-targeted agents with different mechanisms of action are

combined to increase treatment efficacy, a significant increase in overall survival could not be

achieved. Therefore, there is an urgent need for new strategies to increase the success of

combination chemotherapy in gastric cancer.

In today’s combination protocols, anticancer drugs are being administered concomitantly to the

patients in chemotherapy sessions. Recent studies suggest that application of the anti-cancer

drugs with a time-interval can increase the degree of synergism provided that the appropriate

time-interval and order of application are determined. In this study, we aim to investigate how

the time-interval and order of application affect the efficacy of chemotherapeutic-molecular

targeted agent dual combinations, and determine the sequential application schedules that

achieve maximum synergistic effect in gastric cancer.

To achieve the goals of the study, dual combinations of five different chemotherapeutics from

anthracyclins, platinium derivatives, taxanes, fluoropyrimidines and topoisomerase inhibitors,

and three different molecular-targeted agents targeting EGFR, mTOR or c-Met are being tested

on four different gastric adenocarcinoma cell lines. In addition to concomitant application, the

drug pairs at each combination are being applied according to ten different schedules at which

the drugs are applied with two different orders and five different time-intervals. The degree of

synergism at each schedule is being measured with Chou-Talalay Method.

Our preliminary data suggests that the time-interval and order of application for

chemotherapeutic-molecular targeted agent dual combinations are important determinants of

the degree of synergism in gastric cancer cells. We believe that the treatment schedules that will

be defined in this project can increase the success of chemotherapeutic-molecular targeted agent

combinations in gastric cancer.

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Funding/Supports:“The authors gratefully acknowledge The Scientific and Technological Research

Council of Turkey (TÜBİTAK) for funding by 3501- Career Development Program (CAREER) Grant

(Grant No: KBAG-117Z460) and use of the services and facilities of the Koç University Research

Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of

Strategy and Budget. The content is solely the responsibility of the authors and does not necessarily

represent the official views of the TÜBİTAK and the Presidency of Strategy and Budget.”

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Targeting TACC3 as a novel therapeutic strategy for breast cancer treatment

Ö. Akbulut1*, D. Lengerli2, Ö. Saatci3, E. D. Ergül4, U. Ö. Ş. Şeker4, B. Çalışkan2, E.

Banoğlu2, Ö. Şahin1,3,4* 1Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University,

Ankara, Turkey 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara,

Turkey 3Department of Drug Discovery and Biomedical Sciences, University of South Carolina,

Colombia, SC, USA 4UNAM-National Nanotechnology Research Center, Institute of Material Science and

Nanotechnology, Bilkent University, Ankara, Turkey *Corresponding author; [email protected]

Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is an essential microtubule-

associated protein which is concentrated at centrosomes where it ensures chromosomal

segregation and microtubule stability. Abnormal expression is frequently observed in a

variety of cancers highlighting its importance to be used as a therapeutic target. KHS101 and

SPL-B are two small molecule inhibitors targeting TACC3, shown to suppress tumor growth

of glioblastoma and ovarian cancer xenografts, respectively. However, the clinical arm of

these inhibitors is still missing.

Here, we combined rational drug design approaches with screening methods where some

chemical fragments of already available TACC3 inhibitors were changed with their isosteric

equivalents to improve potency as well as drug-like properties. We identified a novel TACC3

inhibitor, BO-264, which showed higher potency (in nanomolar range) in in vitro and in vivo

systems and can be used as a mitotic blocker in breast cancer. Inhibitor-TACC3 binding was

validated through target engagement assay, drug affinity responsive target stability and

isothermal titration calorimetry methods. Compared to other available inhibitors, it showed

superior effects on the cellular processes such as mitotic progression, DNA repair and cell

viability, recapitulating the siRNA effect used against TACC3. Notably, BO-264 had

remarkable cytotoxicity effect on several cancer cell lines in NCI-60 human tumor cell line

panel while it doesn’t affect non-cancerous cells. Importantly, oral administration of BO-264

significantly suppressed the tumor growth in BC xenografts both in immunocompromised

and immunocompetent mice models which also increased survival. Finally, kinome profiling

was performed to test the specificity of BO-264 for TACC3 and further validated by state-

of-art binding assays.


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High potency independent from subtype and tissue and minimum toxicity of BO-264

highlight its potential use as mitotic blocker and a novel TACC3 inhibitor not only in breast

cancer, but also in other cancers.

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DNA Damaging Activities of Terbuthylazine

Özlem Demirci

University of Dicle, Faculty of Science, Biology Department, Diyarbakır

Pesticides and their metabolites are important xenobiotics that cause environmental pollution

and lead to very important ecological and health problems. Pesticide residues are present in

water areas, soils, fruits, and vegetables. Although, their application is based on selective

toxicity for certain organisms yet it has resulted in serious effects on many non-target organisms

as well. The use of pesticides has created a type of chemical environment which is proving

harmful to the living systems. Investigation of the interaction of macromolecules with

compounds that cannot be eliminated by means of detoxification mechanisms in organisms

exposed to xenobiotics such as pesticides is a valuable tool for revealing toxic damage.

Persons are exposed to pesticides directly not only when they are applied to vegetables, but also

through the metabolites that are stored in different structures which may turn out to be more

dangerous. Plants can bio-concentrate these environmental agents and convert pro-mutagens

into toxic metabolites. This fact raises the concern that plant systems might also activate

agrochemicals and environmental agents, thereby introducing new mutagens into the human

food chain. For this reason, the present study designed to investigate DNA damaging activity

of terbuthylazine.

Terbutylazine is a herbicide that is widely used in agricultural fields that act as photosynthesis

II inhibitors in target plants. In this study, DNA damaging activity of terbuthylazine was

investigated by using Agarose Gel Electrophoresis.

It was observed that this insecticide have ability to damage DNA.

Keywords: DNA Damage, terbuthylazine, pesticide pollution

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hBD-1 ve hBD-3 Antimikrobiyal Peptidlerinin Ürotelyal Kanser Dokularındaki

Ekspresyonları

Pınar Kaygın1, Serpil Oğuztüzün1, Gülçin Güler Şimşek2, Şeyma Özkan2, Sezen Yılmaz Sarıaltın3, Arzu Kaya Koçdoğan4, Onur Dirican1, Sema Çetin1, Muharrem Atlı1

1Kırıkkale Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Kırıkkale, Türkiye 2Sağlık Bilimleri Üniversitesi, Keçiören Eğitim ve Araştırma Hastanesi, Patoloji Bölümü, Ankara,

Türkiye 3Ankara Üniversitesi, Eczacılık Fakültesi, Toksikoloji Ana Bilim Dalı, Ankara, Türkiye

Antimikrobiyal Peptidler (AMP), bakteri, virüs, mantar gibi patojenlere karşı antimikrobiyal

aktivite göstererek vücudu koruma görevi üstlenmiş, bağışıklık sisteminin doğal elemanlarıdır.

Antimikrobiyal peptidlerin iki geniş ailesini oluşturan Human Beta Defensin (hBD)’ler ve

katelisidin (LL-37/hCAP-18) peptidleri, memelilerde epitel hücreleri, makrofaj ve

nötrofillerden eksprese edilmektedir. Bu peptidlerin antimikrobiyal etkilerinin yanında kanser

biyolojisinde de çeşitli rollerinin olduğu bildirilmiştir. Katelisidin ve defensinler, memelilerde

eksprese edilen AMP’lerin iki büyük üyesidirler. Ürotel kanserinin etiyolojisinde yeni bulgular

immun sistemin bu hastalıktaki önemini vurgulayan niteliktedir. Ancak ürotel kanserlerinin

baskılanmasında immun sistemin rolü hakkında çok az şey bilinmektedir. Bu çalışmada; Ürotel

kanser gelişiminde, hastalardan alınan ürotel karsinomlu ve benign ürotel epiteli dokularda,

hBD-1 ve hBD-3 antimikrobiyal peptidlerinin ekspresyon farklılıklarının incelenmesi

amaçlanmıştır. Ankara Keçiören Eğitim Araştırma Hastanesi Patoloji Kliniği arşivinden 69

hastadan alınan ve bu kişilere ait olan ürotel karsinomlu ve benign ürotel epiteli dokuların

belirtilen antikorlar ile immunohistokimyasal yöntemle boyanmış ve sonra boyama şiddetine

göre incelenmiştir. Negatif boyanma (0), hafif şiddette boyanma (+1), orta şiddette boyanma

(+2) ve şiddetli boyanma (+3) şeklinde değerlendirilen boyama sonuçlarına göre dokularda

peptidlerin ekspresyonları arasındaki farklılıklar SPSS istatistik analiz programında Anova

Testleri kullanılarak; peptidlerle hastaların klinik ve demografik veriler arasındaki ilişkiler ise

aynı programda Pearson Correlation testi ile belirtilmiştir. Buna göre, ürotel karsinomlu

dokularda hBD-1 peptidinin, benign ürotel epiteli dokularında hBD-3 peptidinin daha fazla

eksprese olduğu istatistiksel olarak anlamlı bulundu (p<0.05). Sonuç olarak, belirtilen

antimikrobiyal peptidlerin ürotel kanser biyolojisinde rol oynayabileceği ilk olarak bu çalışma

ile gösterilmiştir. Buna ek olarak bu peptidlerin ürotel kanserindeki rollerinin, daha fazla

örneklem ile çalışılarak ve kanser yolağındaki diğer gen ve proteinlerle karşılaştırılarak

aydınlatılması gerektiği düşüncesindeyiz.

Anahtar Kelime: Ürotel Kanser, Antimikrobiyal Peptidler, hBD-1, hBD-3

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Investigation of the relationship between cellular adaptation and therapeutic resistance

in chronic myeloid leukemia cells under imatinib pressure

Seda Baykal-Köse1, Ahmet Sinan Yavuz2, Eda Açıkgöz3-4 ,Öykü Gönül-Geyik1, Halil Ateş5,

Uğur Sezerman6, Güner Hayri Özsan5 and Zeynep Yüce1,*

1Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey

2Department of Molecular Biology, Genetics and Bioengineering, Faculty of Engineering and Natural

Sciences, Sabanci University, Istanbul, Turkey

3Department of Histology and Embryology, Faculty of Medicine, Ege University, Izmir, Turkey

4Department of Histology and Embryology, Faculty of Medicine, Yuzuncu Yil University, Van,

Turkey

5Department of Hematology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey

6Department of Biostatistics and Medical Informatics, Faculty of Engineering, Acibadem University,

Istanbul, Turkey

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease characterized by the

t(9;22)(q34;q11) translocation, resulting in the chimeric BCR-ABL oncogene. Despite the

significant success of tyrosine kinase inhibitors (such as imatinib) inhibiting Bcr-Abl

oncoprotein function in the treatment of CML, drug resistance is still a serious and maybe the

most important problem affecting 20-30% of patients.

Our in vitro studies are designed using a TKI resistant K562 subclone (K562-Ir) which does not

harbour any BCR-ABL amplification or kinase domain mutation. The K562-Ir subclone was

developed in our laboratory by natural selection under imatinib pressure. We performed

immunofluorescence, Western blot and mRNA microarray (transcriptome) techniques for gene

expression analyses; three-dimensional cell culture studies; cell differentiation experiments;

different TKIs applications with cell death analysis; flow cytometric cell membrane marker

screening; proliferation assays; senescence assays; Q-PCR for amplification and expression

confirmation studies.

We observed that unlike K562 wild type cells, K562-Ir cells were adherent and resistant to

nilotinib, dasatinib, bosutinib and ponatinib in addition to imatinib. K562-Ir cells are more

resistant to cell death and proliferate slower. They are capable of forming tumor spheroids in

three-dimensional cell culture studies. We also showed a significant differences in the

expression of genes that regulate cell-tissue-organ differentiation and development processes

in K562-Ir cells. There is an increase in the expression of embryonic and cancer stem cell

surface markers and reweal that these cells have drifted away from their hematological origin.

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In addition miRNAs which are responsible of cell differentiation, proliferation and cell death

resistance are differentially expressed in the K562-Ir cells.

In conclusion, we present a new TKI resistance model suggesting that under imatinib stress a

sub-clone of the leukemic cells develop transcriptional instability resulting in a Bcr-Abl-

independent/aggressive population with phenotypic adaptation capacity. We believe that the

new model will contribute to a better understanding of drug resistance and relapse problems in

CML biology and the emergence of new therapeutic approaches.

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Neutrophil-to-Lymphocyte Ratio and Platelet Distribution Width Predicts

Invasiveness of Bladder Carcinoma

Selim Yalçın1, Ercan Yuvanç2

1Kırıkkale University Faculty of Medicine, Department of Medical Oncology

2 Kırıkkale University Faculty of Medicine, Department of Urology

Aim: Bladder cancer (BC) is a common tumor in the urinary tract and 75-85% of urothelial

carcinomas (UC) are non-muscle-invasive while 15-25% are invasive. Intracavitary BCG

therapy is one of the prophylaxis and treatment options and one of the anti-tumor mechanisms

of BCG is related to local immunological responses on urothelial cells. Inflammatory cells

around the tumor cells play a significant role in the progress and prognosis of tumors.

Patients and Methods: For this retrospective study, we reviewed the surgical and

pathological reports of 250 patients with UC operated between 2013 and 2019. Samples for

full blood count analysis were collected preoperatively.

Statistics: Factors analyzed were patient age, gender, lifestyle, occupational environment,

tumor stage, tumor size, tumor multiplicity, MPV, neutrophil count, lymphocyte count, platelet

count, platelet distribution width (PDW) and neutrophil/lymphocyte ratio (NLR).

Results: The study population was divided into three groups, i.e. muscle-invasive urothelial

tumors (n=107) and non-muscle-invasive urothelial tumors (n = 143) and healty individuals

(n=150). NLR had the most significant p value in correlation with pathological stage (p=0.005)

while the platelet count had the most risk (OR= 4.2746). When we sorted out the hazard ratios

of these parameters from strongest to weakest, it could be summarized as platelet count > PDW

> female gender > NLR > age (4.2746 – 4.1254 – 3.9734 – 2.8787 – 2.5434, OR value,

respectively). In that study, increased lymphocyte counts were shown in 78% of the patients

with non-muscle-invasive bladder carcinoma but in only 45% of those with infiltrating tumor.

Healthy individuals with a high NLR ratio have a significantly lower NK activity than those

with a low NLR ratio. The platelet count was found to significantly correlate with tumor size,

tumor stage and surgical margin status (p= 0.038 – 0.042 – 0.031, respectively).

Conclusions: The degree of systemic inflammation reflects the local tumor burden and reported

a correlation between lymphocyte reactivity against bladder tumor cells and clinical stage. The

ability of tumor invasion and metastasis is dependent both on the intrinsic characteristics of the

tumor cells and on the environment around the tumor. Patients with high preoperative NLR

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should be considered candidates for additional therapies after resection. Increase of neutrophils

and decrease of lymphocytes is important for determination of the prognosis. In our study, we

found a correlation between pathological stage and age, PDW, NLR, female gender and platelet

count with logistic regression analysis. These parameters can be used to predict the pathological

stage of the tumor before the treatment. NLR is an easily measurable parameter of systemic

inflammation, which can predict pathological stage. The abnormal phenotype of the tumor may

stimulate an influx of inflammatory lymphocytes into tissues surrounding the tumor. The

systemic inflammatory response also features changes in the relative levels of circulating white

blood cells. Predicting the prognosis of patients with cancer by examination of peripheral blood

leukocytes would seem to be an easier and useful procedure. Thrombocytosis may adversely

affect survival by facilitating cell invasion and metastasis. There is evidence that platelets

protect tumor cells by shielding them from the host’s immune system.

Keywords: Urothelial carcinoma, Neutrophil-to-lymphocyte ratio, Platelet distribution width

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Picoa Ve Terfezia Türlerinin Anti-Kanser Etkilerinin Araştırılması

Semih Dalkılıç, Sevda Kırbağ, Lütfiye Kadıoğlu Dalkılıç, Şule İNCİ, İsmail Korkmaz

Fırat Üniversitesi Fen Fakültesi Biyoloji Bölümü ELAZIĞ

İnsanlık tarihi boyunca, bazı mantarlar hem beslenmek için hem de hastalıkları önlemek için

fonksiyonel gıdalar olarak kullanılmıştır. Tüm yenilebilir mantarlar arasında yer mantarı bir

dizi ayırt edici özelliğe sahiptir ve diğer mantarlardan daha fazla dikkat çekmiştir. Yer altında

yetişen bu mantarlar, lezzetli tadı ve misk aroması nedeniyle çok miktarda tüketilir.

Terfezia ve Picoa türleri geleneksel olarak hem beslenmek, hem de iyileştirici özellikleri,

afrodizyak özellikleri için Elazığ-Malatya bölgesi civarında toplanmaktadır. Bu çalışmada, yarı

kurak, kurak veya çöl türüflerinin antikanser aktivitelerinin belirlenmesi amaçlanmıştır.

Yapılan çalışmada Terfezia ve Picoa türleri (Terfezia boudieri, T. claveryi, T. olbiensis, Picoa

juniperi ve P. lefebvrei) gibi çöl mantarlarından elde edilen ekstreler kullanılmıştır.

Terfezia ve Picoa türlerinin su ve metanol ekstrelerinin antikanser etkisi, MDA-MB-231 meme

kanseri hücre hattı ve PC3 prostat kanseri hücre hatları kullanılarak MTT (3- (4,5-dimetiltiazol-

2-il) -2,5-difeniltetrazolyum bromür) Assay ile test edildi. Mantarların Metanol ve su ile elde

edilen ekstreleri kanser hücre hatları üzerine 50 -100-200 ve 400 mg/ml konsantrasyonda 72

saat süresince uygulandı.

Çöl trüflerinin metanol ve su ile elde edilen ekstreleri, kanser hücrelerinin canlılığını farklı

oranlarda etkilediği gözlendi. Özellikle T. claveryi ve T. olbiensis'in ekstrelerinin anlamlı

düzeyde antikanser etki gösterdiği belirlenmiştir.

Key words: Yer mantarı, Ekstre, Anti-kanser, MTT

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The effects of Mackia amurensis leukoagglutinin on the expressions of some extracellular

matrix proteins in anaplastic thyroid carcinoma cell line, 8505C

Serap Sancar-Baş, Engin Kaptan Istanbul University, Faculty of Science, Department of Biology, Section of Molecular Biology

Vezneciler 34134 İstanbul/Turkey

Introduction: Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancy and

most of the patients are diagnosed with advanced and metastatic disease. Therefore, the

preventative therapies could be important for metastatic progression of ATC. The extracellular

matrix proteins regulate the remodelling and deposition of ECM and also effect cell motility,

adhesion and survival. High stromal expression of secreted protein acidic and cysteine -rich

(SPARC) have been reported and it has been observed that lysyl-oxidase (LOX) activity leads

to cell invasion, migration and high metastatic activity in ATC. On the other hand, osteopontin

(OPN), an important glycoprotein related to cell-cell and cell-ECM interactions, has been

associated with invasiveness of papillary thyroid carcinoma. In this study we aimed to

investigate the effect of Maackia amurensis leukoagglutinin (MAL-II), which is spesific for α-

2,6 sialylated glycans, on the levels of these metastasis related proteins in ATC cell line, 8505C.

Material-Method: Anaplastic cells were cultured and treated with 0.25 µM MAL-II for 24 h

at the non-lethal dose and duration according to our previous experiments. MAL-II treated cells

were lysed and RNA and proteins were isolated. The relative mRNA expression levels of

SPARC, LOX and OPN genes were determined with quantitative Real-time PCR and the

measurement of protein levels were studied with Western blotting.

Results: MAL-II treatment decreased the expressions of SPARC, LOX and osteopontin genes.

The protein levels of LOX and SPARC were also decreased with MAL-II, but no changes

osteopontin levels was observed.

Discussion: These results have pointed out that MAL-II treatment alters the expressions of

some extracellular matrix proteins of ATC cells. When considering MAL-II glycan specifity,

targeting α-2,6 sialylated glycans in ATC can be important strategy for the prevention of its

metastatic properties. In conclusion, MAL-II may have a therapeutic potential for treatment of

metastatic ATC.

Keywords: Anaplastic thyroid carcinoma, Mackia amurensis-II lectin, Metastasis, LOX, SPARC,

Osteopontin.

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An Investigation of the Antitumoral Activity of Phycocyanin Derived from Spirulina

(Arthrospira) Platensis on Mice

Sibel Bayıl Oğuzkan, İzzettin Güler, Mehmet Özaslan, Kemal Bakır, Betül Kut Güroy

1Department of Medical Services and Techniques, Vocational School of Health Services, University of

Gaziantep, Turkey

2Department of Biology, Molecular Biology and Genetic, University of Gaziantep, Turkey

3Department of Pathology, Medicine Faculty, University of Sanko, Gaziantep,Turkey

4Central Research Laboratory, University of Yalova, Turkey

Spiruluna is the most important and economically important phycocyanin that is a blue

photosynthetic pigment that is water-soluble and strong fluorescent properties. In this current

study, following the administration of phycocyanin by gavage feeding to balb-c mice with

Ehrlich Ascites Tumor (EAT), its antitumoral effect was studied in vivo. Phycocyanin was

administered to three different experimental groups; i.e. treatment, control and prophylaxis

(n=6) in different concentrations and the antitumoral activity in each group in terms of dosage

effect was detected. At the end of the treatment, cardiac blood was collected from every animal

to determine the oxidant and antioxidant status and liver enzymes parameters (TAL, TOL, ALT

and AST). The subjects were sacrificed under ether anesthesia, the kidney, stomach, small

intestine, and large intestine tissue was removed and pathologically evaluated for tumor

development.. As a result, while the TAL value of the prophylaxis groups was found to be

statistically significant compared with the control group (p<0.05), no statistically significant

difference was found in the other parameters (p<0.05). When the prophylaxis groups were

statistically compared between each other, while a significant increase was observed in values

as the dose increased, a statistically significant difference was found in terms of antioxidant

activity between the prophylaxis group, which was administered the lowest phycocyanin dose.

When the TAL, TOL, ALT and AST values of the treatment groups were statistically compared

with the control group, it was detected that the treatment group (200 mg) had the highest TAL

value, with a statistical significance (p<0.05). Likewise, there was a statistically significant

difference between treatment group and control group in terms of the ALT values, and the same

results were also found to be similar in the comparison by dosage difference. The

histopathological evaluations demonstrated that dosage difference does not have a preventive

effect against tumor formation.

Keywords: phycocyanin, EAT, TAS, TOS, ALT, AST

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Effects of Maackia amurensis leukoaglutinin treatment on the transcriptome of human

anaplastic thyroid cancer cell line 8505C

Suna Bektas, Engin Kaptan

Istanbul University Faculty of Science Department of Biology, Section of Molecular Biology, 34134

Vezneciler, Istanbul, Turkey

Lectins are bioactive molecules and have highly important roles in various cellular process.

They can bind specifically and irreversibly to free sugars and sugar residues of glycoconjugates.

Effects of plant lectins on normal and malignant cells and the underlying molecular mechanisms

have been subject of many researches. Although it is known that Maackia amurensis

leukoagglutin-II (MAL) decreases the tumorigenic and malignant capabilities of human

anaplastic thyroid cancer cells, its molecular mechanism has not been known yet. In this study,

it was amed to investigate the possible molecular mechanisms playing role in suppressing

malignant and tumorigenic characteristics of anaplastic thyroid cancer cells through the

treatment of MAL. For this purpose, whole transcriptome analysis was performed using RNA-

Seq in total RNA isolates of 8505C human anaplastic thyroid cancer cells treated with 0.25μM

MAL during 24 hours. Gene expression profile of MAL treated 8505C cells exhibited

significant changes in the expression of various genes related to the tumorigenic and malignant

characteristics of cancer cells such as LGR5, GPC5, PTGFR, CHFR. Our findings indicated

that the expression pattern of these genes were found to be associated with Wnt/β-catenin

pathway, which was an important signal transduction pathway in proliferation, stem cell control

and carcinogenesis. Besides, MAL treatment were also affected to G-protein-mediated receptor

signalling processes. Our data also pointed out that MAL is an effective bioactive molecule for

the suppression of the tumorigenic and malignant characteristics of anaplastic thyroid cancer.

In addition, our findings indicate that MAL has a significant potential for the treatment of

anaplastic thyroid cancer. However, in order to elucidate specific mechanisms for the effect of

MAL on 8505C cells, the functionality of the related genes and pathways needs to be tested by

conventional methods.

Keywords: Lectins, MAL, Thyroid cancer, RNA sequencing

Funding information: This study was supported by Scientific Research Projects Coordination Unit

of Istanbul University with the project number 32144.

Oral Presentation

119

Cytogenetic Anomalies in Lymphomas: A single center study

Süreyya Bozkurt

Department of Medical Biology, Faculty of Medicine, Istinye University, Istanbul

Lymphomas are clonal tumors originating from T or B lymphocytes and natural killer (NK)

cells. Lymphomas show different morphological, immunological and clinical traits. Although

there are differences in distribution according to geographical regions and etiologic factors,

75% of all lymphomas are non-Hodgkin and 25% are Hodghkin lymphomas. Chromosomal

translocations is one of the etiologic factor in lymphomas (1,2,3).

This study included 150 lymphoma patients who came to cytogenetic laboratory for

conventional karyotyping between 2008-2016. Fifty-five patients were female and 95 were

male. Karyotyping could not be performed in 34 patients (%24) of the bone marrow samples

and 114 (%76) of them were karyotyped. Fifteen patients had Hodgkin lymphoma and 46 had

non-Hodgkin lymphoma. Of the non-Hodgkin's lymphomas, 7 were T-cell lymphoma, 16 were

B-cell lymphoma, 5 were Mantle cell lymphoma and 8 were Burkitt lymphoma. Bone marrow

of the remaining 89 patients came to the laboratory with the diagnosis of lymphoma alone

without any sub-classification information.When the karyotype results are examined, it is seen

that 33 of the patients with NHL have normal karyotype and 9 of them have abnormal

karyotype. Karyotyping could not be performed in 33 patients. In patients with Hodgkin

lymphoma, normal karyotype was found in 10 patients and no results were obtained in 5

patients. None of the patients had abnormal karyotype in Hodgkin lymphoma. Numerical and

structural abnormalities have been identified in NHL patients. As structural anomaly;

t(8;14)(q24;q32), del(1p), del(9p), dup(1q) and del(3q) were found. As numerical anomaly

monosomy of chromosome 6, 8, 11, 14 were detected.

Refences

1-Jiang M, Bennani N N and Feldman A L. Lymphoma classification update: T-cell lymphomas,

Hodgkin lymphmas, and histiocytic/dendritic cell neoplasm .Expert Rev Hematol.

2017.March;10(3):239-249.

2-Lynch R C, Gratzinger D, Advani R H.Clinical impact of the 2016 update to the WHO lymphoma

Classification. Curr.Treat.Options in Oncol.(2017) 18:45.

3-Jaffe E S.Diagnosis and classification of lymphoma: Impact of technical advances.Seminars in

Hematology.(2019) 56:30-36.

Oral Presentation

120

The Role of Nuclear P-glycoproteins in Nuclear Sparing and Chemoresistance to

Doxorubicin in Gastric Adenocarcinoma Cell Models

Tevriz Dilan Demir1,2 †‡, Hande Özkan1,2‡, Gülnihal Özcan2,3*

1Koc University Graduate School of Health Sciences, Istanbul, Turkey 2Koc University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey

3Koc University School of Medicine, Istanbul, Turkey

†Presenting Author, ‡Equal contribution to the study

*Corresponding author, [email protected]

Doxorubicin is one of the most potent and most widely used chemotherapeutics in cancer

treatment. Its’ primary mechanism of action is DNA damage through binding to DNA and

inhibition of topoisomerase II. Therefore, intra-nuclear concentration of doxorubicin is an

important determinant for its anti-cancer efficacy. In sensitive cancer cells doxorubicin

predominantly accumulates at the nucleus leading to cell death. On the other hand, nuclear

accumulation could not be achieved in resistant cells despite the presence of the drug at the

cytoplasm. This phenomenon is known as “nuclear sparing” in the literature. However, the

underlying mechanism for the nuclear sparing and its causal relationship with chemoresistance

is unknown yet. Here, we aim to investigate the role of nuclear p-glycoprotein in nuclear-

sparing and resistance to doxorubicin. P-glycoprotein (p-gp), is a drug efflux pump localized

on the cell membrane which decreases the intracytoplasmic concentration of various

chemotherapeutics including doxorubicin. Recent studies and our findings showed that p-gp’s

can also be localized on the nuclear membrane. It was also suggested that p-gp’s on the cell

membrane can translocate to the nuclear membrane after exposure to doxorubicin. We

hypothesized that increased nuclear expression of p-gp may lead to nuclear sparing and may be

an acquired mechanism of resistance to doxorubicin. To test our hypothesis, we established

doxorubicin resistant gastric adenocarcinoma cell lines by continuous exposure to doxorubicin.

In immunofluorescence and subcellular fractionation studies we observed that the nuclear

expression of p-gp increased with increasing degree of resistance. The nuclear accumulation of

doxorubicin decreased significantly in resistant cells compared to age-matched control cells

investigated with fluorescence imager-reader and confocal microscope. These findings suggest

that increased expression of nuclear p-gp’s may be an important mechanism for acquired

resistance to doxorubicin via decreasing the accumulation of the drug within the nucleus of

gastric adenocarcinoma cells.


Oral Presentation

121

Funding/Supports:

“The authors gratefully acknowledge The Scientific and Technological Research Council of Turkey

(TÜBİTAK) for funding by 3001 Starting R&D Projects Funding Program (Grant No: SBAG-217S342) and

use of the services and facilities of the Koç University Research Center for Translational Medicine

(KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is

solely the responsibility of the authors and does not necessarily represent the official views of the

TÜBİTAK and the Presidency of Strategy and Budget.”

Oral Presentation

122

Synthesis, Antiinflammatory and Anticancer Effects of Novel Amide Containing

Compounds

Tuğba Güngör1, Adem Özleyen2, Y. Berkay Yılmaz2, Tuğba Taşkın Tok3, Mehmet Ay1, Tuğba Boyuneğmez Tümer4

1Department of Chemistry, Faculty of Arts and Science, Natural Products and Drug Research

Laboratory, Çanakkale Onsekiz Mart University, Çanakkale, Turkey 2Graduate Program of Biomolecular Sciences, Institute of Natural and Applied Sciences, Çanakkale

Onsekiz Mart University, Canakkale, Turkey 3Department of Chemistry, Faculty of Arts and Science, Gaziantep University, Gaziantep, Turkey 4Department of Molecular Biology and Genetics, Faculty of Arts and Science, Çanakkale Onsekiz

Mart University, Çanakkale, Turkey

[email protected]

Non-Steroidal Anti-inflammatory Drugs (NSAIDs) are the most widely used drug classes all

over the world. The best-known examples of NSAIDs are aspirin, celecoxib, nimesulide,

diclofenac, ibuprofen, etodolac, naproxen etc. Despite the widespread usage of these drugs on

multifunctional diseases such as inflammation, type II diabetes, metabolic syndrome and

cancer, their long term usage are associated with serious gastrointestinal, cardiac and renal side

effects. To overcome these problems, there is a great need to develop new superior anti-

inflammatory drugs.

In this study, nimesulide, N-(4-nitro-2-phenoxyphenyl)methanesulfonamide, which inhibits

COX-2 selectively and shows analgesic and antipyretic effects was selected as a reference

compound1-3. In recent years, it is reported that nimesulide can be used as an anticancer agent

since it suppresses the proliferation of cancer cells and induces apoptosis2,3. Herein, we

designed and synthesized a series of novel amide containing nimesulide derivatives with

sequential multistep organic reactions and characterized their structures by using melting point,

FT-IR, 1H NMR, 13C NMR and MS analysis. The compounds were screened for their selective

COX-2 inhibitory properties and secondary anticancer effects by using in vitro and cell based

assays. Among the synthesized compounds, five of them demonstrated better inhibitory COX-

2 activity as compared to nimesulide. Two compounds demonstrated moderate antiproliferative

effects against colon cancer cell line. Results showed that lead compounds can be further

developed by using in silico design and synthesis studies for in vivo experiments.

Keywords: Nimesulide, Anticancer, Antiinflammatory, Synthesis, In silico design

*This study was funded by TUBITAK, Grant No.117Z398.


Oral Presentation

123

References

1. Akarca U. S. 2005. “Gastrointestinal Effects of Selective and Non-Selective Non-Steroidal Anti-Inflammatory Drugs”, Current Pharmaceutical Design, 11, 1779-1793.

2. Hida T., Kozaki K., Muramatsu H., Masuda A., Shimizu S., Mitsudomi T., Sugiura T., Ogawa M., Takahashi T. 2000. “Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines”, Clinical Cancer Research, 6 (5),

2006-2011.

3. Renard J. F., Julémont F., de Leval X., Pirotte B. 2006. “The use of nimesulide and its analogues in cancer chemoprevention”, Anti-Cancer Agents in Medicinal Chemistry, 6 (3), 233-237.

Oral Presentation

124

As a potential overall-biomarker of cancer stem cells CD90: on drug resistance

perspective

Utku Ozbey1,2, Nur Ekimci Gurcan1,2, Negar Taghavi Pourianazar1,2, Emre Can Tuysuz1,2, Melike Bayindir Bilgic1,2, Aysegul Kuskucu2, Omer Faruk Bayrak2

1Department of Genetics and Bioengineering, Yeditepe University, 34755, Istanbul, Turkey

2Department of Medical Genetics, Yeditepe University Medical School, 34755, Istanbul, Turkey

Cancer stem cells (CSCs) also known as tumor initiating cells (TICs) are like normal stem cells

and they show the same pattern in tumor. According to recent studies, CSCs are associated with

tumor progression, recurrence and prognosis. Therefore, targeting CSCs in cancer therapy is

important challenge in researches.

CD90 (THY-1) is a membrane glycoprotein which is founded in several cancers. Therefore,

CD90 expression is associated with stemness properties in cancer cells. As an example, small

populations of CD90 expressing HCC, GBM and gastric cancers Epithelial-Mesenchymal

Transition (EMT) profiles, ALDH1 activities and stemness biomarker expressions are

upregulated.

In this study, we perfomed CRISPR/Cas9 technology for silencing THY-1 gene in AGS,

HGC27, A549 and PANC1 cancer cell lines. CD90-/- populations are sorted via BD FACS Aria

III system and stable cell line of knockout cells has been generated and CSC related gene

expressions (Sox2, Oct4, CDH1, Twist, ABCG2) have been studied which are also associated

with drug resistance in cancers. After that, drug resistance studies with cisplatin, cytarabine,

etoposite and taxol on both knock-out and parental cells had been continued over a year. After

that, resistant cells CD90 expressions and profiling of molecular weight of CD90 protein via

western blot. Our findings suggest that, CD90 is potential CSC biomarker in these cancers and

CSC associated drug resistance. Additinaly, molecular weight differentiation of CD90 protein

in drug resistant cells is associated with glycosylation pattern of CD90 is related with drug

resistance in these cell lines.

Consequently, CD90 protein is associated with CSC properties in several cancers and targeting

of CD90 is effecting development of drug resistance in cancers. Therefore, glycosylation

pattern of CD90 might be effective on drug resistance in cancer cells. Finally, according to these

findings, targeting of CD90 in cancer could be new important therapy strategy in drug resistance

inhibition manner.

Oral Presentation

125

The Role of c-Met Neddylation in Hepatocellular Carcinoma

Yeliz Yılmaz1, 2, Bora Güloğlu1,3, Hande Topel1, Ezgi Karaca1, 4, Neşe Atabey1

1Izmir Biomedicine and Genome Center, Izmir, Turkey 2Department of Medical Biology and Genetics, Institute of Health Sciences, Dokuz Eylul University,

Izmir, Turkey 3Department of Biochemistry, University of Oxford, Oxford, United Kingdom

4Izmir International Biomedicine and Genome Institute, Izmir, Turkey

Neddylation is the covalent binding of NEDD8 protein to lysine residues of target proteins.

Neddylation causes alteration of activation, stabilization and localization of proteins. Receptor

tyrosine kinase c-Met has elevated expression and activation in cancer. Enlightening the

mechanisms of sustained expression and activation is of great importance. Only two receptor

kinases were shown to be neddylated. We hypothesize that c-Met might also be prone to

neddylation which might render the kinase more stable and active, resulting in more aggressive

hepatocellular carcinoma (HCC) clones. To conduct our hypothesis, we first investigated total

neddylation status in several HCC cell lines and tumor tissue microarrays, NEDD8 is

ubiquitously expressed and both c-Met and NEDD8 stainings are positively correlated.

Immunoprecipitation of c-Met from lysates treated with c-Met ligand hepatocyte growth factor

and/or c-Met inhibitor SU11274 revealed that c-Met is neddylated upon ligand activation and

neddylation is diminished with inhibitor treatment. c-Met activation is also decreased when

cells are treated with MLN4924 with/without SU11274. The use of SU11274 and MLN4924 in

combination also decreases HCC migration and proliferation. Next we aim to identify potential

neddylation sites through computational analysis. We focused on c-Met’s C-terminal tail and

identified two lysine residues as possible targets of E3 ligases. When we looked at the crystal

structure, we identified K1360 on the tail region as conformationally available. When we

focused on E3 ubiquitin ligase c-Cbl as a known interactor of c-Met, three possible

ubiquitination sites were identified via UbPred as K27, K104 and K1232. Since K27 and K104

belong to the extracellular moiety, K1232 was chosen as the other candidate for neddylation. In

conclusion, we identified a novel post-translational modification of c-Met which alters its

activation and biological responses that leads the cells more invasive and metastatic. NEDD8:c-

Met interaction might be a target for the treatment of hepatocellular carcinoma.

Oral Presentation

126

Keton Cisimlerinin In Vıtro İnsan Meme Kanseri Hücrelerinin Canlılığını Azaltıcı Etkisi

Zelal Zuhal Kaya1, Ayşe Mine Yılmaz2, Gökhan Biçim2, Seval Kaya3, A. Suha Yalçın2

1Tıbbi Biyokimya Bölümü, Tıp Fakültesi, Acıbadem Mehmet Ali Aydınlar Üniversitesi, 34752,

İstanbul, Türkiye 2Tıbbi Biyokimya Bölümü, Tıp Fakültesi, Marmara Üniversitesi, 34854, İstanbul, Türkiye

3Histoloji ve Embriyoloji Bölümü, Tıp Fakültesi, Dicle Üniversitesi, 21280, Diyarbakır, Türkiye

Glikoz katabolizmasının indüklenmesi ve pirüvatın laktik aside dönüştürülmesi, oksijen

varlığında bile kanser hücrelerinde başlıca metabolik modülasyonlardır. Bu fenomen Warburg

etkisi olarak bilinir. Önceki çalışmalar kanser hücrelerinin glikoz bağımlılığı nedeniyle keton

cisimlerini birincil enerji kaynağı olarak kullanamadıklarını göstermiştir. Bu çalışmada, keton

cisimlerininin farklı meme kanseri hücrelerinin canlılığını ve çoğalmasını in vitro olarak inhibe

edip etmeyeceğini araştırdık. İnsan meme kanseri hücreleri (MCF-7, MDA-MB-231, MDA-

MB-435) ve insan foreskin fibroblastları (kontrol), yüksek glikozlu standart ortam, glikozsuz

standart ortam ve glikozsuz ortama ek olarak 10 mM ve 20 mM asetoasetat veya 10 mM ve 20

mM β-hidroksibütirat içerisinde büyütüldü. Asetoasetat ve β-hidroksibütiratın hücre canlılığı

üzerindeki doza bağlı etkisi MTT testi ile belirlenirken, morfolojik değişiklikler ışık

mikroskobu ile gözlenmiştir. Apoptotik hücre miktarı, Annexin V-FITC / PI apoptoz saptama

kiti ile ölçülmüştür. Keton cisimlerinin uygulanması, glikoz varlığına kıyasla, tüm meme

kanseri hücrelerinde hücre çoğalmasını ve canlılığını önemli ölçüde azaltmıştır. Aksine, kontrol

hücrelerinin canlılığı glikoz varlığında / yokluğunda değişmedi. Ek olarak, keton cisimlerinin

hücre canlılığı üzerindeki etkisinin şiddeti, üç meme kanseri hücresinde farklıydı.

Sonuçlarımızın meme kanserinde yeni tedavi yaklaşımlarının gelişimine katkı sağlayacağına

inanıyoruz. Bununla birlikte, bu farkın moleküler mekanizmasını açıklığa kavuşturmak için

daha fazla çalışmaya ihtiyaç vardır.

Anahtar Sözcükler: Keton cisimleri, Asetoasetat, β-hidroksibütirat, Meme kanseri hücreleri

Bu çalışma, Marmara Üniversitesi Bilimsel Araştırma Projeleri Birimi (Proje no: SAG-C-YLP-

081117-0614) tarafından finansal olarak desteklenmiştir.

Oral Presentation

127

Effect of Doxorubicin on Expression of RNA m6A Methylation Enzymes in Leukemia

Cell Lines

Zeliha Emrence1, Melda Sarıman2, Burcu Salman1, Neslihan Abacı1, Sema Sırma Ekmekci1

1Department of Genetics, Istanbul University, Aziz Sancar Institute of Experimental Medicine,

Istanbul, Turkey. 2Molecular Cancer Research Center, Istinye University, Istanbul, Turkey

Up to date, more than 100 chemical modifications of RNA have been identified. N6-

metyladenosine (m6A) is the most commonly observed modification in mRNA, modulating

gene expression by altering features such as structure, maturation, stability, splicing, export,

translation, decay, and affecting cell fate decision, cell cycle regulation, cell differentiation, and

circadian rythim. RNA modification enzymes have been associated in many diseases, including

cancer. It has been shown to play a role in cancer development, diagnosis and treatment by

effects such as proliferation, migration and invasion, and its mechanisms are not fully

elucidated. We aimed to investigate the expression of m6A writers (WTAP, METTL3,

METTL14) and m6A eraser (ALKBH5) via treatment Doxorubicine, a chemotherapeutic agent,

in acute lymphoblastic cell line, REH, and acute promyelocytic cell line, NB4. For this purpose,

500nM doxorubicine was applied to REH and NB4 cells and incubated for 24 hours. At the end

of the incubation period, RNA was isolated from the cells, and cDNA was synthesized. Gene

expression was determined using quantitative real time PCR and analyzed using the 2^ ct

method. Demethylase ALKBH5 expression in NB4 and REH did not change compared to

control. In methylase enzymes, WTAP expression was decreased 1.6 fold in NB4, 1.3 fold in

REH and METTL3 expression was decreased 2.9 fold in REH while did not change in NB4;

METTL14 was decreased 1,8 fold in NB4 and 3 fold in REH. Our results releaved that

administration of doxorubicin ensue decreased expression of methylase enzymes in REH and

NB4 cell lines. These results suggest that doxorubicin may contributes to treatment by affecting

RNA m6A methylation, besides the previously known effects.

128

POSTER PRESENTATIONS

Poster Presentation

129

Plants Used Against Cancer in Folk Medicine in Turkey

Meltem Güleç1, Ayşegül Çalışkan2, Ayşe Şeyma Büyük3

1Istinye University Faculty of Pharmacy, Department of Pharmacognosy, Istanbul, Turkey

2Istinye University Faculty of Pharmacy, Department of Analytical Chemistry, Istanbul, Turkey

3Istinye University Faculty of Pharmacy, Department of Clinical Pharmacy, Istanbul, Turkey

Since time immemorial humankind benefits from plants as nutritient, medicine, cosmetic or to

make tools and shelters for itself (Hoppe 1958). Through history, traditional knowledge was

gathered by trial and error method, practices were developed and sustained (Sezik 1991). It has

been passed on from generation to generation to reach our day. While indigenous people

change their lifestyle, knowledge and traditional medicine based on plants was abandoned and

replaced mostly with conventional medicine (Walter 2003). This study aims to gather

information on medicinal plants used traditionally against cancer in Turkey before it is lost

forever. 124 taxa has been identified and given as a table including their scientific and local

names, used parts and usage against cancer.

More studies are needed to provide scientific evidences to folklore use. It may be helpful in the

development of future medicines or treatments.

Keywords: Cancer, Traditional medicine, Ethnobotany, Turkey, Medicinal plant

References

Hoppe, H.A. 1958. Drogenkunde: Handbuch der Pflanzlichen und Tierichen Rohstoffe, Cram, De

Gruyter und Co., Hamburg.

Sezik, E. 1991. Dünya’da bitkilerle tedavi yaygınlaşıyor, Bilim ve Teknik Dergisi 21:278.

Walter, H.L. 2003. Pharmaceutical Discoveries Based on Ethnomedicinal Plants: 1985 to 2000 and

Beyond, Economic Botany 57(1):126-134.

Poster Presentation

130

Targetıng Trem2 Reduces Prolıferatıon, Induces Cell Death On Breast Cancer Cells*

Ayten Kılınçlı, İbrahim Tekedereli

Department of Medical Biology and Genetics, Inonu University, Malatya

Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor on human

monocyte-derived dentritic cells. TREM transmembrane proteins play vital roles in regulating

inflammation and immune response through their association with adaptor proteins. TREM2

is considered to be a protective negative regulator of inflammation. Besides playing a critical

role in immune responses, TREM2 was involved in a variety of other biological processes,

including osteoclastogenesis, brain homeostasis, and phagocytosis. To date, studies on whether

there is any relationship between TREM2 and cancer development are limited. The aim of this

study is to investigate the role of blocking TREM2 on breast cancer cell lines.

This study was conducted on MCF10A, MDA-MB-231 and MCF7 cells. Western Blot was

used for analysis the expression of proteins. After knocking down of TREM2 by siRNA, cell

proliferation, colony forming ability and cell death rates were tested by MTS, colony formation

assay, trypan blue exclusion assay, respectively.

TREM2 expression was higher in MCF10A than MDA-MB-231 and MCF7 cells. After

treatment with siRNA, cell proliferation was reduced by 60 % in MDA-MB-231, 50 % in

MCF7 cells. Colony forming ability was decreased by 90 % in MDA-MB-231, 80 % in MCF7

cells. Cell death rates were 45% in MDA-MB-231 and 40 % in MCF7.

In this study, for the first time TREM2 expression was analyzed in breast cancer cells and

immortalized human mammary epithelial cells in vitro. TREM2 suppression reduced cell

proliferation and increased cell death. This study suggested that targeting TREM2 could be a

new hope for the treatment of breast cancer.

This work is supported by The Scientific and Technological Research Council of Turkey (TUBITAK) project number 114S979

Poster Presentation

131

DNA Degradation by Water and Ethanol extracts of Allium tuncelianum in the

Presence of Copper Ions: Implications for Anticancer Properties

Selim Gün1, Bircan Çeken Toptancı2, Göksel Kızıl2, Murat Kızıl2, Süleyman Kızıl3

1University of Van Yüzüncü Yıl, Faculty of Science, Chemistry Department, Van, Turkey 2University of Dicle, Faculty of Science, Chemistry Department, Diyarbakır, Turkey

3University of Dicle, Faculty of Agriculture, Department of Field Crops, Diyarbakir, Turkey

There has been an increasing interest in the potential cancer chemopreventive properties of

diet-derived phytochemical agents. Therefore, dietary antioxidants derived plant have attracted

considerable interest for their ability to induce apoptosis and regression of tumors in animal

models. While it is believed that the antioxidant properties of these agents may contribute to

lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account

for apoptosis induction and chemotherapeutic observations.

Most studies on anticancer mechanisms of plant polyphenols invoke the induction of cell cycle

arrest and modulation of transcription factors that lead to anti-neoplastic effects [1]. Dietary

antioxidants can alternatively switch to a prooxidant action in the presence of transition metals

such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth

inhibition in cancer cells. Copper is an essential metal found in chromatin, and observed to be

elevated in a number of malignancies [2]. Further, the cellular DNA breakage and anticancer

effects were found to be significantly enhanced in the presence of copper ions.

In the present study, we tested the ability of water and ethanol extracts of Allium tuncelianum

which is grown Ovacık district of Tunceli province, to cause DNA strand breaks on pBluescript

M13+ plasmid DNA system, both in the absence and the presence of Cu(II), as measured by

standard an agarose gel electrophoresis assay. Both extract tested caused some breakage of

cellular DNA, the degree of such breakage is enhanced in the presence of copper.

The results clearly indicate that extracts of Allium tuncelianum can alternately behave as

prooxidants in the presence of Cu(II) leading to cleavage of plasmid DNA that such DNA

breakage is physiologically feasible and could be of biological significance that have great

potential as putative chemopreventive or therapeutic agents.

1) Kuo, M. L., Huang, T. S., & Lin, J. K. (1996). Curcumin, an antioxidant and antitumor promoter, induces apoptosis in human leukemia cells. Biochimica et Biophysica Acta, 1317, 95-100.

2) Linder M.C (2012). The relationship of copper to DNA damage and damage prevention in humans. Mutation Research, 733(1-2):83-91.

Poster Presentation

132

Role of NEK2A in PLK4 Induced Multipolar Cell Divisions

Selahattin Can Özcan1, Batuhan Mert Kalkan2, Erkam Keskin1, Ceyda Açılan Ayhan1

1Koc University, School of Medicine, Istanbul, Turkey

2Koc University, Graduate School of Health Sciences, Istanbul, Turkey

Introduction: Centrosomes are the major microtubule organizing centers of animal cells, and

they play important roles in organizing the mitotic spindle poles. Centrosomes consist of two

centrioles; a mother and a daughter centriole, surrounded by pericentriolar matrix. Polo like

kinase 4 (PLK4) is important in initiation of centriole duplication and over-expression of PLK4

leads to multiple rounds of centriole duplication in a single cell cycle. Furthermore, PLK4 over-

expression produces “rosette” shaped centrosomes, which consist of several centriols

surrounding the mother centriole. NIMA like kinase (NEK2) is responsible from centrosome

seperation by phosphorylating centrosome cohesion proteins like C-Nap-1, Rootletin and

Cep68. Centriolar links in rosette structures is currently undefined and to understand the role

of Nek2 on PLK4 induced centrosomes, we hypothesised that co-expression of PLK4 and

NEK2 could synergically contribute to centrosome amplification and multipolar spindles

(MPS).

Material and Method: Protein expressions of Nek2 and PLK4-GFP was confirmed by

Western blotting. PLK4 mediated centrosome amplification was visualized by Centrin-2

staining with a confocal microscope. GFP positive metaphase and interphase cells were scored

manually using a fluorescent microscope.

Results: Overexpression of PLK4-GFP in HEK293T cells resulted in centrosome

amplification as determined by confocal microscopy. Metaphase cells were scored in PLK4

over-expressing HEK293T and HEK293T+NEK2 cells. We observed that PLK4

overexpression increased the rate of MPS, and NEK2 over-expression further increased

multipolarity. Also we scored interphase cells for centrosome numbers and observed a similar

phenomenon.

It is known that rosettes generated by PLK4 expression could contribute to multipolarity more

in second cell division (48H) due to disengagement of centriols from rosette centrosomes. But

the observation of increased multipolarity in NEK2+PLK4 cells in the first cell division (24H),

compared to WT+PLK4 cells (%14 to %22) may be attributed to the role of NEK2 on centriolar

links in rosettes.

Poster Presentation

133

Conclusion and future plans: PLK4 over-expression increases the frequency of MPS in

HEK293T cells and NEK2 further contributes to this increase. While the structure of PLK4

induced rosette centrosomes is defined, centriolar links in rosettes remain unknown. To

understand whether this links are similar with normal centrosomes, high-end microscopy

techniques like STED imaging is required. Our most important future plan is to characterize

centriolar links in rosettes and to find out whether NEK2 could phosphorylate these links and

seperates centriols from rosette centrosomes.

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Investigation of Nek2 Kinase Targets with Focus on Centrosomal Unclustering

Batuhan M. Kalkan1, S. Can Ozcan2, Ceyda Açılan Ayhan2

1Koc University, Graduate School of Health Sciences, Istanbul, Turkey


Background and Purpose: The chemotherapeutics used today mostly suffer from the

destructive side effects on healthy tissues as a result of failure to selectively target tumor cells.

Unlike normal cells, tumor cells frequently exhibit extra centrosomes, which tend to form

multipolar spindles (MPS), leaving one/some of the cells with less genetic material triggering

death pathways. Nevertheless, cancer cells divide successfully by clustering their extra

centrosomes into two poles. Nek2A kinase is a key molecule regulating mitotic processes,

including centrosome separation, and we have shown that Nek2 overexpression leads to

centrosomal unclustering. In this project, we aim to investigate how and through which

molecules Nek2A accomplishes its unclustering activity. For this reason, we started with

known Nek2A targets with relevant function and assess their involvement in centrosomal

unclustering. The targets that can efficiently uncluster centrosomes and leads to cell death will

be put forward as novel molecules with a chemotherapeutic potential.

Method: A total of 4 candidate Nek2A targets, (C-NAP1, Rootletin, Centlein, Trf1) will be

tested in different cancer cell lines containing extra centrosomes and exhibiting efficient

centrosome clustering. For candidates, whose silencing can cluster multiple centrosomes and,

the effect of Nek2A upregulation will be tested to understand if Nek2A unclustering

mechanism depends on these targets.

Results: Our previous data showed that overexpression of Nek2A induces unclustering of

centrosomes and MPS, while its disruption provides clustering of extra centrosomes leading

bipolar divisions. Moreover, we have observed a significant reduction in relative cell viability

after overexpression of Nek2A in the cells with supernumerary centrosomes, which is probably

due to unclustering of extra centrosomes leading to MPS. We have previously designed three

guide RNAs for each Nek2A targets and successfully cloned into LentiCRISPR-v2 vectors,

followed by viral packaging.

Future Directions: Beside its known targets, novel Nek2A targets will be investigated using

a phosphoproteomic approach comparing Nek2A overexpressing cells and inactivated cells

using a dominant negative form of Nek2A, knock-out and knock-down separately. Proteins,

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135

which have a known function in mitosis, will be confirmed through western blotting,

coimmunoprecipitation, colocolization studies and kinase assays. Following characterization,

the newly established Nek2A targets will be tested for their ability to override Nek2A

reclustering phenotype when Nek2A is silenced. Lastly, cell death following inhibition of

centrosomal unclustering via silencing of candidate molecules will be determined.

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136

New Therapeutıc Approaches Targetıng Mll-Af9 Leukemıa Through Epıgenetıc

Reprogrammıng

İpek Bulut1, Buse Cevatemre2, Adam Lee3, Arasu Ganesan3, Ceyda Açılan Ayhan1, 4

1Koc University, Graduate School of Science and Engineering, Istanbul, Turkey

2Koc University, Research Center for Translational Medicine, Istanbul, Turkey

3University of East Anglia, School of Pharmacy, Norwich, England


Leukemia is a highly complex disorder of blood and bone marrow and characterized by

inhibition of differentiation during hematopoiesis and leading to an uncontrolled abnormal cell

proliferation. Mixed lineage leukemia (MLL) is a form of acute leukemia which represents

poor prognosis and due to chromosomal translocation, resulting in a hyperactive MLL fusion

protein. It has been indicated that MLL leukemias are largely based on epigenetic irregulations

rather than genomic instability. A transcription factor (AF9) that fuses with MLL plays a role

in the uncontrolled proliferation of MLL cell lines.

Chromatin modifying enzymes are aberrantly expressed in leukemias. Targeting these

regulators such as the Lysine-specific demethylase (LSD1) and Histone deacetylase (HDAC)

has been considered as novel treatment modality. In MLL-AF9 leukemias recruiting of these

enzymes due to translocation leads the activation of several genes that inhibit differentiation

and cause uncontrolled cell proliferation.

In this study, novel compounds which synthesized to inhibit LSD1, HDAC6 and both LSD1&

HDAC6 (Dual Inhibitor) are characterized. In our study, we showed that the compounds inhibit

target enzymes by in vitro enzyme activity tests, we also showed that the compounds inhibit

target enzymes in the cell by cellular thermal shift assay (CETSA). Toxic effects of the

compounds on leukemia cells were shown with ATP-dependent cell viability tests. Increased

histone methylation and acetylation levels after inhibition of target enzymes were indicated

with Western blot. Increased expression of downstream genes due to enzyme inhibiton were

showed using RT-qPCR. The synergistic effect of the inhibitors with the therapeutic drugs used

in the clinic was investigated. We showed that the death response could be increased with

combination of Doxorubicin and our compounds.

Defects in epigenetic pathways involving increased expression levels or abnormal patterns of

activity are one of the key drivers of cell proliferation in cancer. Targeting MLL-AF9 is an

attractive strategy for therapeutic intervention in patients with this genetic translocation. Since

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our inhibitors are more effective on MLL-AF9 leukemias, we hope that our study will result in

the characterization of targeted drugs for use in leukemia therapy.

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138

Investigation of the free amino acid profile of different breast cancer cell lines (MCF-7,

MDA-MB-231, and CRL-4010)

İsmail Koyuncu, Ebru Temız, Ataman Gönel, Mevlüt Bahçevan, Nihayet Bayraktar


Cancer is one of the leading causes of death worldwide, and its early diagnosis improves the

chances of survival for patients. Affecting one out of every twenty women, breast cancer is the

most common cancer type. Due to the increasing incidence of cancer worldwide, genomic,

proteomic and metabolic methods are used to develop a new technology, which has high

specificity and is faster and more sensitive. Metabolomic is a powerful analytic tool based on

new biomarkers and therapeutic targets as well as has a capable of changing the production,

usage, and level of many metabolites. The present study aims to compare the free amino acid

profile of breast and normal cell lines, which have different characteristics, and to investigate

their biomarker potentials for the diagnosis and treatment of breast cancer. In this framework,

intracellular free amino acid profiles of MDA-MB-231 (triple-negative), MCF-7 (ER+) breast

cancer and CRL-4010 normal cells were investigated by the method of LC-MS/MS. According

to the obtained results, trans-4-OH-proline and 4-OH-proline (p<0,05*), ortophospo-serine,

citrulline, aspartate, 3-amino-isobutyric acid (p<0,01**), and GABA (p<0,001***) were found

to be statistically increased in MDA-MB-231 line compared to the CRL4010; however,

isoleucine, losin, valine, tyrosine, tryptophan, threonine, lysine, histidine, glutamine, aspartate,

1-MHIS (p<0,05*), norvaline, carnitine, serine, asparagine, arginine, alanine (p<0,01**), and

argininosuccinic acid, sarcosin, trans-4-OH-proline, cystathionine, 4-oh-proline (p <0.001 ***)

were found to be significantly increased in MCF-7 line compared to the CRL 4010.

Consequently, our data show that the levels of some amino acids are changed in the pathogenies

of the tumor. At this stage, we can speculate that these amino acids can play roles in tumor

progression and drug activity (sensitive/resistant) but detailed amino acid level manipulating

studies are required to elucidate the relationship between amino acid profile and cancer

pathogenesis.

Keywords: Breast cancer, MCF-7, MDA-MB-231, CRL-4010, metabolomics, LC-MS/MS

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139

Hepatoselüler Karsinoma, Mide ve Kolorektal Kanser Hastalarında Tedavi Öncesi ve

Sonrası Oksidatif Stresin Araştırılması

Elif Bozbulut1, Mustafa Gökçe2, Muhammed Yunus Bektay3, Murat Serilmez 4, Mustafa Uzun5, Turgut Aksoy5, Abdurrahim Kocyigit1, Eray Metin Guler1

1 Bezmialem Vakıf Üniversitesi Tıp Fakültesi Tıbbi Biyokimya A.D. Fatih – İstanbul 2 Bezmialem Vakıf Üniversitesi Eczacılık Fakültesi Farmakoloji A.D. Fatih – İstanbul

3 Bezmialem Vakıf Üniversitesi Eczacılık Fakültesi Klinik Eczacılık A.D. Fatih – İstanbul

4 İstanbul Üniversitesi Onkoloji Enstitüsü Fatih – İstanbul

5 Bezmialem Vakıf Üniversitesi Tıp Fakültesi Fatih – İstanbul

Dünya genelinde her sene 1 milyondan fazla yeni kolorektal kanser (KRK) vakası teşhis

edilmektedir. KRK dünya genelinde en sık görülen üçüncü kanser ve dördüncü en yaygın

kanserden ölüm sebebidir. KRK özellikle sanayi ülkelerinde yaygın ve kanser ölümlerinde

ikinci sıradadır. Mide kanseri dünya genelinde en sık görülen dördüncü kanser türü olup son

yıllarda görülme sıklığının azalmasına rağmen, kanserle ilişkili ölüm nedenlerinin başında

gelmektedir. Hepatoselüler karsinoma (HSK) karaciğerin sık rastlanan malin tümörüdür ve en

çok Afrika, Çin ve Avrupa’da gözlenmektedir. Son yılların verilerine göre kanserler arasında

sıklık açısından beşinci sırada yer alırken kanserden ölüm sıklıkları arasında üçüncü sırada

bulunmaktadır. HCC gelişiminde birçok faktörün önemli rol oynadığı bilinmekte; siroz, alkol

kullanımı, oksidatif stres ve hepatit B, C virüsleri risk faktörleri arasında değerlendirilmektedir.

Hepatokarsinogenez patogenezinde oksidatif stresin artışı, proto-onkogenler, growth faktörler

ve tümör baskılayıcı genlerde mutasyonların rol oynadığı düşünülmektedir. Kanserin ortaya

çıkmasında enfeksiyon ve enflamasyona bağlı oksidatif stresin rol oynadığı her ne kadar bilinse

de, bu ilişki şimdiye kadar tam olarak aydınlatılamamıştır.

Kanser tanısı konan 45-65 yaş arası 150 hasta ile demografik açıdan aynı özellikteki 50 sağlıklı

kontrol gruplarından alınan venöz kan örnekleri santrifüj edilerek serumları ayrıldı. Serum

örneklerinden total tiyol, natif tiyol, total antioksidan seviyeleri (TAS) ve total oksidan

seviyeleri (TOS) fotometrik yöntem ile ölçüldü. Oksidatif stres indeks (OSI) seviyeleri ve

disülfit ise hesapla bulundu.

Bu çalışmada amacımız, mide kanseri, kolorektal kanser ve hepatoselüler karsinoma teşhisi

konan hastaların tedavi öncesi ve ilk kemoterapi sonrası oksidatif stres durumunun

değerlendirilmesidir. Sonuç olarak, tüm kanser türlerinde tedavi öncesi kontrole göre yüksek

çıkan oksidatif stres seviyeleri tedavi sonrası da daha da arttığı gözlemlenmiştir.

Anahtar Kelimeler: Mide kanseri, kolorektal kanser, hepatoselüler karsinoma, oksidatif stres

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Cross-talk between tumor and stem cells: determination of conditioned media cytokine

levels

Gamze Tanriover*, Sayra Dilmac*, Mustafa Gokhan Ertosun**, Gulperi Oktem***, Altug

Yavasoglu***

*Akdeniz University, School of Medicine, Department of Histology and Embryology

**Akdeniz University, School of Medicine, Department of Plastic and Reconstructive Surgery

***Ege University, School of Medicine, Department of Histology and Embryology

Tumor and surrounding microenvironment cells are closely related and interact constantly. The

mutual communication between these cells may affect some factors such as cytokines that

involved in tumor progression. It is known that the interactions between stem cells and their

niche are reciprocal; since secrete some factors. We hypothesized that depending on

microenvironment cytokines can modulate to stem cells response.

DPSC (dental pulp stem cells) which is confirmed for co-culture experiments and MDA-MB-

231 cells were used in this study. Firstly, we established single and co-cultures using MDA-

MB-231 and DPSC cells and obtained conditioned media (CM). As a control conditioned

media were prepared from single cell line cultures. Secondly, TGF 1 and GDF15 levels were

determined in CM using ELISA.

Surprisingly, the CM from MDA-MB-231 incubate with DPSC cells and CM from DPSC

incubate with MDA-MB-231 cells have increased TGF 1 and GDF15 expressions but, the

mediums from individual cell lines did not identify any sensible change for the cytokines. It

was suggested that increasing these cytokines might be related with the cells cross talk’s.

An improved understanding of this microenvironment will help us to cross-talk the tumor and

stem cells or to develop better therapeutic strategies for inhibition of some cytokines. It could

be thought that this relation would improve the survival and quality of life of cancer patients.

Keywords: MDA-MB-231, DPSC, cross-talk, TGF 1, GDF15, ELISA

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Yeni Sentezlenen Naftakinon Türevli Bileşiklerin Üçlü Negatif Meme Kanseri

Hücreleri Üzerindeki Sitotoksik Etkilerinin İncelenmesi

Hatice Dinçer, Didem Karakaş, Zeliha Gökmen, Engin Ulukaya

Kanser Biyolojisi ve Farmakolojisi, Sağlık Bilimleri Enstitüsü, İstinye Üniversitesi Cevizlibağ,

İstanbul

Kanser ve kanser nedenli ölümlerin her geçen gün artış göstermesi günümüz dünyasının en

önemli problemlerinden biri olarak karşımıza çıkmaktadır. Meme kanseri kadınlarda en sık

görülen kanser türleri arasında ilk sırada yer almaktadır. Üçlü negatif meme kanseri (TNBC)

östrojen reseptörü, progesteron reseptörü ve insan epidermal büyüme faktörü reseptörü II

geninin eksprese olmadığı bir tümör grubudur. İlaçla hedeflenebilir reseptörlerin eksikliği

nedeniyle kemoterapi, hastalık sonuçlarını iyileştirmek için önerilen tek sistemik tedavidir.

Ancak klinikte kullanılan kemoterapötik ajanlar henüz TNBC hastalarına kalıcı bir çözüm

sunmamaktadır. Bunun sebebi kemoterapi sonrası hücrelerde gelişen direnç ve relapstır.

Metastatik relapstan sonra sağkalım diğer meme kanseri alt tiplerine göre daha kısa, tedavi

seçenekleri az, cevap oranları düşüktür. Bu nedenle TNBC'nin tedavi sonucunu iyileştirmek

için, güvenilir yeni ilaçlar ve tedaviler geliştirilmelidir.

Doğada bulunan kinonik bileşiklerin antibakteriyel, antifungal ve antitümorijenik özelliklere

sahip olduğu bilinmektedir. Naftokinonlar, kinon ailesinin üyeleridir ve umut verici özellikleri

nedeniyle araştırmalarda yaygın olarak kullanılırlar. Bu doğrultuda kinon çekirdeği içeren

klinik olarak önemli birçok antitümör ilaç temel alınarak yeni naftakinon türevli bileşikler

sentezlenmiştir.

Bu çalışmada yeni sentezlenmiş olan naftakinon türevli bileşiklerin (ZP-2, ZP-3, ZP-4, ZP-5,

ZP-11) belirli dozlarının (50-25-12,5-6,25-3,125-1,562 µM) MDA-MB-231 TNBC hücre

hattındaki 48. saatteki sitotoksik etkilerine bakılmıştır. Yapılan SRB sonuçlarına göre en etkili

bulunan 2 bileşiğin (ZP-2, ZP-4) IC90 değerleri belirlenerek 12 ve 24. saatlerdeki sitotoksik

etkileri MUSE Flow Sitometri cihazında Annexin-V/PI ve Caspase 3/7 testleri ile

gösterilmiştir.

Anahtar Kelimeler: Üçlü negatif meme kanseri, MDA-MB-231, naftakinon, sitotoksisite, apoptoz

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The Importance of FOXP3 C/A (rs:3761548) Genetic Variants and Protein Expressions

in tumoral tissues of patients with Non-Small Cell Lung Cancer

Dilara Sönmez1, Şeyda Demirkol1,2, Merve Uzunoğlu1, Cem Horozoğlu3, Akif Turna 4, Volkan Kara4, Ebru Nur Ay1,5, Mehmet Tolgahan Hakan1,6, Özlem Küçükhüseyin1, İlhan

Yaylım1

1Molecular Medicine Department, Aziz Sancar Institute of Experimental Medicine / Istanbul Medical Faculty at Istanbul University, Istanbul- TURKEY.

2Istanbul Biruni University, Vocational School of Health Services, Istanbul- TURKEY. 3Department of Pathology Laboratory Techniques, Vocational School of Health Services, Istanbul

Gelisim University, Istanbul, Turkey 4Department of Thoracic Surgery, İstanbul University-Cerrahpaşa, Cerrahpaşa School of Medicine,

Fatih, 34096, İstanbul-TURKEY. 5Istinye University, Vocational School of Health Services, İstanbul, Turkey

6Hitit University, Art and Science Faculty, Department of Biology, Çorum-TURKEY

Regulatory FOXP3 + T-cells are known to cause low lymphocyte infiltration by controlling

tumor stromal infiltration in non-small cell lung cancer (NSCLC) patients. Therefore, in

previous studies, the increase of FOXP3 + T-cells in tumor environment has been associated

with poor prognosis in NSCLC. A single nucleotide polymorphism (SNP) in the FOXP3 gene

(rs3761548 in the promoter region) has been found to be associated with susceptibility to

NSCLC.

This study was conducted on 48 patients and 124 healthy controls diagnosed as non-small-cell-

lung cancer in İstanbul Cerrahpaşa University Training and Research Hospital. In order to

determine the different gene variants of FOXP3 C/A(rs:3761548) molecule in the blood

samples of the laryngeal cancer and healthy controls, DNA isolation was performed and then

polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) was

applied. And then also, proteins isolated from the control and tumor tissues of 22 patients with

trizol (Ambion) were measured by western blot protein determination method. Statistical

analysis of the study was determined by SPSS 13 program.

As a result of our study, FOXP3 rs3761548 (A/C) polymorphism was associated with a

significantly increased cancer risk for non-small-cell lung cancer (p<0.001). In addition, our

study demonstrated that the frequencies of CC and AA/CC genotypes significantly increased

in NSCLC patients (for both results; p<0.01) [ respectively, odds ratio (OR)=3.495; 95%

confidence interval (CI)=2.055-5.944, OR=2.087; 95% CI=1.653-2.634]. When

histopathological data of our lung cancer patients were evaluated, we found increased

frequencies of CA genotype (p=0,044) and A allele for our patients with the presence of

vascular invasion (p=0.024 OR=2.656; 95% CI=1.040-6.781) than those with absence of

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143

vascular invasion. We didn’t find any correlation between FOXP3 rs3761548 genotypes and

FOXP3 protein expressions in the tumoral tissues of the NSCLC patients.

According to our results, we have indicated the modulating effect of rs3761548 in the promoter

region may be an important parameter for the risk and tumor progression for NSCLC. Our data

also investigated possible correlation between FOXP3 SNP (C/A - rs:3761548) and FOXP3

protein expression on the etiopathogenesis of NSCLC.

The present work was supported by the Research Fund of Istanbul University. Project No. 24517

Poster Presentation

144

Investigation of the Cell Cycle Specifity of Zn(II) 5,5-Diethylbarbiturate Complex on

A-549 Human Lung Cancer Cell Line

Mehmet Emin Köse1*, Buse Cevatemre2, Ferda Arı3

1Department of Biology, Faculty of Arts and Sciences, Uludag University, 16120, Bursa, Turkey

Current address: Cancer Biology and Pharmacology, Health Sciences Institute, Istinye University, 34010, İstanbul, Turkey

2Koç University Research Center for Translational Medicine, 34450, İstanbul, Turkey 3Department of Biology, Faculty of Arts and Sciences, Uludag University, 16120, Bursa, Turkey

Lung cancer is a significant health problem and the survival rate is still not satisfactory despite

the discoveries of new treatments. Survival time (5 years) fails by 60-70% and in advanced

stages to 5%. Many scientists are focused on the cell cycle because of the promising effect of

the combination of cytostatic and cytotoxic agents. Therefore, this study was conducted to

determine that at whether newly synthesized [Zn(barb-κN)2(phen-κN,N’)]·H2O complex was

sensitive and/or specific to the cell cycle in A549 lung cancer cells. Synchronization process

carried out by using the cell cycle specific agents [aphidicolin (2 µg/mL) and mimosine (0,5

mM), thymidine (2 mM), nocodazole (40 ng/mL) and colcemid (20 ng/mL) Using the cell cycle

specific agents, aphidicolin and mimosine, thymidine, nocodazole and colcemid which are

specific for G1 phase, S phase, G2/M phase, respectively.]. Following the verification of the

synchronization by Flow Cytometry (Muse) Cell analyzer, A-549 lung cancer cells were

treated with Zn(II) complex and then subjected to sulforhodamine B assay to determine cell

viability at 48h. After synchronization, no significant effect on viability was observed

compared to Zn (II) and its combinations. In conclusion, effect of Zn(II) complex was found

regardless of cell cycle status.

Keywords: Lung cancer, anti-cancer activity, cell cycle, synchronization, [Zn(barb-κN)2(phen-

κN,N’)]·H2O complex.

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145

Is Notch1 Associated with LeptinR, IL-6 and TNF-α in the CRC Tumorigenesis?

Erkasap N1, Ozyurt R1, Ozkurt M1, Erkasap S2, Yaşar F2, İhtiyar E2, Canaz F3, Yılmaz E3,

Çolak E4.

1Department of Physiology, 2General Surgery, 3Pathology, 4Statistics, Eskisehir Osmangazi

University Medical Faculty, Eskisehir / Turkey

The aim of this study is to investigate the interaction of Notch, leptinR, IL-6 and TNF-α in

cancerous and adjacent normal colorectal tissues and the effects of various related genes on

colorectal cancer (CRC) development mechanism. One of the important intracellular pathways

involved in CRC is the Notch signaling pathway. Notch has an important role in regulating the

balance between proliferation, differentiation and apoptosis. Recent studies in several cancers

have reported that one of the Notch regulators is leptin. Most cancer studies have shown

changes in blood leptin levels. Such as leptin another adipokines, IL-6 and TNF-α have been

reported to have proangiogenic activity and increase tumor neovascularization. All these data

indicate that adipokines play an important role in the molecular regulation of cancer

development. However, the role of these four molecules has not yet been fully understood in

the CRC. Notch1, leptinR, IL-6 and TNF-α expressions were detected by qRT-PCR in

cancerous (n:40) and adjacent normal (n:40) colorectal tissues at a distance of 10 cm.

According to qRT-PCR results, in cancerous tissue comparison with normal tissues, mRNA

levels of Notch1, leptinR, IL-6 and TNF-α increased respectively (p<0.05), (p<0.001),

(p<0.001) and (p<0.001). The results of this study give information about Notch1, leptinR, IL-

6 and TNF-α related intracellular pathways involved in the formation of colorectal cancer and

may provide new treatment methods to the literature.

This study is supported by a grant from The Scientific And Technological Research Council Of Turkey

(TUBITAK 1001, no: 116S628).

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146

Effects of phosphorylation changes of E2F1 from S235 on proliferation and migration

Sayra Dilmac*, Mustafa Gokhan Ertosun**, Osman Nidai Ozes***, Gamze Tanriover*

*Akdeniz University, School of Medicine, Department of Histology and Embryology

**Akdeniz University, School of Medicine, Department of Plastic and Reconstructive Surgery ***Akdeniz University, School of Medicine, Department of Medical Biology and Genetics

Breast cancer is one of the causes of death in women due to the lack of effective treatment

methods. The basic stage of tumor development is the uncontrolled proliferation of tumor cells.

Controlling of cell proliferation depends on the limited progression of the cell cycle. Many

factors are involved in the cell cycle regulation. One of these factors, E2F1 which is the

transcription factor, is the regulation of cell cycle and plays a role in apoptosis. E2F1 is

necessary the progression of the cell cycle. In addition to, E2F1 could be affect motility of the

tumor cells. We aimed to determine if, E2F1 phosphorylation effects in cell proliferation and

migration in tumor cells.

E2F1 S235 (Serine 235) phosphorylation changes was achieved by Site-Directed Mutagenesis

(SDM) methods. We changed serine aminoacid at position 235 to Alanine (A) (inhibition form

of phosphorylation) and Glutamic acid (E) (phosphomimetic form). We used NT-4T1 (Non-

Treated), WT (Wild-type E2F1) and E2F1 S235 mutant forms (S235A, S235E) 4T1 cells. 4T1

cells (1000/per well) were seeded 94-well plate for MTT assay. Cells were treated 24, 48 and

72 hours for MTT. 4T1 cells were seeded (9x105 cells/per well) 6-well plate for migration

assay. A straight scratch was made on the cell layer using a 200 μl sterile pipette tip. 4T1 cells

were photographed at 0 and 48 hours. Wound healing was measured with using ImageJ

Changed to from E2F1-Serin235 to Alanine cell viability was increase. On the contrary to, if

changed this Serine to Glutamic Acid cell viability was decreased. Similarly, 4T1 cells

migration have increased with E2F1-S235A. Migration ability of 4T1 cells have decreased with

E2F1-S235E.

Our results suggested that the phosphorylation of E2F1 on S235, could decrease proliferation

and migration on 4T1 cells. These results indicate that S235 phosphorylation of E2F1 is very

important for tumor cells progression.

Keywords: E2F1, S235 phosphorylation, 4T1, MTT, migration

Poster Presentation

147

The Role of p62 in Pancreatic Cancer Development and Metastasis

Kivanc Görgülü1, Kalliope N. Diakopoulos1, Selin Ulukaya1, Jiaoyu Ai1, Derya Kabacaoglu1,

Katrin Ciecielski1, Ezgi Kaya-Aksoy1, Dietrich A. Ruess1, Götz Hartleben2, Stephan Herzig2,

Roland M. Schmid1, Bruno Sainz Jr3, Marina Lesina1 and Hana Algül1

1II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

2Institute for Diabetes and Cancer, German Center for Diabetes Research (DZD), Neuherberg, Germany

3Department of Biochemistry, School of Medicine, Autónoma University of Madrid, Madrid, Spain

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas

with 5-year survival rate. Therapeutic approaches are still limited for patients due to the

cancer’s aggressive nature and late diagnosis.. Recently, inhibition of autophagy in PDAC has

shown promising results in pancreatic cancer patients. Autophagy is a pivotal player to

maintain cellular homeostasis in physiological and pathological conditions. Here we show that

p62/SQSTM1, is a degradation marker of autophagy, has multiple roles in pancreatic cancer

development and progression. In order to reveal the function of p62 in pancreatic cancer, we

generated mice that express oncogenic Kras in primary pancreatic cancer cells and have

homozygous disruption of p62 (p62;Kras) and compared them with mice with only oncogenic

Kras (controls). Pancreas specific deletion of p62 attenuated pancreatic cancer development.

However, once tumor developed homozygous deletion of p62 enhanced metastasis in mice. To

understand the mechanism of in vivo findings, primary tumor cells were isolated and used to

perform several analyses. We observed that Kras cells had more proliferative, organoid

forming and colony forming capacity compared to p62;Kras cells. Having morphological

differences in mitochondria via transmission electron microscopy analysis, metabolic profiling

of cancer cells has shown that p62;Kras cells had higher glycolytic flux. To confirm, metastatic

phenotype of p62;Kras mouse model, intra-tail vein injection of cancer cells is performed,

p62;Kras cells had more metastatic dissemination compared to Kras cells. RPPA analysis of

cancer cells depicted that epigenetic regulation and mitochondrial membrane organization

related pathways may be underlying mechanism of these changes in p62;Kras cells. Therefore,

particular functions of autophagy related proteins in pancreatic cancer should be conceived

when planning therapeutic strategies targeting autophagy in pancreatic cancer.

Poster Presentation

148

Expression levels of glutathione S transferase isoenzymes in pleomorphic adenoma of

the parotid gland and normal gland.

Sedat Aydin1, Arzu Kaya Koçdoğan2, Mehmet Gokhan Demir3, Serpil Oguztuzun4, Murat Kilic5, Nagehan Barışık6, Nuran Topal4, Pınar Kaygın4

1Department of Ear Nose Throat and Oral Maxillofacial Surgeon, Istanbul University, Istanbul,

Turkey 2Istanbul Gelisim University, Vocational School of Health Sevices, Patology Laboratory Techniques,

Istanbul, Turkey 3Department of Otorhinolaryngology, Kartal Dr. Lutfi Kirdar Kartal Education and Research

Hospital, Istanbul, Turkey 4Department of Biology, Kirikkale University, Faculty of Arts and Sciences, Kirikkale, Turkey 5Department of Pharmacy Services, Ankara University Vocational School of Health Services,

Ankara, Turkey 6Department of Patology, Kartal Dr. Lutfi Kirdar Kartal Education and Research Hospital,

Istanbul, Turkey

Pleomorphic adenoma is the most common salivary gland tumor that has detected on parotid

gland. Enzymatic and genetic markers are of considerable significance in understanding the

mechanisms of tumorigenesis and in the identification, diagnosis, prevention and possible

treatment of tumors and prognosis and follow-up. Glutathione S-transferases (GSTs) are a well

known enzyme family which role in detoxification, either by conjugating glutathione with

electrophilic compounds or by direct binding with carcinogens. GSTs have been divided into

several subgroups. GSTs enzymes might be used as tumor markers to diagnose neoplastic

lesions. We have known that parotid gland tumors are diagnosed via fine neeble aspiration

biopsy. Sometimes diagnostic difficulties has arisen and recurrent fine needle aspiration is

needed. The tumor markers might be used also in this situation. The normal distribution of the

GST in salivary gland was investigated and reported the higher GST activity in adenoma than

normal tissue. In this study, we investigated the distribution of GST in parotid gland

pleomorphic adenoma and normal parotid gland tissue . The sudy group is composed of 52

patients (25 female, 27 male) aged between 18-77 (mean 40.2) who were operated due

pleomorphic adenoma of the parotid gland. It was found that GSTP1 expression was higher in

the normal tissues of 14 patients (42.31 %) compared to tumor tissues and 22 (26.92%). GSTK1

expression was higher in the normal tissues of 12 patients (50%) compared to tumor tissues

and 26 (23%). Higher levels of GSTK1 expression in normal tissues in comparison to tumor

tissues was found to be statistically significant (p<0,05). There was no statistically significant

correlation between expression of GST isoenzymes and clinical information of patients. Our

findings showed the decreased GSTK1 staining in tumor cells which is significantly different

from normal parotid cells. GSTK1 might be useful tool to differentiate the normal cell from

Poster Presentation

149

pleomorphic adenoma. We think that this information will reduce the confusion that may be

encountered in the differentiation of malignant and benign tumors especially in fine needle

aspiration biopsy.

Keywords: Parotid gland pleomorphic adenoma, Glutathione-S-transferase, immunohistochemistry,

fine needle aspiration biopsy

Poster Presentation

150

New Orthotopic Mouse Colorectal Cancer Model

Sibel Gökşen1, Sefa Özdemir2, Süleyman Can Öztürk2, Güneş Esendağlı2

1Department of Medical and Surgical Research, Hacettepe University Institute of Health Sciences,

Ankara, Turkey. 2Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey.

Mouse cancer models are the important milestone for drug testing and screening tumor

development. Orthotopic models give an opportunity to observe tumor growth on its own tumor

microenviroment. In our study, we developed a new method for mouse colorectal cancer model

for orthotopic tumor engraftment from cell culture to animal.

CT26 murine colon carcinoma cells were grown until they reach to 70% confluency using full

RPMI medium (10% FBS, 1% penicilline/streptomycine antibiotics and 1% L-glutamine) at

37 °C and 5 % CO2. For harvest, after trypsinization the cells were washed with PBS and

centrifuged (400g x 5 minutes). CT26 cells (2x106/100μl) in PBS coated with Matrigel matrix

(1:10) and prepared for engraftment to 8 weeks old male BALB/c mouse. Anesthesia was

performed using ketamine/xylazine method. The cecum was delivered through 15 mm incision,

firmly scratched with a 28G needle and then returned back to abdomen. Prepared cells were

poured on the small wounds of scratching and incision was closed using 5/0 monofilament

absorbable suture. 0,5 ml sterile 0.9 % NaCl was injected subcutaneously to prevent

dehydration. On 10th day after surgery, mouse was euthanized and dissected. Primary tumors

were observed only on cecum and colon.

In our new methodology there is no need to do cecotomy or direct injection of the cells into

colon. This our new mouse colorectal cancer model can be used for drug treatment and

development, tumor screening and cancer biogenesis. Also, it will give new insights for cancer

studies due to its orthotopic usage.

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FULL TEXT

Full text of oral presentation

152

The importance of dysregulated miRNAs on ovarian cysts and tumors

Ece Gumusoglu1, Tuba Gunel1, Mohammad Kazem Hosseini1, Nogayhan Seymen2, Taylan Senol3, Uğur Sezerman2, Samet Topuz4, Kılıç Aydınlı5

1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul,

Turkey.

2Acıbadem University, School of Medicine, Department of Basic Sciences, Biostatistics, Istanbul

Turkey.

3Bağcılar Training and Research Hospital, Department of Obstetrics and Gynecology, İstanbul,

Turkey.

4Istanbul University, Department of Obstetrics and Gynecology, Istanbul Medical Faculty, Istanbul,

Turkey.

5Medicus Healthcare Centre, Istanbul, Turkey

Abstract


the most lethal gynecologic malignancy. Epithelial ovarian cancer (EOC) is asymptomatic and

few screening tests are available. In our study, we aimed to find the importance of dysregulated

miRNAs in ovarian cyst and their expression profile difference between ovarian cancer cases.

Another goal of our study is to identify novel circulating miRNAs to be used as therapeutic

prediction of EOC. In this study, we studied three different samples: serums of EOC patients,

healthy individuals (HI) and benign ovarian cysts (BOC). Their miRNA expressions have been

compared by microarray. Microarray data were analyzed according to miRNA expressions the

relation between miRNAs target genes and EOC were examined by bioinformatic tools. 75 and

66 significantly dysregulated miRNAs were identified by microarray in BOC vs. EOC and

BOC vs. HI comparison, respectively. Then, we focused on common miRNAs that found in

both comparison and finally 46 important miRNAs were detected which can represent the only

common sample group, BOC. After these findings, we also considered miRNA profiling in

EOC and HI, and surprisingly any common miRNAs were found with these 46 miRNAs. Thus,

we analyzed them depending on their potential importance on BOC pathogenesis. After

bioinformatic analysis, our findings indicated that there are several biological processes and

pathways which can be considered to be related BOC development.

Introduction


the most lethal gynecologic malignancy (Srivastava et al, 2017). Among the types of ovarian

cancer, epithelial ovarian cancer (EOC) is the most lethal ovarian cancer type (Srivastava et al,

2017). EOC is usually asymptomatic and few screening tests are available, almost 70% of

women present with advanced-stage (stage III or IV) disease, with 5-year survival rates of less

than 30% (Retamales et al, 2017). Patients who are diagnosed with stage I disease have a 5-

year survival rate of up to 90%, and patients with stage (stage III or IV) disease have a 5-year


153

survival rate of up to 70%. Therefore, early detection of EOC has a great importance in clinic

(Retamales et al, 2017).

An ovarian cyst is a type of body surrounded by a very thin wall and filled with fluid secreted

from the local microenvironment, tumor cells, and stroma (Onur et al, 2013). Ovarian cysts can

be divided into 2 groups: functional cysts and non-functional cysts. Most ovarian cysts belongs

to non-functional class and they are asymptomatic (Lee et al, 2000), which includes polycystic

ovary syndrome (PCOS) (Woo et al, 2013).

MicroRNAs (miRNAs) are small regulatory RNA family found in plasma and used for

diagnosis of human diseases. miRNAs are non-coding RNAs that represses specific mRNAs

and regulates post-transcriptional gene expression (Lan et al, 2015). Although miRNA

expression is irregular in ovarian cancer, mutations are found rarely in cancer related miRNAs.

Several studies showed that different miRNA profile between normal ovarian epithel and

ovarian cancer (Lan et al, 2015).

In this study, miRNA expression profiles have been compared between patients suffering

ovarian cancer, simple ovarian cysts and healthy individuals. In this study, we aimed to detect

significantly dysregulated serum miRNAs with the comparison of their expression levels

between patients (BOC and EOC) and healthy individuals. Thus, we aim to illuminate

pathogenesis of BOC and to make contribution to the difference from ovarian cancer in terms

of molecular mechanisms. Furthermore, we can make a small step to develop novel therapeutic

approaches for cystic ovarian syndromes.

Material Methods

This study is ethical approved by Istanbul University Faculty of Medicine Clinical Researches

Ethics Committee (Permission no: 2014/1175) on 08/08/2014. Scientific Research Projects

Coordination Unit of Istanbul University also funded our study under the projects numbered as

47803 and 51472. Two main steps were followed in this study: serum miRNA profiling by

microarray approaches and bioinformatic analysis of target genes and pathways.

Sample collection

In this study, 10 epithelial ovarian cancer (EOC) serum samples which are valuable in common

clinical practice, from patients, 11 benign ovarian cyst (BOC) serum samples and 15 serum

samples from HI were collected to compare miRNA expression levels by microarray. The

samples were collected by Department of Obstetrics and Gynaecology, Istanbul Medical

Faculty in Istanbul University. All volunteers have been asked to confirm and sign the informed

consent approved by the ethical committee. Before cyst or tumor removal surgery, peripheral

blood samples were collected from each group in clot activator tube (10 ml) and centrifuged

immediately at 3500 rpm for 15 min at 4 °C. The supernatant fluid, serum, was transferred to

RNase-free tubes after centrifugation. Following this step, serum samples were immediately

stored at − 80 °C until studied.


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Total RNA extraction

Total RNA was extracted with the mirVana™ miRNA Isolation kit (Ambion, life technology,

USA) for microarray analysis following to the manufacturer’s protocols. In the final step of

extraction, total RNAs were eluted into 35 μl mirVana elution solution.

Quality Control of Extracted RNAs

The quantity control step was performed on extracted all RNAs by NanoDrop ND-2000

Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 8 samples with the highest

quality were selected from each group (EOC, BOC and HI) for microarray study. All total RNA

samples were stored in -80°C until laboratory preparation.

MiRNA expression analysis with microarray

The miRNA expressions from all sample types were analyzed by Agilent miRNA microarray

chips (Agilent SurePrint G3 Human miRNA r21 8x60K). The miRNA Labeling and

Hybridization Kit (Agilent Technologies) was used according to the manufacturer’s

instructions. Cyanine 3-cytidine bisphosphate (pCp-Cy3) was used for each total RNA (which

included small RNAs) labeling. After this step, for hybridization was done for 24 h on Human

miRNA Microarray Version 16 (Agilent Technologies) slides which include 1368 miRNAs

encoded by genes located across all chromosome. These slides were scanned by an Agilent

SureScan Microarray Scanner (Model G2600D) and “AgilentFeature Extraction” software

(v.12.0) was used for image analysis. Tissue and serum derived RNAs from each group (EOC

and BOC) were analyzed in single microarray chip, then compared each other. The target genes

and pathways of these miRNAs were analyzed with KEGG (Kyoto Encyclopedia of Genes and

Genomes) and GO (Gene Ontology) databases with p value < 0,005.

Results

Sample characterization

The median age of EOC and BOC, the mean of CA-125 (cancer antigen-125) level, the

consumption of alcohol and tobacco profiles, histological types and stages, the number of

patients with metastasis or other diseases and their survival situations were shown in Table 1.

None of the patients had taken neoadjuvant chemotherapy, all patients had undergone primary

cytoreductive surgery.


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Table 1. Demographic and clinical characteristic of BOC and EOC.

BOC EOC

Number of Samples 11 10

The median of age 40 47

The mean of CA-125 (U/ml) 85,3 1520,2

Alcohol N N

Cigarette N N

Histological types and stages 4 cases are endometrioid cysts, 2 5 cases are stage 3C grade 3

cases are dermoid cysts, 2 cases serous cancers, 2 cases are stage

are mucinous cystadenoma and

2 3B grade 3 serous cancers, 1 case

cases are serous cystadenoma (No is 2B grade 1 serous cancer, 1

valid staging system for cyst case 2A endometrioid and 1 case

patients) is 1A grade 3 serous cancers

The number of patients with 1 patient developed borderline 9 patients developed metastasis

metastasis or other diseases ovarian cancer

occurred after sample

collection

Survival All still alive 8 patients alive, two patients

passed away (23 and 32

months)

*N: No usage

Microarray results

MiRNA expression profiling results has shown that there were several significantly changed

miRNAs in serum samples. All dysregulated miRNAs were selected based on p values (< 0.05)

and fold changes (> 2). 75 miRNAs were found as dysregulated in BOC compared to EOC

samples. Otherwise, from BOC vs HI comparison, 66 miRNAs were found as significantly


156

dysregulated. After analysis of these two results, it was found that 46 miRNAs were common

in both group and their expression alteration also similar (up- or downregulation). (Table 2)

According to these results, 4 miRNAs were down and 2 miRNAs were up regulated in BOC

tissue samples.

Table 2. Dysregulated common miRNAs in serum samples in BOC vs. EOC and BOC vs. HI

comparison

BOC vs. EOC BOC vs. HI

miRNA logFC adj.P.Val logFC adj.P.Val

hsa-miR-1207- -1.362 0.01307976 -1.864 0.003935395

5p

hsa-miR-1227- -2.742 2.43E-05 -2.484 0.000243633

5p

hsa-miR-1915- -3.134 2.29E-06 -2.374 0.000281523

3p

hsa-miR-2861 -2.127 0.000104813 -1.659 0.003935395

hsa-miR-3162- 1.479 0.004755918 1.515 0.012906392

3p

hsa-miR-328- -1.331 0.047903462 -2.06 0.010634825

5p

hsa-miR-34b- 2.099 1.23E-05 1.27 0.008383498

3p

hsa-miR-3591- 1.884 0.000136467 1.313 0.013365004

3p

hsa-miR-3656 -1.851 8.38E-05 -2.133 2.92E-05

hsa-miR-3663- -1.452 0.027134019 -1.621 0.0404849

3p

hsa-miR-3665 -2.544 4.85E-08 -1.792 4.21E-05

hsa-miR-371b- -3.304 1.79E-06 -3.006 1.15E-05

5p

hsa-miR-3940- -1.748 5.35E-06 -1.978 1.76E-06

5p

hsa-miR-3960 -1.402 0.005525347 -1.508 0.010634825


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hsa-miR-4254 1.594 5.80E-06 1.119 0.00155371

hsa-miR-4281 -2.097 0.000110739 -2.498 2.92E-05

hsa-miR- -1.074 0.018695511 -1.717 0.001791038

4433a-3p

hsa-miR-4446- 1.379 0.000149864 1.314 0.000851001

5p

hsa-miR-4466 -2.632 2.29E-06 -2.357 2.85E-05

hsa-miR-451a 2.521 0.016837998 2.95 0.01841656

hsa-miR-451b 2.183 0.002086641 2.283 0.004670585

hsa-miR-4530 -1.21 0.031159443 -1.639 0.013973395

hsa-miR-466 1.627 1.34E-05 1.006 0.007591278

hsa-miR-4730 2.335 0.005006931 2.411 0.012906392

hsa-miR-4763- -2.552 4.94E-05 -2.447 0.000228055

3p

hsa-miR-4787- -2.245 0.000129136 -2.117 0.000805023

5p

hsa-miR-5001- -3.01 1.54E-05 -2.602 0.000281987

5p

hsa-miR-5010- 1.729 0.000217645 1.771 0.000639126

3p

hsa-miR-572 -1.297 0.011149386 -1.377 0.022997867

hsa-miR-6068 -2.639 0.00010944 -2.249 0.002020182

hsa-miR-6087 -1.782 0.003970873 -1.823 0.010951788

hsa-miR-6088 -2.439 3.78E-06 -2.488 5.61E-06

hsa-miR-6090 -1.296 0.019690327 -2.38 0.000421357

hsa-miR-6125 -2.39 0.000110739 -2.218 0.000796256

hsa-miR-638 -1.564 0.00162682 -1.175 0.039770036


158

hsa-miR-6514- 2.233 0.000109411 1.564 0.010301901

3p

hsa-miR-6716- 1.986 0.009854892 2.357 0.009626385

3p

hsa-miR-6724- -1.957 0.000297636 -2.356 0.000122162

5p

hsa-miR-6791- -3.662 1.16E-08 -3.163 5.42E-07

5p

hsa-miR-6800- -2.58 4.77E-06 -1.977 0.000454228

5p

hsa-miR-6821- -2.452 5.80E-06 -2.08 0.000184515

5p

hsa-miR-6834- 1.704 0.000804102 1.292 0.023323819

3p

hsa-miR-6850- -2.207 0.005629416 -2.264 0.014502295

5p

hsa-miR-7108- -2.25 0.000117511 -2.478 0.000122162

5p

hsa-miR-7704 -1.668 0.003856916 -1.687 0.011303091

hsa-miR-8485 2.08 0.001038117 1.65 0.021095792

According to target gene and pathway analysis of these 46 miRNAs, Table 3 showed that

3 pathways were detected with KEGG analysis.

Table 3: The pathways and target genes of common 46 miRNAs from KEGG database

Pathways miRNAs P value Target Genes

hsa-miR-1915-3p 2,46E-18 FUT3,FUT6

Glycosphingolipid biosynthesis

hsa-miR-34b-3p 0,0020494 FUT9

hsa-miR-3665 9,08E-27 FUT3,FUT6

- lacto and neolacto series

hsa-miR-4530 8,07E-08 FUT3,B4GALT3

hsa-miR-6514-3p 1,14E-08 B3GALT5,FUT9

GALNT13, GALNT1,

hsa-miR-34b-3p 1,02E-07 C1GALT1

hsa-miR-4466 8,44E-12 GALNT14


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Mucin type O-Glycan

POC1B-GALNT4, B4GALT5,

biosynthesis

hsa-miR-5010-3p 0,000405 GALNT4, GALNT1, GCNT1

hsa-miR-6088 0,0042684 GALNT7,WBSCR17

hsa-miR-6090 5,53E-07 GALNT8

hsa-miR-6724-5p 6,17E-11 GALNT6,GALNT8

COL27A1, COL3A1, COL1A1,

hsa-miR-3663-3p 1,34E-13 COL4A4, COL11A1

ECM-receptor interaction

COL24A1, COL27A1,

hsa-miR-4254 1,18E-11 COL6A3, COL4A6

hsa-miR-6088 0,0049984 LAMA3, COL24A1, COL1A1

hsa-miR-7704 3,77E-06 COL27A1

Discussion

The physiological and pathological roles of altered miRNAs have been demonstrated in many

tumor types and may play an important role in the diagnosis, prognosis, and treatment of cancer

(Wan et al, 2014). In our study, we aimed to find a new perspective to illuminate the pathogenesis

of ovarian cyst and cancer. Therefore, we focused on miRNA expressions in both group and also

HI group for control. After microarray and bioinformatics analysis of results, 46 miRNAs were

selected because of their common expression level alteration in both comparisons. When they were

evaluated due to their target genes and pathways, we have found 3 pathways which are also

important in some other cancer developments. For instance, Mucin Type O-Glycan Biosynthesis

pathway is an evolutionarily conserved protein modification present on membrane-bound and

secreted proteins (Tran et al, 2013). Aberrations in O-glycosylation causes some human diseases

and are related with disease risk factors (Tran et al, 2013). There are researches about this

pathway and cancer relation. Broukhausen indicated that O-Glycan structures are often

unusual or abnormal in cancer, and greatly contribute to the phenotype and biology of

cancer cells. Some of the mechanisms of changes in O-glycosylation pathways have been

determined in cancer model systems. Therefore, this pathway can be effective in cyst

formation. We suggested that these genes and pathways preferred to be analyzed with

further investigations to prove their certain role in cyst and cancer formation. Also, we

recommend also their role in malignant transformation whether it is effected in cyst to

cancer transformation. Besides all these, our miRNA findings derive the understanding of

ovarian cyst and cancer mechanism.


160

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doi:10.5483/bmbrep.2013.46.7.151


161

In vitro Self-renewal Properties in A549 and H1299 Non-Small Cell Lung Cancer Cells

Hiba H. A. Khair, Isik Didem Karagoz

Department of Biology, Faculty of Art and Science, Gaziantep University, Gaziantep, Turkey

Abstract:

Lung cancer remains at the top of the list of cancers with the highest incidence and mortality

rates worldwide. The cancer stem cell paradigm proposes that a small subpopulation of cancer

cells called cancer stem cells (CSCs) can self-renew, recapitulate tumor heterogeneity by

differentiating into different lineages of cells that constitute the bulk of the tumor and

substantially lead to tumor recurrence. Accumulating evidence suggests that a small

population of stem-like cells are responsible for the initiation, progression, and metastasis of

lung cancer. This study aims to assess cancer stem-like properties in A549 and H1299 non-

small cell lung cancer cells. The spheroid formation assay was used to assess self-renewal

ability indicated by tumorspheres formation and to calculate sphere-forming efficiency in

vitro. The limiting dilution assay was used to calculate the minimum number of cells needed

to be cultured to see at least one spheroid in vitro. Cells derived from both cell lines could

form tumor spheroids. The sphere-forming efficiencies of A549 and H1299 cells were 4.14 ±

1.06% and 5.81 ± 1.28%, respectively. However, H1299 cells produced more and larger

tumorspheres when compared to A549 cells. The mean diameter of tumor spheroids in H1299

and A549 cells were 95.25 ± 41.03 and 80.70 ± 31.72 μm, respectively (p=0.00003). In

limiting dilution assay, A549 and H1299 single cells produced spheroids at 23.5 ± 43.7% and

62.5 ± 49.4%, respectively. The frequency of sphere-forming cells (stem-like cells) was found

to be 6.06-fold higher for H1299 cells than those for A549 cells. These results suggest that the

self-renewal ability of H1299 cells is higher and more pronounced compared to A549 cells.

As a conclusion, both A549 and H1299 cells comprise stem-like cells with self-renewal ability.

Keywords: Lung cancer, Cancer stem-like properties, Self-renewal.

Introduction:

Lung cancer is the main cause of cancer deaths worldwide with both high mortality and

morbidity rates due to the limited therapeutic options (Kalemkerian et al., 2013; Siegel et al.,

2016). Approximately 80% of lung cancers are non-small cell lung cancers, which are non-

neuroendocrine tumors including adenocarcinomas, large cell carcinoma and squamous cell

carcinoma (Parsons et al., 2010; Siegel et al., 2016). Even with the most advanced imaging,

staging, surgical strategies, chemotherapy, radiotherapy, as well as personalized treatments

with targeted therapies such as EGFR tyrosine kinase inhibitors and ALK inhibitors in specific

patients group, the five-year survival rate for patients is only 5–15% (Kato et al., 2004; Winton

et al., 2005; Leon et al.,2016).

The stem cell model of carcinogenesis suggests that cancers arise from cells with stem-like

characteristics as a result of the impaired regulation of self-renewal pathways. Such


162

dysregulation stimulates the expansion of this cell population that then may undergo additional

genetic or epigenetic modifications to turn into a fully transformed clone (Wicha et al., 2006).

Tumor heterogeneity and the presence of small subsets of cells with stem-like properties called cancer stem cells (CSCs) have been reported in almost all malignancies during the past decade

(Leon et al.,2016). CSCs typically exhibit many features of embryonic or tissue stem cells including slow growth rates and insensitivity to chemotherapy and/or radiation therapy,

therefore new treatment modalities that selectively target these cells are needed to control stem cell survival, proliferation, and differentiation (Jordan et al., 2006).

Increasing evidence proposes that lung cancer incorporates a cancer stem cell subpopulation

responsible for the onset, spread, and metastasis of the disease (Ho et al.,2007; Eramo et

al.,2008; Levina et al.,2008; Bertolini et al.,2009; Jiang et al.,2009, O’Flaherty et al.,2012;

Wang et al.,2013). Due to the fact that CSCs cells have been implicated in tumorigenicity,

cancer progression, and therapeutic resistance, the study of cancer stem cells in lung cancer

would provide important insights into the understanding of CSCs biology, expansion, and

maintenance, as well as for the development of novel diagnostic and prognostic tests and

exploring novel therapeutic targets for combating this disease (Pine et al.,2008; Peacock and

Watkins ,2008; Miyata et al.,2015; Zakaria et al.,2017). However, each type of tumor

comprises different progenitor or stem cell types that are controlled by various molecular

pathways, which results in variation in the expression of markers in lung cancer subtypes

making these cells more difficult to be identified and targeted (Zakaria et al.,2017).

The ability to self-renew and produce differentiated progeny constitutes the basic features of

cancer stem cells (Ponti et al., 2005; Eramo et al., 2008). Tumorspheroids are a typical

descriptor of cancer stem cells that can maintain stemness profiles in proliferation,

differentiation and under serum-free culturing conditions (Weiswald et al., 2015). Sphere

formation assay has been widely used as a surrogate assay for the enrichment of cancer stem

cells or cancer cells with stem-like characteristics from solid tumors. Tumorspheroids culture

systems are based on the ability of stem/progenitor cells to survive and form floating 3-

dimensional spheroid bodies in semisolid media, such as collagen or Matrigel or in low

attachment plates, in serum-free growth medium containing epidermal growth factor and basic

fibroblast growth factor (Ishiguro et al.,2017). Spheroid cultures as a method of enrichment

for cancer stem cells have been used in many human cancers and cancer cell lines including

glioma, melanoma, sarcoma, lung cancer, renal cancer, and rhabdomyosarcoma (Singh et al.,

2003;Fang et al., 2005;Gibbs et al., 2005; Eramo et al., 2008; Zhong et al., 2010 ; Walter et

al., 2011).

The in vitro extreme limiting dilution assay is used to determine the frequency of cancer stem

cells (CSCs) enriched suspension cultures obtained from a primary tumor sample or a cell line

and grown in a serum-free medium containing growth factors. In addition, in vitro and in vivo

limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or

genetic knockdown strategy on the cancer stem cell frequency (CSC) in a cancer cell

population of a given cell line or a primer tumor sample (Agro and O'Brien, 2015).

In view of all that has been mentioned so far, this study aims to assess cancer stem-like properties in A549 and H1299 non-small cell lung cancer cells.


163

Materials and Methods:

Cell culture:

A549 and H1299 non-small cell lung cancer cell lines were maintained in RPMI-1640 medium

supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Cells were

grown in a humidified incubator at 37°C and 5% CO2 (Zakaria et al., 2015).

Spheroid formation assay:

To enrich for A549 and H1299 CSCs, they were plated at ~100 cells per well in ultra- low

attachment 96-well plates (Corning, New York, USA) in spheroid culture medium containing

serum-free medium DMEM-F12K (1:1) supplemented with 20 ng/ml EGF, 10 ng/ml bFGF,

1x B27 supplement, 1x insulin, 0.4% Bovine Serum Albumin and 1 % PenStrep. To prevent

cell aggregation, methylcellulose was added at a concentration of 1.5% to the culture medium.

The cells were incubated at 37°C for 14 days according to the previously published protocol

(Leung et al., 2010) with slight modifications. At the end of the incubation period; spheroids

were counted using an inverted microscope. The area, diameter, and size of the spheres were

analyzed using Image J-NIH software.

Extreme Limiting dilution assay:

Extreme Limiting Dilution Analysis (ELDA) was performed as described by Hu and Smyth

(2009). Briefly, cells derived from A549 and H1299 NSCLC cell lines were plated with 100

µl of serum-free medium per well at 100, 100, 10 and 1 cell seeding densities (24 wells per

dilution) on ultra- low attachment 96-well plates and incubated for 14 days. At the end of the

14 days, the number of wells showing the lung tumor spheroids formation was calculated. The

frequency of the spheroid-forming cells in the A549 and H1299 cell lines was calculated using

the ELDA webtool at http://bioinf.wehi.edu.au/software/elda (Bioinf.wehi.edu.au, 2018)

(Kanwar et al., 2010).

Statistical Analysis:

All data are expressed as mean ± SD were and calculated using Office Excel 2016 (Microsoft

Corporation, USA) The statistical difference between groups were evaluated for significance

using the Student's t test. Those with a P value equal 0.05 or less were considered statistically significant (Han et al., 2012; Jiang et al., 2016).

Results:

Spheroid formation assay results:

To determine whether a population of self-renewing cancer stem cells is present in A549 and

H1299 non-small cell lung cancer cell lines, the cancer stem-like cell population was enriched

in these cell lines. For this purpose, cells were cultured in a serum-free sphere formation

inducing conditions using a medium consisting of DEMEM /F12 (1:1) supplemented with

0.4% BSA, 1x B27, 20ng/ml EGF, 10ng/ml bFGF and 1x insulin and were seeded in culture

plates with ultra-low adhesion properties. The growth potential of floating spheroid colonies

of A549 and H1299 stem-like cells was examined. A few of the cells remaining after the first


164

cell death rapidly proliferated and formed lung tumor spheroids. The medium was changed

four times a week during the 14 days incubation period. After 10-14 days of culture, ball-like

spheres were observed. Images of tumor spheroids were taken with an inverted microscope

using 4x, 10x, 20x and 40x objective lenses to observe and analyze spheroids (Figure 1 and

Figure 2). The spheroid analysis was performed using image processing algorithms of the

ImageJ 1.51r software program.

To optimize the seeding density of each cell line, the number of cells required to form spheroids in preliminary tests was determined. Spheroid formation was observed by an inverted microscope at the following cell densities: 1x104, 5x10 3, 3x103, 1x103, 500 and 200 cells/ml. The seeding density to form H1299 and A549 spheroids with a mean diameter of 95.25 ± 41.03 and 80.70 ± 31.72μm, respectively, is about 1000 cells/ml.

A549 and H1299 cell lines were seeded at a density of 100 cells/well in a total of 24 wells

from an ultra-low adherent 96 well plates to obtain new spheroids. The percentage of the

spheroid formation efficiency (SFE%) was calculated by dividing the total number of formed

spheroids by the total number of seeded living cells and multiplying by 100. The sphere

forming efficiencies of A549 and H1299 parental cells were approximately 4.14 ± 1.06% and

5.81 ± 1.28%, respectively. Cells derived from both cell lines had the ability to form tumor

spheroids. However, the number of tumorspheroids and their size were different. The H1299

cell line produced more and larger tumorspheres when compared to the A549 cell line. This

suggests that each cell line contains a different number of stem-like cells. The spheroids were

round, with regular borders and a larger size than the cancer cells. As a result, both A549 and

H1299 cell lines have self-renewal abilities; however, we observe that this ability is more

pronounced and higher in the H1299 cell line.

(A) (B)

(C) (D)

1


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Figure 1. (A) H1299 adherent monolayer parental cells. Scale bar is 100μm. Original magnification 20x. (B), (C) and (D) Tumor spheroids derived from H1299 cells after 14 days

of culture under serum-free conditions. The seeding density was 1000 cells/ml per well. Scale bar is 100μm. Original magnification is 10x. (B) The diameters of the first, second and third spheres

are 76.82,115.45 and 71.47μm, respectively. (C) The diameters of the first and second spheres are 78.66, and 86.42μm, respectively. (D) The diameters of the first and second spheres are 74.57, and

92.80μm, respectively.

(A) (B)

1

(C) (D)

1 2

1

2 3

Figure 2. (A) A549 adherent monolayer parental cells. Scale bar is 100μm. Original

magnification 20x. (B), (C) and (D) Tumor spheroids derived from A549 cells after 14 days of

culture under serum-free conditions. The seeding density was 1000 cells/ml per well. Scale bar

is 100μm. Original magnification is 10x. (B) The diameter of the sphere is 87.14μm. (C) The

diameters of the first and second spheres are 87.1 and 98.56μm, respectively. (D) The diameters of

the first, second and third spheres are 76.53,78.04 and 108.79μm, respectively.

Extreme Limiting dilution assay results:

A549 and H1299 cells were examined for their ability to regenerate themselves using

limiting dilution analysis (Table 1). Typical spheroids were formed after 14 days when

A549 and H1299 cells were grown in a 96-well culture plate at extreme limiting dilutions.

A549 and H1299 single cells (24 single cells per experiment) formed new spheroids at 23.5

± 43.7% and 62.5 ± 49.4%, respectively. The frequency of sphere-forming cells (stem-like

cells) was 6.06-fold higher for H1299 than that of A549 cells (Table 1, and Figure 3).


166

As a result, these observations show that both A549 and H1299 spheroids are composed of a heterogeneous cell population with self-renewal capacity (stem-like cells) or without (non-stem-like cells or differentiated cells).

Discussion:

Increasing evidence has shown that NSCLC enhanced tumorigenicity, as in other solid cancer

types, is driven by a tumor cell population that exhibits stem cell-like characteristics such as

self-renewal, differentiation, cell mobility and therapy resistance, and referred to as cancer

stem cells (CSC) or tumor-initiating cells (TIC) (Eramo et al.,2008; Bertolini et al.,2009;

Magee et al.,2012;Visvader et al.,2012). Further investigation of the CSCs population would

provide a deeper understanding of the regulatory mechanisms involved in their maintenance

and facilitate the development of therapeutic strategies to control lung cancer in the long term

(Liu et al.,2013).

Table1. The frequency of tumor spheroids formed by A549 and H1299 cells. 95% CI: 95% confidence interval.

Cell number/well Number of wells

Number of wells showing

spheroids

A549 H1299 A549 H1299

1000 24 24 24 24

100 24 24 21 24

10 24 24 19 21

1 17 24 4 15

Spheroid-forming frequency 1/19.65 1/ 3.24

(% 95 CI)

P value 1.79e-11=0,0000000000179

Table 2. The diameters of spheroids formed by A549 and H1299 cells.

A549 H1299

Diameter Mean (μm) 80.70 ± 31.72μm 95.25 ± 41.03μm

P value 0.00003


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H1299

A549

Figure 3. A log-fraction graph of the limiting dilution model fitting the data in Table 4.1. The slope of the line is the log-active cell fraction. The dotted lines give the 95% confidence interval. The data value with zero negative response at dose 1000 for A549 cells and the data value with zero negative response at dose 100 for H1299 cells are represented by triangular downward values.

To establish a suitable system for the study of CSCs in NSCLC, we used the sphere formation

assay, which is a marker-independent approach. For this purpose, we compared the sphere-

forming capacities of A549 and H1299 cells. In serum-free and non-adherent culture

conditions, the non-CSCs undergo anoikis but CSCs survive and give rise to tumorspheres

(Charpentier and Martin,2013) thus, theoretically, the number of spheroids in the culture is

reflective of the number of CSCs in the sample.

Sphere numbers at different cell seeding densities showed a good correlation for both A549

and H1299 cell lines. However, H1299 cells were more potent than A549 cells in sphere

formation. In terms of the frequency of sphere-forming cells (stem-like cells), it was found to

be 6.06-fold higher for H1299 cells compared to A549 cells (Table 1). H1299 cells formed

spheres of an average of about 95.25 ± 41.03μm in diameter, whereas A549 cells formed

spheres of an average of about 80.70 ± 31.72μm in diameter (Table 2). Overall, the frequency

of sphere-forming cells in H1299 cells was significantly higher than that of A549 cells (Table

1), indicating a higher cancer stem cell content in this cell line. These results are in accordance

with recent studies, reporting decreased sphere formation potential (Chung et al.,2015;

Holmboe et al.,2017) and in vivo tumorigenicity of A549 compared to H1299 cells (Chung et


168

al.,2015). Taken together, these results suggest that H1299 cells could be a suitable model

system for studying CSCs in NSCLC.

Conclusion:

Recently the cancer stem cells are implicated in tumor initiation, progression, invasion,

metastasis, relapse and resistance to chemotherapy and radiotherapy. Since the resistance to

conventional chemotherapeutic agents is becoming a growing phenomenon, there is an urgent

need to develop novel drugs that specifically target the cancer stem cell populations in tumors.

Thus, it is of great value to conduct future studies to broaden our current knowledge of the

biology of cancer stem cells and the underlying molecular regulatory mechanisms of their

behavior.

Acknowledgments:

Financial support from Gaziantep university BAP (Project no.FEF. DT.17.12) is gratefully acknowledged.

Conflicts of interest:

The authors declare no conflicts of interest.

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Investigation of Apoptotic Effect of Betulinic Acid in Renal Cancer Cells

Merve Nur Ataş1, Barış Ertuğrul1, Elif Sinem İplik2, Bedia Çakmakoğlu1, Arzu Ergen1

1Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Molecular

Medicine, Istanbul, Turkey 2Istanbul Yeni Yuzyil University, Faculty of Pharmacy, Istanbul, Turkey


ABSTRACT

Kidney cancer or renal cancer is the fourteenth most frequent cancer in the world. With its

increasing incidence in the last few years, the renal cell carcinoma is the most common kidney

cancer type. Apart from the environmental factors, increasing activities of expressions of genes

and/or their pathways make cells become cancerous. AKT-1 and mTOR promote cell growth,

proliferation and survival, participate in cellular homeostasis and have ability to suppress

apoptosis in various ways.

Betulinic acid which is a lupane-type pentacyclic triterpenoid is derived from various plants.

Betulinic acid exhibits wide range biological activities such as anti-inflammatory, anti-viral,

anti-bacterial and particularly anti-cancer activities. It is known that betulinic acid promotes

apoptosis in cancer cells along with no toxic effect in normal cells. This effect makes betulinic

acid a potential anti-cancer drug.

In our study, clear cell renal carcinoma cell line CAKI-2 and healthy cell line MRC-5 are used

to research the effect of betulinic acid on apoptosis and gene expression levels. In order to

identify the toxic effect of betulinic acid, WST-1 and to detect apoptosis Annexin-V were

conducted. Expression analysis of AKT-1 and mTOR genes were conducted with Real-Time

PCR to measure the effect of betulinic acid in gene expressions levels.

As a result of this study, the apoptotic activity of betulinic acid on kidney cancer cell line was

detected with Annexin-V. In gene expression analysis, there was statistically significant

decrease in AKT-1 expression level while mTOR expression level was increased. Betulinic

acid with its apoptotic effect on renal cancer cell line and non-toxic effect on normal cell line

is a potential anti-cancer drug promising for future studies.

Keywords: Betulinic acid, renal cancer, apoptosis

Acknowledgment: The present work was supported by the Research Fund of Istanbul University.

Project No: 31565


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INTRODUCTION

Renal cancer accounts for about 2-3% of all adult malignant tumors [1]. The most common type

of kidney cancer is renal cell carcinoma (RCC), which is %90 of all renal cancer cases and

occurs in renal tubular epithelial cells [2]. Considering the continuous increase in the incidence

of renal cell carcinoma due to risk factors such as smoking, alcohol use, hypertension and

obesity [3], it has become extremely important to establish new treatment options to improve

the quality of life and, more importantly, survival ratio of patients after diagnosis.

The treatment options of RCC are, surgery and nephrectomy in the treatment of localized RCC

and systemic treatment for metastatic RCC or in cases of recrudescence after local treatment

[4]. Systemic treatment is designated according to the molecular biology of the RCC subtypes

that patients have. Immune checkpoint inhibitors, mTOR inhibitors, and approved drugs are

among the systemic treatment options [5].

AKT/mTOR pathway which plays a key role in regulation of cell growth, proliferation,

survival, metabolism and cellular homeostasis, is one of the pathways with overactivation in

various types of cancer including renal cell carcinoma [6]. AKT-1 and mTOR are members of

this pathway [7]. Any mutation occurring in the components of AKT/mTOR pathway may lead

to tumor development [8].

Betulinic acid is a lupane-type pentacyclic triterpenoid, which consists of six isoprene units,

and is isolated from various plants [9]. It has various biological activities such as anti-viral,

anti-bacterial, anti-inflammatory, particularly anti-cancer activities [10]. Although betulinic

acid is known to exhibit apoptotic effect through the mitochondria-dependent pathway,

studying different molecules and pathways will help to improve its known effects.

In this context, the purpose of this study is the investigation of apoptotic activity of betulinic

acid in renal cell carcinoma cells and examination of its effects on apoptosis-associated genes,

AKT-1 and mTOR.

MATERIALS AND METHODS

Cell culture

The CAKI-2 clear cell renal cell carcinoma and MRC-5 lung fibroblast cell lines were acquired

from American Type Culture Collection (ATCC, USA). The CAKI-2 (ATCC® HTB-47™) and

MRC-5 (ATCC® CCL-171™) cell lines were cultured in McCoy’s 5A and EMEM medium


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respectively, and supplemented with %1 Penicilin-Streptomycin and %10 Fetal Bovine Serum

(FBS) at 37 oC in %5 CO2

Reagents and assay kits

All cell culture reagents were purchased from Thermo Fisher Scientific (USA). Betulinic acid

and WST-1 cell proliferation assay kit were purchased form Sigma-Aldrich (USA) and Roche

Life Sciences (Germany), respectively. Annexin-V kit was purchased from Merck Millipore

(USA) and The RNA isolation, cDNA synthesis and qPCR assay kits were acquired from Jena

Bioscience (Germany).

Cell proliferation analysis

The cell lines were seeded at a density of 104 cells per well in 96-well plates. After 24 h

incubation, were treated with increasing concentration of betulinic acid (1, 2.5, 5, 7.5, 10, 25

and 50µM) for 24, 48 and 72 h. WST-1 solution (10µl) was applied to each well and the well

plates were incubated for 4 hours at 37oC. The reaction was measured at 460 nm using

microplate ELISA reader (Thermo Fisher Scientific, Germany).

The analysis of apoptotic activity

The cells were seeded at density of 105 per well in 6-well plates and were treated with different

concentrations of betulinic acid (25 and 50µM for 24h). At the end of 24h, the cells were

suspended with 1xbinding buffer at a concentration of 1x106 cells/mL. Following this, 5 µl

Annexin V-FITC and 5 µl PI were added to 100 µl of suspension cells. The cells were incubated

for 20 min at room temperature in dark. The results were analyzed using flow cytometry.

qPCR gene expression analysis

After each cell line was treated with determined concentrations of betulinic acid (25 and 50µM

for 24h), total RNA was extracted from cells and cDNA synthesis was performed from RNA

samples. It was determined fold changes in mRNA levels of AKT-1, mTOR and housekeeping

gene (GAPDH). The fold change was calculated as 2-∆∆CT.

Statistical analysis

Statistical analysis was performed using SPSS 21.0 program. The significance limit was

accepted as p <0.05. Mann-Whitney-U test was used for inter-group evaluation.


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RESULTS

Betulinic acid reduced cell viability in renal cancer cells

We investigated the cytotoxic effects of betulinic acid on CAKI-2 and MRC-5 cell lines using

the WST-1 colorimetric cell proliferation assay. The cells were seeded and incubated with

increasing concentration (1-50µM) of betulinic acid for 24, 48 and 72h. WST-1 assay was

performed at the end of each incubation period. The analysis showed that cell viability of CAKI-

2 cells treated with 25 and 50 µM betulinic acid decreased significantly. Nevertheless, it was

observed that betulinic acid treatment had no significant effect on cell viability of MRC-5 cells.

Betulinic acid enhanced apoptosis in renal cancer cells

In order to detect apoptosis and necrosis in betulinic acid treated cells, we performed Annexin

V assay. In CAKI-2 cell line, apoptotic cell death was observed %7.5 at 25 µM and %14.25 at

50 µM doses of betulinic acid for 24h incubation period (p<0.05). In the healthy cell line MRC-

5, it was observed that betulinic acid treatment had no effect on apoptosis of cells.

Betulinic acid changed AKT-1 and mTOR gene expression levels

In CAKI-2 cells treated with 25 µM betulinic acid, 0.41-fold change in AKT-1 expression

(p=0,034) and 1.61-fold change in mTOR gene expression (p=0,037) was observed. On the

other hand, in 50 µM betulinic acid treatment, 0.38-fold change in AKT-1gene expression

(p=0,037) and 1.94-fold change in mTOR gene expression was detected. These results showed

that betulinic acid caused a statistically significant decrease in AKT-1 gene expression while

increasing mTOR gene expression.

DISCUSSION

Renal cancer is the most lethal type of cancer among urological cancers. In recent years there

has been an increasing interest in natural compounds derived from plants in search of new

treatment options. Betulinic acid, a natural compound obtained from the bark of various plants,

is a member of the triterpenoids group and has many biological activities. Betulinic acid

performs the anti-cancer activity, mainly through the mitochondrial pathway [11].

Recently, many studies have been conducted to identify the anti-cancer activity of betulinic

acid. Lee et al. extracted potential anti-cancer compounds including betulinic acid, from Cornus

walteri. In ovarian cancer cell line A2780, among other compounds, betulinic acid showed the

highest toxic effect on cells. Annexin-V analysis showed that betulinic acid induced cell


175

apoptosis in dose-dependent manner and enhanced apoptosis by modulating Bcl-2 protein levels

[12].

Yang et al. RCC cell lines 786-O and ACHN in their study on the anticancer effects of betulinic

acid; It has been found to have an anti-proliferative effect on cells, induce apoptosis via

mitochondrial pathway by decreasing BCL-2 expression and increasing BAX and caspase 3

expression [13].

Apart from its effects to mitochondrial pathway, betulinic acid affects different molecules and

pathways in apoptosis. In cervical cancer cells HeLa, Xu et al. investigated the apoptotic effects

of betulinic acid and found that betulinic acid treatment caused significant decrease in PI3K

(p110a), PI3K (p85), p-AKT(Ser473) and p-AKT (Thr308) in time and dose dependent manner

[14].

In this study, we determined the apoptotic effect of betulinic acid on CAKI-2 renal cell

carcinoma cell line. We performed WST-1, Annexin-V, and Real-Time PCR to determine the

effects of betulinic acid on cell viability, apoptosis and AKT-1 and mTOR gene expression

levels in time and dose dependent manner. According to the results, betulinic acid reduced cell

viability, induced cell apoptosis and significantly reduced AKT-1 gene expression.

In conclusion, apoptotic activity of betulinic acid and its potential effect on AKT/mTOR

pathway suggest that betulinic acid could be a potential anti-cancer drug. With further studies,

using different analysis methods, the effect of betulinic acid on the AKT / mTOR pathway in

RCC or in different cancer types can be better elucidated.

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176

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2017.

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177

Identification of lncRNA molecules involved in the regulation of EGF-induced of cellular

proliferation

EGF ile indüklenen hücresel proliferasyonda rol oynayan lncRNA moleküllerinin

belirlenmesi

Esra Bozgeyik

Tekirdag Namik Kemal University, Faculty of Medicine, Department of Medical Biology, Tekirdag,

Turkey.

Özet

Akciğer kanseri, dünya çapında kansere bağlı ölümlerin önde gelen nedenlerinden biridir. 2019

kanser istatistiklerine göre, 230000'den fazla yeni akciğer kanseri vakasının ortaya çıkacağı ve

143000 kişinin akciğer kanseri nedeniyle öleceği tahmin edilmektedir . Ayrıca, lncRNA'lar

(long non-coding RNA), protein kodlama kapasitesine sahip olmayan ve çoğalma, büyüme,

bölünme, farklılaşma, hayatta kalma ve hücrelerin göçü gibi hayati hücresel süreçlerde önemli

rol oynayan 200 nükleotitten daha uzun fonksiyonel RNA molekülleridir. Çok çeşitli biyolojik

fonksiyonlarından dolayı, bu transkriptlerin deregülasyonu, akciğer kanseri de dahil olmak

üzere bütün kanserlerin moleküler patogenezine büyük ölçüde katkıda bulunur. EGF, hücre

proliferasyonunun düzenlenmesinde rol oynayan anahtar faktördür. EGF, normal fizyolojik

işlemlerin sürdürülmesi için çok temel bir protein olduğundan, EGF tarafından indüklenen

sinyal yolaklarının düzenlenmesinde meydana gelen bozukluklar, insan kanserlerinin bazı

belirgin özelliklerine katkıda bulunur. Bu nedenle, EGF ile indüklenen hücresel proliferasyonda

rol oynayan lncRNA moleküllerinin belirlenmesi, insan kanserlerinin moleküler patolojisinin

aydınlatılması adına önem arz etmektedir. Bu nedenle bu çalışmada biz EGF ile indüklenen

hücresel proliferasyonda farklı eksprese edilen lncRNA moleküllerini belirlemeyi amaçladık.

Sonuç olarak, MALAT1, ANRIL ve MCM3AP-AS1 lncRNA'ların ekspresyon seviyelerinin

arttığı, LOC105372161 ve MEG3'ün ekspresyon seviyelerinin azaldığı bulundu. Ancak,

yalnızca LOC105372161'in ifadesindeki değişikliklerin istatistiksel olarak anlamlı olduğu

bulundu. Bu sonuçlar, LOC105372161'in EGF tarafından indüklenen hücre proliferasyonun

düzenlenmesinde rol oynayabileceğini ve BCL2'nin LOC105372161'in varsayılan hedefi

olabileceğini göstermektedir.


178

Abstract


cancer statistics, it is estimated that over 230000 new lung cancer cases will occur and 143000

people will die due to lung cancer. Moreover, lncRNAs (long non-coding RNA) are functional

RNA molecules longer than 200 nucleotides that do not have protein coding capacity and play

important roles in the vital cellular processes such as proliferation, growth, division,

differentiation, survival, and migration of cells. Due to their wide range of biological functions,

deregulation of these transcripts highly contributes to molecular pathogenesis of cancers

including lung cancer. EGF is key factor that is involved in the regulation of cell proliferation.

Since EGF is a very chief protein for the maintenance of normal physiological processes,

deregulation of EGF-induced signaling significantly contributes to the several hallmark

capabilities of human cancers. Thus, determination of lncRNA molecules involved in the

regulation of EGF-induced promotion of cellular proliferation is of great interest to illuminate

the molecular pathology of human cancers. Accordingly, in this study we aimed to identify

differentially expressed lncRNAs involved in the regulation of EGF-induced stimulation of

cellular proliferation. As a result, expression levels of MALAT1, ANRIL and MCM3AP-AS1

lncRNAs was found to be increased whereas expression levels of LOC105372161 and MEG3

were found to be decreased. However, only changes in the expression of LOC105372161 were

found to be statistically significant, suggesting that LOC105372161 is involved in the

regulation of EGF-induced cell proliferation and BCL2 might be the putative target of

LOC105372161.

Keywords: EGF, lncRNA, lunc cancer, VIM-AS1


179

Introduction


cancer statistics, it is estimated that approximately 230000 people will be diagnosed with lung

cancer and 143000 people will die due to lung cancer [1]. Moreover, lncRNAs (long non-coding

RNA) are functional RNA molecules longer than 200 nucleotides that do not have protein

coding capacity. LncRNAs can interact with certain proteins with their specific secondary and

tertiary structures and bind to certain regions of the DNA to influence the expression of genes

in the relevant location. Studies show that lncRNAs play important roles in normal

physiological processes as well as in the pathogenesis of complex diseases such as cancer [2].

Epidermal growth factor (EGF) is a growth factor that plays important roles in proliferation,

growth, division, differentiation, survival, and migration of cells. EGF is a ligand for the

epidermal growth factor receptor (EGFR) and binding of EGF to EGFR leads to growth factor

induced autophosphorylation of the receptor and activation of downstream signaling cascades

promoting cell proliferation. Although EGF is a very important protein for the maintenance of

normal physiological processes, deregulation of EGF-induced signaling significantly

contributes to the several hallmark capabilities of cancers [3, 4]. Cancer cells may acquire

increased expression of growth factors to stimulate cognate receptors for autocrine signaling.

In an alternative way, they may also stimulate neighboring cells to supply these growth factor

ligands themselves for sustaining proliferation. Accordingly, an increased level of EGF

provides a good model for stimulation of cell proliferation and studying mediators of cellular

proliferation. It is also evident that several lncRNAs are involved in the cellular proliferation

and deregulation of these molecules leads to increased and sustained proliferation of cancer

cells.

Thus, identification of lncRNA molecules involved in the regulation of EGF-induced

stimulation of cellular proliferation is of great interest to understand better about the molecular

pathology of human cancers. Accordingly, here we aimed to determine lncRNAs involved in

the regulation of EGF-induced stimulation of cellular proliferation.

Material and Methods

Cell culture


180

The lung cancer cell line, A549, was cultured in a carbon dioxide incubator at 37 °C, containing

5 % CO2 and in 95% humidified medium. For the propagation of cells, DMEM (Dulbecco's

Modified Eagle's Medium, Sigma-Aldrich, Germany) containing 10 % FBS (fetal bovine

serum, Sigma-Aldrich, Germany) was used.

EGF treatment

For the EGF treatments, cells were seeded to 6-well plates at a concentration of 106 per milliliter

and wells were treated with the increasing concentrations of EGF as 50 ng/ml, 100 ng/ml, 200

ng/ml, 400 ng/ml. after 24 hours of incubation cells were collected using Trypsin-EDTA

solution.

RNA isolation and qPCR

RNA isolation was performed with the Qiagen RNeasy RNA isolation kit (Qiagen, Germany).

RNA concentrations were determined with NanoDrop® ND-1000 spectrophotometer. RT2

First Strand Kit (Qiagen, Germany) was then used to synthesize single chain cDNA and

recommended protocols of manufacturer were followed. miScript SYBR® Green PCR Kit

(Qiagen, Germany) was used in combination with gene-specific primers for each lncRNA

molecule to determine lncRNA expression levels between the groups. GAPDH

(Glyceraldehyde-3-Phosphate Dehydrogenase) and ACTB (beta (β)-actin) were used as

reference genes.

Statistical analysis

Ct (cycle threshold) values obtained from quantitative PCR were calculated and analyzed by 2-

ΔCt method where ΔCt = Hedef gen – Referans Gen [5]. The t test was used for comparisons,

and Mann Whitney U test was used for samples that did not show normal distribution. Also,

fold-change analysis was performed. For the fold-change analysis, 2-ΔΔCt (ΔΔCt=[ Treated

(Target gene - Reference gene) - Control (Target gene – Reference gene)]) [5]. For all results,

p <0.05 was considered statistically significant.

Results

Differential expressions of lncRNAs after EGF treatments


181

After treatment of A549 cells with different concentrations of EGF, lncRNA expression levels

were analyzed. As a result, while expression levels of LOC105372161 and MEG3 decreased

with increasing EGF concentrations, MALAT1, ANRIL and MCM3AP-AS1 lncRNAs

expression levels was found to be not significantly altered (Figure 1A, 2B).

Figure 1. Expression of lncRNAs in EGF-treated A549 cells. Heatmap (A), fold change (B).

Particularly, the expression level of MEG3 was found to be decreased in cells treated with 400

ng/ml and 200 ng/ml of EGF compared to untreated samples. However, this change was not

statistically significant (p400 ng/ml EGF =0.0728, p200 ng/ml EGF = 0.0676). In addition,

ANRIL expression levels was also found to be decreased with increasing EGF concentrations,

but this change was not statistically significant (p400 ng/ml EGF = 0.8958) (Figure 2). The

expression level LOC105372161 was found to be lower in cells treated with 400 ng/ml and 200

ng/ml EGF compared to untreated cells and this change was statistically significant (p400 ng/ml

EGF = 0.0042, p200 ng/ml EGF = 0.0095) (Figure 2).

Discussion

Lung cancer is the most common cause of cancer-related deaths in the world. Therefore, there

is an urgent need to fully elucidate the molecular mechanisms of this disease. Accumulating

evidence suggest lncRNAs play a chief role in the development and progression of human

malignancies including lung cancer. Recent studies have shown that lncRNAs are important

regulatory molecules that play a role in controlling various cellular activities [6]. Therefore,

determination and/or identification of non-coding counterparts of coding genes in molecular

pathogenesis of lung cancer are very important for developing more efficient targeted


182

therapeutics. Here, lncRNA expression levels in EGF-stimulated A549 lung cancer cells were evaluated

and LOC105372161 and MEG3 lncRNAs were found to be differentially expressed with the increasing

EGF concentrations.

Figure 2. Relative gene expression changes of lncRNAs according to different EGF concentrations.

However, only changes in LOC105372161 expression was found to be statistically significant.

Low expression of MEG3 was consistent with the previous findings but this change was not

enough to be statistically significant. However, changes in LOC105372161 expression was

found to be statistically significant. Reduction of LOC105372161, which is transcribed from

opposite strand of BCL2, expression levels in EGF-induced cell proliferation indicates that this

lncRNA might be involved in the negative regulation of BCL2 anti-apoptotic gene. Yet, more

comprehensive studies are needed to delineate the functions of this lncRNA in tumor formation.

References

• Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2019. CA: a cancer journal

for clinicians, 2019. 69(1): p. 7-34.

• Bhan, A., M. Soleimani, and S.S. Mandal, Long Noncoding RNA and Cancer: A New

Paradigm. Cancer Res, 2017. 77(15): p. 3965-3981.


183

• Johnson, L., et al., Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proceedings of the National Academy of Sciences, 1980. 77(1): p. 394-398.

• Jorissen, R.N., et al., Epidermal growth factor receptor: mechanisms of activation and signalling, in The EGF receptor family. 2003, Elsevier. p. 33-55.

• Schmittgen, T.D. and K.J. Livak, Analyzing real-time PCR data by the comparative C T method. Nature protocols, 2008. 3(6): p. 1101.

• Cech, T.R. and J.A. Steitz, The noncoding RNA revolution—trashing old rules to forge new ones. Cell, 2014. 157(1): p. 77-94.

• Tano, K., et al., MALAT‐1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility‐related genes. FEBS letters, 2010. 584(22): p.

4575-4580.


184

Yeni antikanser ilaç keşfinde genomik ve metabolomik: Fırsatlar ve zorluklar

Ekrem Kum, Süleyman Özakın, Ebru İnce*

Dicle Üniversitesi Fen Fakültesi Biyoloji Bölümü 21280 Diyarbakır

*[email protected]

Özet

Elli yıldan uzun bir süredir, mikroorganizmalardan ve bitkilerden elde edilen küçük

organik moleküller çok sayıda kullanışlı kanser kemoterapötik ilacına kaynak teşkil etmiştir.

Doğal ürünlerin bu tip öncü bileşiklerinin tarama çalışmaları son yıllarda ivme kazanmış,

karasal bitki ve mikroorganizmalar kadar, deniz fauna ve florasını oluşturan bileşenlerinin de

anti-kanser aktiviteleri araştırılmaya başlanmıştır. Doğal ürünlerin biyosentezlerinin daha iyi

anlaşılması ile beraber, geleneksel ilaç veya ilaç öncülü tarama çalışmaları yerini genomik

bilginin kimyasal verilerle kombine edilmesi prensibe dayalı farklı stratejilere bırakmıştır. Anti-

kanser dahil olmak üzere farklı biyolojik aktivite ve emsalsiz kimyasal çeşitliliğe sahip

mikrobiyal doğal ürünlerin üreticisi olan Aktinomisetler kompleks bir yaşam döngüsüne ve

diğer bakteriler ile kıyaslandığında büyük genomlara sahiptirler. Yeni nesil dizileme

teknolojileri sayesinde genomlarında biyoaktif doğal ürün sentezinden sorumlu yüksek

miktarda biyosentetik gen kümesine sahip oldukları bulunmuştur. Bu makalede; mikrobiyal

kaynaklı yeni biyoaktif bileşik keşfinde genomik ve metabolomik tarama stratejileri, kullanılan

biyoinformatik programlar, avantaj veya zorluklarına değinilerek, son yıllarda bu teknoloji ile

elde edilmiş yeni anti-kanser ilaçların altı çizilmiştir.

Anahtar kelimeler: Doğal ürünler, anti-kanser , genomik, metabolomik, LC-MS/MS.

• Giriş

Doğal ürünler; mikroorganizmalar, bitkiler ve omurgasızlar tarafından üretilen küçük

organik moleküllerdir. Aynı zamanda sekonder metabolit olarak adlandırılan doğal ürünler

çeşitli ve olağandışı kimyasal yapılara ve küçük moleküler kütlelere sahiptirler. Hem kimyasal

yapı çeşitliliği hem de farklı biyolojik aktivitelere sahip olmalarından ötürü şu an klinikte

kullanılan birçok ilaç doğal ürün veya onların kimyasal modifikasyonlarından elde

edilmektedir.

Doğal ürünler alkaloidler, terpenoidler, flavonoidler, nükleositler, ribozomal olmayan

peptidler (NRP), poliketidler (PK) olmak üzere çeşitli temel gruplara ayrılmaktadır. Bilinen


185

doğal ürünlerin yaklaşık % 60’ını terpenoidler oluşturmaktadır. Terpenoidler bitkiler, mantarlar

ve mikroorganizmalarca sentezlenen biyoaktif metabolitlerdir. Terpenoidlerin biyosentezi

izopentil ünitelerinin substrat olarak kullanılmasıyla gerçekleştirilmektedir. Klinikte güncel

olarak kullanılan antikanser ilaç taxol (Paclitaxol®) en iyi bilinen terpenoidlerdendir.

Mikrobiyal doğal ürünler biyoaktif bileşiklerin en önemli sınıfını oluşturmaktadır.

Bunlar arasında antibiyotikler, antifungallar, antitümör ajanlar, toksinler, antihelmitikler,

immunbaskılayıcılar ve sideroforları da içeren, işlevsel olarak birbirinden oldukça farklı

biyokimyasal sınıflara dahil bileşikler mevcuttur. Mikrobiyal kaynaklı doğal ürünlerin en

önemli üreticilerinden biri aktinomisetlerdir. Örneğin, anti-kanser kemoterapötik ilaçlar olan

bleomycin (Blenoxane®) (1) Streptomyces verticillus, doxorubicin (Adriamycin) (2)

Streptomyces peucetius tarafından üretilen doğal ürünlerdir.

(1) (2)

Farmakolojik ve biyolojik açıdan önemli onbinlerce PK ve NRP keşfedilmiş olmasına

rağmen, yalnızca 350 antimikrobiyal molekül bu pazarda yerini bulmuştur (Demain 2009). Son

kırk yıl içerisinde, mikroorganizmalardan markete ulaşabilen ürünlerin sayısı hatırı sayılır bir

biçimde azalmıştır. Bundan dolayı yeni doğal ürünlerin keşfi ve alternatif stratejilerle bu doğal

ürünlerin kimyasal potansiyelini arttırmak önem arz etmektedir.

Geleneksel yöntemler biyolojik aktivite temelli olup, organik ekstraktlardan analitik

yöntemlerle biyoaktif bileşiklerin izolasyonu ve karakterizasyonu prensibine dayanır.

Ekstraktlarda bilinen metabolitlerin yeniden izolasyonu dereplikasyon olarak

adlandırılmaktadır. Dereplikasyon biyolojik aktivite temelli doğal ürün araştırmalarında

karşılaşılan önemli sorunlardan biridir. Özellikle 1990’lı yıllardan günümüze kadar yeni

metabolit keşif oranlarının düşmesinin temel nedeni dereplikasyon sorunudur. Doğal ürün

araştırmalarında yeni yöntemlerin geliştirilmesinin temel amaçlarından biri dereplikasyon

sorunun üstesinden gelmektir. Özellikle bilinen metabolitlerin saflaştırma aşamasına geçmeden

daha ekstrakt aşamasında tespiti için çeşitli veritabanları ve informatik programlar

geliştirilmektedir.


186

(4) Genomik Yaklaşımlarla İlaç Taramaları

Yeni genomların keşfedilmesi neticesinde çoğu mikroorganizmanın şimdiye kadar

üretmiş olduğu doğal ürünlerden daha fazlasını üretme potansiyeline sahip olduğu

anlaşılmaktadır. Zira bu mikroorganizmalar standart laboratuvar koşullarında kültüre

edildiklerinde doğal ürünleri sentezleyen biyosentetik gen kümeleri ya aktifleşememekte

veyahut çok düşük seviyelerde ifade olabilmektedirler. Uygun fiziksel veya kimyasal uyarıcılar

olmadığı takdirde uyku halinde olup klasik kültür koşullarında ifade edilemeyen bu genler

kriptik olarak adlandırılırlar. Özellikle aktinomisetlerin genomları incelendiğinde, kompleks

yapılı çok sayıda doğal ürün biyosentezinden sorumlu kriptik gen kümesinin varlığı ortaya

çıkarılmıştır (Bentley ve ark. 2002, Hosaka ve ark. 2013).

Genomik araştırmaları ile değişik varyetede birçok organizmadan elde edilen büyük

miktarda DNA sekans verisi ulusal veri tabanlarında depolanmaktadır. Araştırıcıların erişimine

açık olan DNA sekans verileri yeni ilaç veya ilaç öncülü bileşiklerin keşfi için yeni

biyoinformatik araçlardan faydalanılmasının yolu açmaktadır. Genomik temelli yaklaşımlar,

daha önce standart fermentasyon koşulları altında keşfedilemeyen yeni doğal ürünlerin açığa

çıkarılması için geliştirilmektedir. Dizilenen bu genomlar sonucunda yeni doğal ürünlerin

keşfedilmesi, genom madenciliği vasıtasıyla ise bunların biyosentetik yolaklarına ulaşılması

hedeflenmektedir. Genomda izi sürülen kriptik genlerin, aynı zamanda uygun ortam

koşullarında ifadesi sağlanmaktadır (Şekil 1).

Şekil 1. Genom madenciliği (Choi ve ark. 2015)

Sıra dışı habitatlara adaptasyon sağlamış mikroorganizmaların standart laboratuvar

koşullarında kültüre edilmeleri oldukça zordur. Bu amaçla yeni kültür metotları geliştirilmesine

rağmen mikroorganizmaların ancak %1’i standart koşullarda kültüre edilebilmektedir.

Genomik teknolojiler, sayesinde kültüre edilebilen veya edilemeyen mikroorganizmaların

böyle bileşiklerin sentezi için yüksek biyosentetik kapasiteye sahip olduğunu ortaya koymuştur.

Genom tarama yönteminin en temel bileşenleri biyoinformatik programlardır. Son dönemde

geliştirilen ‘antiSMASH ve ‘NapDOS’ biyoinformatik programları genomdaki doğal ürün

genlerinden yola çıkarak bileşiğin iskelet yapısı hakkında önemli bilgiler vermektedir. Buna en


187

güzel örnek miksobakteri Sorangium cellulosum’da tanımlanan anti-mitotik ve anti-neoplastik

bir ajan olan epothilone’un yapısının biyoinformatik programlarca ana iskelet yapıya yüksek

benzerlikte tahmin edilmiş olmasıdır (Li ve ark. 2009) (3)-Tahmini Yapı, (4)-Gerçek Yapı.

(3) (4)

Çeşitli veri tabanlarında analiz edilmeyi bekleyen binlerce genom dizilimi bulunmaktadır.

Bu dizilimlerin analizlerinin kimyasal verilerle desteklenmesi doğal ürün araştırmalarındaki en

yeni stratejidir.

28. Metabolomik Yaklaşımlarla İlaç Taramaları

Biyolojik sistemlerde bulunan küçük moleküler ağırlığa sahip metabolitleri global

seviyede analiz edebilen metabolomik çalışmalarında kütle spektroskopisi (MS) en yaygın

kullanılan yaklaşımlardan biridir. Bir ekstrakt içinde çok düşük konsantrasyonlardaki

metabolitlerin tespitinde, hem oldukça hassas olması hem de bileşiğin kimyasal yapısı hakkında

bilgi vermesinden dolayı, yeni bileşik araştırmalarında geniş bir kullanım alanına sahiptir.

Genel olarak metabolomik; hedefe odaklı analizler (targeted) ve herhangi bir hedefe

odaklanmayan analizler (non-targeted) olmak üzere ikiye ayrılır. Hedefe odaklı metabolomik,

ilgili metabolik yolaklarda kesin miktar tayini yapılmış metabolitleri içerirken, herhangi bir

hedefe odaklanmayan metabolomik ön yargısız bir şekilde birçok metabolitin ölçümünde

kullanılan bir yaklaşımdır. Kütle spektroskopisi; sıvı kromatografi (LC-MS), nükleer manyetik

rezonans (LC-MS-NMR), gaz kromatografisi (GC-MS) gibi yöntemler ile kombine olma

özelliğine sahiptir. İkili kütle spektrometresi (MS/MS) ise en az iki aşamada kütle analizini

gerçekleştiren genel bir yaklaşımdır.

Doğal ürünlerin toplu moleküler ağı (Global Natural Products Social Molekuler

Network-GNPS) en yaygın ve kullanışlı veri tabanlarından biridir.

(5) Moleküler ağ analizleri

Moleküler ağ analizi geçtiğimiz on yıl içinde geliştirilmiş yeni bir dereplikasyon ve

metabolomiks yöntemidir. Bu yöntem herhangi bir ekstrakt ve biyolojik örnek içinde bulunan

metabolitlerin MS/MS verilerinin analizine dayanmaktadır. MS/MS, analiz edilen iyonların

fragmentasyonuna neden olduğundan bileşiklerin kimyasal yapısı hakkında oldukça önemli


188

bilgi vermektedir. Bu sebepten dolayı doğal ürün araştırmalarında MS/MS analizleri önemli

bir kullanım alanına sahiptir. Hibrit MS sistemindeki ilk analizör tarafından tespit edilen iyona

ait fragment iyonlar, ikinci analizör tarafından tespit edilerek sıralı MS (tandem MS) veya

MS/MS verisi olarak çeşitli veri tabanlarında ve programlarda analiz edilmek üzere

kullanılmaktadır. GNPS (Global Natural Products Social Molekuler Network), dereplikasyon,

bilinen bileşiklerin yeni analoglarının bulunması ve aynı zamanda yeni bileşik keşfi için en

yaygın kullanılan veri tabanlarından biridir (Tablo 1).

Tablo 1. Doğal ürün tarama veri tabanları.

Veri Tabanı Web Adresi

GNPS www.gnps.ucsd.edu

Norine www.bioinfo.lifl.fr

Scifinder www.cas.org

Metlin www.scripps.edu

Massbank www.massbank.jp

AntiBase www.user.gwdg.de

Marinlit www.chem.canterbury.ac.zn

Drugbank www.drugbank.ca

HMDB www.hmdb.ca

MS/MS analizi sonucunda benzer fragmentasyona sahip iyonlar kimyasal yapı olarak

birbirine benzerlik gösterir. Şekil 2’de görüldügü gibi moleküler network analiziyle benzer

fragmentasyona sahip iyonlar network içinde birlikte gruplandırılmaktadır. Bu gruplandırma

benzer fragment iyon sayısı ve MS/MS pik yoğunluğuna göre program tarafından vektörel

büyüklük (kozin değeri) olarak hesaplanır.

Şekil 2. GNPS ile moleküler ağ analizi (Wang ve ark. 2016).

http://www.gnps.ucsd.edu/

http://www.bioinfo.lifl.fr/

http://www.cas.org/

http://www.scripps.edu/

http://www.massbank.jp/

http://www.user.gwdg.de/

http://www.chem.canterbury.ac.zn/

http://www.drugbank.ca/

http://www.hmdb.ca/


189

1 MS rehberli genom madenciliği

Son yıllarda doğal ürün araştırmalarında genomik bilginin kimyasal verilerle kombine

edilmesi prensibine dayalı farklı stratejiler geliştirilmiştir. Özellikle MS/MS analizi sonucunda

elde edilen metabolit fragmentasyon verisinin, biyoinformatik temelli genom taramaları

sonuçlarıyla birleştirilmesiyle ‘peptidogenomik’ ve ‘glikogenomik’ tarama stratejileri

geliştirilmiştir. Her iki stratejinin temelinde, MS verisiyle genomik bilginin değerlendirilmesi

amaçlanmaktadır.

MS/MS fragmentasyonu sonucunda peptidler (ribozomal ve ribozomal olmayan) daha

küçük peptidlere parçalanmaktadır. Bu parçalanma sonucunda peptid yapısından kopan her

aminoasit spesifik kütle kayma değerine sahip olmaktadır. Bu değere bağlı olarak peptidin

yapısındaki aminoasitler belirlenebilmektedir. Fragmentasyon temelli aminoasit bilgisi,

biyoinformatik programlarca peptid sentezinde olası subsrat olarak kullanılan aminoasit

sonuçlarıyla karşılaştırılıp doğal ürün biyosentezinden sorumlu gen kümesine ulaşılmaktadır.

Peptidogenomik olarak adlandırılan bu yöntem ile stenothricins örneğinde olduğu gibi yeni

doğal ürünler keşfedilmektedir (Kersten ve ark. 2011).

Glikozilasyon, doğal ürün biyosentezinde en yaygın görülen modifikasyon

reaksiyonlarındandır. Glikozilasyon reaksiyonlarının metabolitlerin hücresel haberleşme,

moleküler tanıma ve biyolojik aktivite açısından oldukça önemli olduğu bilinmektedir.

Lomaiviticin ve landomycine gibi glikolize doğal ürünlerin biyolojik aktivite potensinin şeker

üniteleriyle ilgili olduğu düşünülmektedir. Ribozomal (nocathiacin), ribozomal olmayan

(vancomycin), alkaloid (staurosporine) (5) poliketid (erythromycin), ve terpen

(phenalinolactone A) gibi birbirinden oldukça farklı yapılara sahip biyoaktif glikolize doğal

ürünler mikroorganizmalarca sentezlenmektedir.

(5) (6)

Glikolize doğal ürünler yapılarında bulunan şeker ünitesine göre MS/MS analizinde

spesifik fragmentasyon sinyali verirler. Bu sinyaller doğrultusunda doğal ürün yapısındaki

şeker ünitesi tespit edilebilmektedir. Glikozil transferaz, 2,3 dehidrataz, metil transferaz,

aminotransferaz ve nükleotit transferaz glikolize doğal ürün biyosentez gen kümelerinde

bulunan spesifik genlerdir. Doğal ürün biyosentezinden sorumlu mikoorganizmanın

genomunda biyoinformatik programlarla bu spesifik genlerin taranması sonucunda olası


190

biyosentez yolakları tespit edilmektedir. MS/MS verisinden glikolize doğal ürün biyosentez

gen kümesine ulaşılması ‘glikogenomik’ olarak adlandırılmaktadır (Kersten ve ark. 2013). Bu

yöntemle son yıllardaki en büyük keşif bir deniz aktinomiseti olan Salinispora tropica

tarafından üretilen salinosporamide A (Marizomib) (6), potansiyel bir antikanser ajanı olarak

saptanan güçlü bir proteazom inhibitörüdür. Multipl miyelomun tedavisi için faz II insan klinik

denemeleri devam etmektedir.

Kaynaklar

Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., Challis, G.L., Thomson, N.R., James, K.D.,

Harris, D.E., Quail, M.A., Kieser, H., Harper, D., Bateman, A., Brown, S., Chandra, G., Chen, C.W.,

Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo, J., Hornsby, T., Howarth, S., Huang, C.H.,

Kieser, T., Larke, L., Murphy, L., Oliver, K., O’Niel, S., Rabbinowitsch, E., Rajandream, M.A.,

Rutherford, K., Rutter, S., Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K.,

Warren, T., Wietzorrek, A., Woodward, J., Barrell, B.G., Parkhill, J., Hopwood, D.A. 2002. Complete

Genome Sequence of the Model Actinomycete Streptomyces coelicolor A3(2). Nature, 417: 141-147.

Choi, S.S., Kim, H.J., Lee, H.S., Kim, P., Kim, E.S. 2015. Genome mining of rare actinomycetes

and cryptic pathway awakening. Process Biochemistry, 50 :1184–1193.

Demain, A.L. 2009. Antibiotics: Natural Products Essential to Human Health. Med Res Rev, 29: 821-

842.

Hosaka, T., Ochi K. 2013. New Strategies for Drug Discovery: Activation of Silent or Weakly

Expressed Microbial Gene Cluster. Apll Microbiol Biotechnol, 97: 87-98.

Kersten RD, Yang YL, Xu Y, Cimermancic P, Nam SJ, Fenical W, Fischbach MA, Moore BS,

Dorrestein PC (2011). A mass spectrometry-guided genome mining approach for natural product

peptidogenomics. Nat Chem Biol 7: 794-802.

Kersten RD, Ziemert N, Gonzalez DJ, Duggan BM, Nizet V, Dorrestein PC, Moore BS (2013).

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated

molecules. P Natl Acad Sci USA 110: 4407-4416.

Li, M.H.T., Ung, P.M.U., Zajkowski, J., Tsodikova, S.G., Sherman, D.H. 2009. Automated

Genome Mining for Natural Product. BMC. Bioinfo. 10: 185-195.

Wang M, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono

CA, Luzzatto-Knaan T et al. (2016). Sharing and community curation of mass spectrometry data

with Global Natural Products Social Molecular Networking. Nat Biotech 34: 828-837.


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A Useful Tool for Distinguishing Gene/Pseudogene Variants Detected via NGS:

Haplotype Analysis

Esra Arslan Ateş1,2, Ahmet İlter Güney2, Tuba GÜNEL1 1Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul.

2Marmara University Pendik Training and Research Hospital, Department of Medical Genetics,

Istanbul.

Correspondence: [email protected]

Abstract

Introduction: Lynch Syndrome is an inherited cancer prone disease mostly associated with

colon, endometrium, ovarian and gastrointestinal tract cancers. Mutations affecting missmatch

repair genes (MLH1, PMS2, MSH2, MSH6) are responsible from Lynch Syndrome. PMS2 has

a psedudogene called PMS2CL showing a high sequence similarity to PMS2 gene.

Here we report a novel mutation in PMS2 gene associated with Lynch Syndrome which was

proven to be on the active gene using haplotype analysis.

Case Report: A 69 year-old woman referred to us because of endometrium cancer. She had a

history of colon cancer diagnosed at the age of 66. Endometrial tumor showed high

microsatellite instability and MLH1 and PMS2 expression loss were detected in

immunohistochemical analysis. There were no other cancer history in her family. BRAF V600E

mutation and MLH1 promoter hypermethyation were not detected in tumor tissue. On the basis

of immunohystochemical findings MLH1 and PMS2 genes were screened for germline

mutations from peripheral blood lymphocytes via next generation sequencing. We detected a

novel heterozygous 47bp insertion (c.1362_1407dup; p.Pro470Valfs*3) in exon 11 of PMS2

gene. The false positive result due to pseudogene was excluded by haplotype analysis using the

SNPs that allow us to distinguish PMS2 and PMS2CL.

Conclusion: PMS2 gene is a MMR system component which has an important role in DNA

replication and homologous recombination, located on chromosome 7p and consist of 15 exons.

There is a pseudogene, PMS2CL, which shows 98% sequence similarity to exons 9 and 11–15

of PMS2 and it is important to distinguish the variations between gene and pseudogene

sequences. NGS data enable us to assess the haplotypes in an amplicon using the distinct SNPs

on gene and psedudogene.



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GİRİŞ

Lynch Sendromu, diğer adıyla Herediter Nonpolipozis Kolon Kanseri (HNPCC) kolorektal

kanserler başta olmak üzere gastrointestinal traktus kanserleri, endometrium kanseri, meme

kanseri gibi kanserlerle karşımıza çıkan kalıtımsal bir kanser yatkınlık sendromudur. Tüm

kolorektal kanserlerin %3’ü ve ednfometrial kanserlerin %3 kadarı Lynch Sendromu ile ilişkili

olarak değerlendirilmiştir [1,2]. Hatalı eşleşme tamir genlerindeki (MMR genleri: MLH1,

MSH2, MSH6, PMS2 ve EPCAM) mutasyonlar Lynch Sendromuna neden olmaktadır [3].

Otozomal dominant kalıtım özelliği dolayısıyla pedigri analizinde vertikal kalıtım özelliği

gösteren aile öyküsü varlığı, tümör dokusunda mikrosatellit instabilitesi ve MMR genlerinde

ekspresyon kaybı saptanması tanıya yönlendiren özelliklerdendir [4].

PMS2 geni Lynch Sendromlu olguların yaklaşık %15’inden sorumlu olup, MMR sisteminin

önemli bir komponentidir. MLH1 ile birlikte bir komleks oluşturarak MMR sürecinde anahtar

bir rol oynar [5]. PMS2 geninin yüksek homoloji gösteren çok sayıda psödogeni bulunmaktadır.

Bu durum moleküler genetik tanıyı zorlaştıran önemli bir problemdir. PMS2CL PMS2 geni

ekzon 9, 10-15 ile yüksek homoloji gösteren bir psödogendir. Bu problemin üstesinden gelmek

için PMS2’ye özgü olan ekzon 10’dan dizayn edilen bir primer kullanmak üzere elde edilen

cDNA’dan Long Range PCR dizaynı kullanılan bir yöntemdir[6].

OLGU

Tarafımıza endometrium kanseri tanısı ile yönlendirilen 69 yaşında kadın olgu polikliniğimizde

değerlendirldi. Olgunun 3 yıl önce kolon kanseri tanısı nedeniyle yapılan rutin takiplerinde

endometrial kitle saptanması üzerine yapılan total abdominal histerektomi öyküsü mevcuttu.

Pedigri analizinde kanser öyküsü bulunmayan hasta izole vaka olarak değerlendirildi. Patolojik

değerlendirmesinde endometrial kanser tanısı alan hastanın tümör dokusunda yüksek

mikrosatellit instabilitesi ve PMS2 ekspresyon kaybı saptandı, MLH1 hipermetilasyonu ve

BRAF V600E mutasyonu saptanmadı. Hastadan bu bulgularla ACMG kriterlerine göre Lynch

Sendromu ön tanısıyla moleküler genetik analiz planlandı [4]. Periferik kandan DNA

izolasyonu (QIAamp DNA Mini Kit, QIAGEN Germantown, MD USA) sonrası MLH1 ve

PMS2 genleri Illumina MiSeq platformunda, HNPCC MASTR Plus (Multiplicom,

Agilent,CA,USA) kiti kullanılarak yeni nesil dizi analizi yöntemiyle değerlendirildi. MLH1

geninde mutasyon saptanmayan olguda PMS2 geni ekzon 11’de heterozigot 47bç bir

duplikasyon saptandı (c.1362_1407dup; p.Pro470Valfs*3) (Şekil 1). Mutasyon veri

tabanlarında tanımlı değildi, çerçeve kaymasına yol açarak erken stop kodonuna sebep olması

beklenmesi dolayısıyla patojenik bir varyant olarak değerlendirildi. Ancak pseödogen


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dolayısıyla tahmin ettiğimiz üzere kopya sayı analizinde ekzon 11’in 4 kopya olduğu

görünmekteydi (Şekil 2). Amplikon değerlendirildiğinde bu bölgede PMS2CL ile PMS2

arasında kısmen düşük bir homoloji olduğu dikkat çekiciydi (%96). Farklı olan nükleotidlerin

değerlendirilmesi ile yapılan haplotip analizinde insersiyonun olduğu okumaların tamamıyla

PMS2 genine ait olduğu ortaya koyuldu (Şekil 3).

Şekil 1: PMS2 geni ekzon 11 47bç insersiyon IGV görüntüsü

Şekil 2: PMS2 CNV analizi


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Şekil 3: PMS2 ve PMS2CL dizileri

Sarı: Farklı nükleotidler Yeşil: Hastada saptanan varyasyonlar

Kırmızı : insersiyon bölgesi

TARTIŞMA

Psödogenler moleküler tanıyı zorlaştıran bir problem olarak karşımıza çıkmaktadır. Gen ile

psödogenler arasındaki minimal farklılıkları kullanarak saptanan mutasyonun gen veya

psödogen üzerinde olduğunu belirlemek ek yöntemlerle gerektirmektedir. Bu durum tanıda

gecikmelere yol açması ve ek olanaklar gerektirmesi bakımından klinik pratikte sıkıntılara yol

açmaktadır. Bu çalışmada yeni nesil dizileme yönteminin tek bir DNA ipliğine ait diziyi ortaya

koyma avantajı kullanılarak amplikondaki tek nükleotid varyasyonları (SNVs) analiz edilmiş

ve amplikonun kopyalandığı DNA molekülünün tamamıyla PMS2 genine ait olduğu

gösterilmiştir. Varyasyonun psödogene ait olma şüphesi durumunda amplikonun bilgi verici

SNV’ler açısısndan değerlendirilmesi ek tetkik gereksinimini ortadan kaldırarak zaman

kazandırmakla birlikte gereksiz harcamaları da azaltacaktır.

KAYNAKLAR

1- Boland, C. R. (2005). Evolution of the nomenclature for the hereditary colorectal cancer

syndromes. Familial Cancer, 4( 3), 211– 218.

2- Lu KH, Dinh M, Kohlmann W, Watson P, Green J, Syngal S, Bandipalliam P, Chen LM, Allen B,

Conrad P, Terdiman J, Sun C, Daniels M, Burke T, Gershenson DM, Lynch H, Lynch P, Broaddus

RR. Gynecologic cancer as a "sentinel cancer" for women with hereditary nonpolyposis colorectal

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3- Kuiper, R. P., Vissers, L. E. L. M., Venkatachalam, R., Bodmer, D., Hoenselaar, E., Goossens, M.,

et al. (2011). Recurrence and variability of germline EPCAM deletions in Lynch syndrome. Human

Mutation, 32( 4), 407– 414.

4- American College of Medical Genetics technical standards and guidelines for genetic testing for

inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated

polyposis).

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(2009a).PMS2 involvement in patients suspected of Lynch syndrome. Genes, Chromosomes &

Cancer, 48( 4),322– 329

6- Nakagawa H, Lockman JC, Frankel WL, Hampel H, Steenblock K, Burgart LJ, Thibodeau SN, de

la Chapelle A. 2004. Mismatch repair gene PMS2: diseasecausing germline mutations are frequent in

patients whose tumors stain negative for PMS2 protein, but paralogous genes obscure mutation

detection and interpretation.

dicle university, diyarbakır, turkeymokad.org/congress.pdf · 2019-10-10 · dicle university,...

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