submitted by dr saima rubab khan dbc-02123003

IMPLICATION OF PROGNOSTIC VARIABLES AND THEIR POTENTIAL ROLES IN THE

DEVELOPMENT OF ORAL SQUAMOUS CELL CARCINOMA (OSCC): A STUDY FROM

LOCAL POPULATION

SUBMITTED BY

DR SAIMA RUBAB KHAN

DBC-02123003

IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

(PH.D) IN BIOCHEMISTRY

INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

THE UNIVERSITY OF LAHORE

2012-2017

i

"Bismillah al rahman al rahim"

In the name of Allah the most gracious the most merciful

http://wahiduddin.net/words/bismillah.htm

ii

SUPERVISrORY COMMITTEE

We, the Supervisory Committee, certify that the contents and form of thesis submitted by DR.

SAIMA RUBAB KHAN (REG. # DBC-02123003) have been found satisfactory after receiving

all the evaluation reports (External/Internal) and recommended it for the award of the degree of

Doctor of Philosophy (PhD) in Biochemistry under title “IMPLICATIONS OF PROGNOSTIC

VARIABLES AND THEIR POTENTIAL ROLE IN THE DEVELOPMENT OF ORAL

SQUAMOUS CELL CARCINOMA (OSCC): STUDY FROM LOCAL POPULATION”.

SUPERVISOR: PROF. DR. ARIF MALIK _________________________

Institute of Molecular Biology and Biotechnology (IMBB/

Center for Research in Molecular Medicine (CRiMM)

The University of Lahore.

CO-SUPERVISOR (I): PROF. DR. M. H. QAZI___________________________




CO-SUPERVISOR (II): PROF. DR. MURAD KHAN ___________________________

Professor of Biochemistry

ABWA Medical College.

Faisalabad.

MEMBER: MS. RABAIL ALAM (BIOSTATICIAN)__________________




iii

EXAMINATION COMMITTEE

The thesis viva DR SAIMA RUBAB KHAN (REG.# DBC-2123003) was held on 17-11-2017

at the Institute of Molecular Biology and Biotechnology/ Center for Research in Molecular

Medicine (CRiMM), The University of Lahore. The Supervisor/Co-Supervisor and the examiner

gave satisfactory remarks on thesis and viva and were approved for the award of the degree of

Doctor of Philosophy (PhD) in Biochemistry under title “IMPLICATIONS OF PROGNOSTIC

VARIABLES AND THEIR POTENTIAL ROLE IN THE DEVELOPMENT OF ORAL

SQUAMOUS CELL CARCINOMA (OSCC): STUDY FROM LOCAL POPULATION”.

______________________________________ ______________________________________

EXTERNAL EXAMINER INTERNAL EXAMINER

PROF. DR. ZAMEER AHMAD PROF. DR. ARIF MALIK

DEPARTMENT OF BIOCHEMISTRY

ALLAMA IQBAL MEDICAL COLLEGE LAHORE INSTITUTE OF MOLECULAR BIOLOGY AND

BIOTECHNOLOGY (IMBB/CENTER FOR

RESEARCH IN MOLECULAR MEDICINE

(CRIMM).

____________________________________________

PROF. DR. M. H. QAZI

DIRECTOR,

INSTITUTE OF MOLECULAR BIOLOGY AND

BIOTECHNOLOGY (IMBB/CENTER FOR

RESEARCH IN MOLECULAR MEDICINE (CRIMM)

iv

ACKNOWLEDGEMENTS

ALL PRAISES FOR ALLAH, WHO GUIDES US FROM DARKNESS TO LIGHT AND HELP

US IN ALL HARDSHIPS, AND ALL MY REVERENCE IS FOR HIS HOLY PROPHET

MOHAMMAD (P.B.U.H) WHO ENABLES US TO RECOGNIZE OUR CREATOR. I AM GRATEFUL

TO THAT SPIRITUAL POWER WHO HELPS ME AT EVERY STAGE OF MY LIFE. GREAT

PERSONALITIES HAVE BROUGHT IMPECCABLE TRANSFORMATION AND AMAZING

CHANGES IN THE USUAL ROUTINES. WHETHER IT IS POLITICS OR ARTS OR SCIENCE,

WHETHER IT IS RELIGIOUS OR PHILOSOPHICAL, WHETHER IT IS BIG OR SMALL, THE

IMPACT MADE BY PEOPLE MADE THEM FAMOUS PERSONALITIES. PROF. DR. ARIF

MALIK “INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY/CENTER FOR

RESEARCH IN MOLECULAR MEDICINE” MY RESPECTED SUPERVISOR; HIS IDEAS HAVE

MANAGED TO COME THROUGH AND HAVE REVOLUTIONIZED THE FACE OF MODERN

SCIENCE. IT IS MY FOREMOST DUTY TO EXPRESS MY HEARTIEST AND SINCEREST

GRATITUDE TO MY RESPECTED CO-SUPERVISOR PROF. DR. M.H. QAZI, DIRECTOR,

INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY FOR THEIR KIND

GUIDANCE DURING MY STUDIES. I AM MUCH OBLIGED TO HIM FOR PRODUCTIVE

GUIDANCE. I ACKNOWLEDGE PROF. DR. SALEEM SHUJA EX-RECTOR OF UNIVERSITY OF

LAHORE, SULAYMAN WAQUAR, SARA ZAHID AND SAEED ISMAIL FOR THEIR HELP IN

THE COMPILATION OF MY WORK. LAST BUT NOT LEAST I OW MY SPECIAL THANKS TO

MY HUSBAND ALI MAQUDDAS KHAN AND MY SONS ALI ABBAS KHAN AND HUSSAIN

ABBAS KHAN FOR THEIR LOVE AND SUPPORT AT EVERY WALK OF MY LIFE.

DR SAIMA RUBAB KHAN

REG.# DBC-02123003

v

DECLARATION OF ORIGINALITY

NAME OF STUDENT: DR. SAIMA RUBAB KHAN

REG.#: (REG.# DBC-02123003)

DECLARATION

1-understand what plagiarism is and I am aware of the University’s policy in this regard.

2-I declare the thesis is my own original work. Where other people’s work has been used (Either from a

printed source, Internet or any sources), this has properly acknowledged and referenced in accordance

with departmental requirements.

3-I have not used work previously produced by another student or any other person to hand in as my own.

4-I have not allowed, and will not allow, anyone to copy my work with the intention of passing it off as

his or her own work.

SIMILARITY INDEX (17%) ON DAY (15-12-2017 AT 05:52 PM)

SIGNATURE OF STUDENT: -----------------------------------------------------------------------------

SIGNATURE OF SUPERVISOR: ------------------------------------------------------------------------

SIGNATURE OF LIBRARIAN: --------------------------------------------------------------------------

vi

DEDICATION

Dedicated

To

MY LOVELY MOTHER,

Whose Prayers and Constant Encouragement Enabled Me to

Complete this Task of Learning

vii

LIST OF ABBREVIATIONS

8-oxodG 8-oxo- 7, 8 dihydro-2’-deoxyguanosine

8-oxoGuo 8-oxo- 7, 8 dihydroguanosine

AGE Advanced glycation end-products

ALT Alanine aminotransferase

AOPP Advanced Oxidative Protein Products

AST Aspartate aminotransferase

AUC Area Under Curve

BUN Blood urea nitrogen

CAT Catalase

CH2-THF 5, 10-Methlenetetrahydrofolate

CH3-THF 5-methltetrahydrofolate

CSβ Cystathionine beta

DIT Di-iodotyrosine

ECM Extracellular Membrane

ELISA Enzyme linked immune-sorbent assay

EGFR Endothelial growth factor receptor

ER Estrogen Receptor

FADPH Flavin adenine dinucleotide Phosphate

Glu Glutamate

Glu-Cys ligase Glutamate-Cysteine ligase

GNMT Glycine N-methyltransferase

GSH Glutathione

GSH Glutathione

GSH synthase Glutathione synthase

GSHPx Glutathione peroxidase

GSHr Glutathione reductase

viii

GSSH Glutathione disulphide

GST Glutamyl S-transferase

H2O2- Hydrogen peroxide free radical

I- Iodide ion

I2 Iodine molecule

IL Interleukin

LPO Lipid peroxidation

LPS Lipopolysaccharides

MATI/II Methionine adenosyltransferase

MDA Malondialdehyde

MMP Matrix metalloproteinases

MIT Mono-iodotyrosine

MPO Myeloperoxidases

MS Methionine synthase

MSR Methionine synthase reductase

MTHFR Methlene tetrahydrofolate reductase

NAC N-acetylcysteine

NFκB Necrosis Factor Kappa Beta

NLR NOD-like receptors

NLRB NOD-like receptors BIR domain

NLRC NOD-like receptors CARD domain

NO Nitric oxide

NOS Nitric oxide synthase

O2- Superoxide free radical

ONOO- Peroxynitrate

OSCC Oral Squamous Cell Carcinoma

PAF Prolactin activating factor

ix

PAMP Pathogen Associated Molecular Pattern

PB Phosphate Buffer

PCR Polymerase Chain Reaction

PIF Prolactin inhibitory factor

PLP Pyridoxal Phosphate

PUFA Polyunsaturated Fatty acids

r-Glu-Cys Gamma-Glutamate-Cysteine

RNS Reactive Nitrogen Species

ROC Receiver Operating Curve

ROS Reactive oxygen specie

RT3 Reverse tri-iodothyronine

SAH S- adenosylhomocysteine

SAHH S- adenosylhomocysteine hydrolase

SAM S-adenosyl methionine

SHMT Serine methylhydroxytransferase

SOD Superoxide dismutase

SOD Superoxide dismutase

T3 Tri-iodothyronine

T4 Tetra-iodothyronine

TAM Tumor Associated Macrophage

TBARS Thiobarbituric acid reactive substances

THF Tetrahydrofolate

TLR Toll-like receptor

TNF Tumor necrosis factor

TPP Total Plasma Peroxidases

TRH Thyrotropin releasing hormone

TSH Thyroid stimulating hormone

x

VIP Vasoactive inhibitory peptide

VitB12 Vitamin B12 (Cobalamine)

VitB6 Vitamin B6 (pyridoxine)

VitB9 Vitamin B9 (Folic acid)

xi

LIST OF SYMBOLS

SYMBOLS NAME

°C Degree centigrade

> Greater than

< Less than

rpm Rotation per minute

µl Micro liter

% Percentage

nm Nano meter

mg/dl Miligram per deciliter

mg/kg Miligram per kilogram

g/dl Gram per deci liter

IU/L International units per liter

xii

TABLE OF CONTENTS

"Bismillah al rahman al rahim"

i

SUPERVISORY COMMITTEE ii

EXAMINATION COMMITTEE iii

ACKNOWLEDGEMENT iv

DECLARATION OF ORIGANALITY v

DEDICATION vi

LIST OF ABBREVIATIONS vii

LIST OF SYMBOLS xi

TABLE OF CONTENTS xii

CHAPTERS DESCRIPTION PAGE #

CHAPTER ONE INTRODUCTION 1

CHAPTER TWO REVIEW OF LITERATURE 5

CHAPTER THREE MATERIALS AND METHODS 20

CHAPTER FOUR RESULTS 24

CHAPTER FIVE DISCUSSION 34

CHAPTER SIX SUMMARY 47

CHAPTER SEVEN CONCLUSION 51

CHAPTER EIGHT LITERATURE CITED 53

http://wahiduddin.net/words/bismillah.htm

xiii

TABLE OF CONTENTS

S.# DESCRIPTION PAGE. #

1.0 INTRODUCTION 01

2.0 REVIEW OF LITERATURE 05

2.1 MATRIX METALLOPROTEINASES (MMPS)

IN OSCC

06

2.2 OXIDATIVE STRESS 11

2.3 NITRIC OXIDE 12

2.4 LIPID PEROXIDATION 13

2.5 CYTOKINES IN OSCC 13

2.6 ANTIOXIDANTS IN OSCC 16

2.6.1 ENZYMATIC ANTIOXIDANTS 18

2.6.2 NON-ENZYMATIC ANTIOXIDANTS 19

3.0 MATERIALS AND

METHODS

20

3.1 SOURCE OF DATA 21

3.2 INCLUSION CRITERIA 21

3.3 EXCLUSION CRITERIA 21

3.4 CHEMICALS 21

3.5 ESTIMATION OF CATALASE (CAT) 21

3.6 ESTIMATION OF THIOBARBITURIC ACID

REACTIVE SUBSTANCES (TBARS)

22

3.7 ESTIMATION OF SUPEROXIDE DISMUTASE

(SOD)

22

xiv

3.8 DETERMINATION OF AOPPs 22

3.9 AGEs PREPARATION AND DETERMINATION 22

3.10 ESTIMATION OF REDUCED GLUTATHIONE

(GSH)

22

3.11 ESTIMATION OF GLUTATHIONE

REDUCTASE (GR)

23

3.12 DETERMINATION OF GLUTATHIONE

PEROXIDASE (GPx)

23

3.13 DETERMINATION OF NITRIC OXIDE (NO) 23

3.14 ESTIMATION OF VITAMIN E 23

3.15 DETERMINATION OF VITAMIN-A 23

3.16 ESTIMATION OF MMP-2,9 and 11 ELISA 23

3.17 ESTIMATION OF IL-1α ELISA 23

3.18 ESTIMATION OF TNF-α ELISA 23

4.0 RESULTS 24

4.1 CIRCULATING STRESS BIOMARKERS

PROFILE OF ORAL CANCER PATIENTS VS

CONTROL

25

4.2 INFLAMMATORY BIOMARKERS PROFILE

OF ORAL CANCER PATIENTS Vs CONTROL

25

4.3 ANTIOXIDANT BIOMARKERS PROFILE OF

ORAL CANCER PATIENTS Vs CONTROL

26

5.0 DISCUSSION 35

6.0 SUMMARY 48

7.0 CONCLUSION 51

8.0 LITERATURE CITED 53

xv

LIST OF TABLES


TABLE-01: LEVEL OF DIFFERENT VARIABLES AND THEIR INTERPLAY IN

THE DEVELOPMENT OF OSCC

27

TABLE-02: PEARSON CORRELATION (TWO-TAILED) IN ORAL SQUAMOUS

CELL CARCINOMA (OSCC)

46

xvi

LIST OF FIGURES


FIGURE 0 1: EXPRESSION OF DIAGNOSTIC VARIABLES OF MEDICAL

IMPORTANCE AND THEIR INTERPLAY IN THE

DEVELOPMENT OF ORAL SQUAMOUS CELL CARCINOMA

(OSCC)

28




(OSCC)

29




(OSCC)

30




(OSCC)

31




(OSCC)

32




(OSCC)

33

FIGURE 0 7: PATHWAY INVOLVED IN PATHOGENESIS OF (OSCC) 45

CHAPTER ONE INTRODUCTION

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INTRODUCTION


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INTRODUCTION

The most frequently occurring mouth cancer, oral squamous cell carcinoma (OSCC),

largely affects the tongue and floor of the oral cavity. It is the development of painless

precancerous lesions appearing in either form of a lump, ulcer with raised fissures, erythroplakia,

leukoplakia or erythroleukoplakia followed by painful symptoms persisting for greater than two

weeks (Neville and Day, 2002). Furthermore, it is majorly asymptomatic which makes it hard to

be detected in initial stages and development of lymph node metastasis in the advanced stages

makes it more problematic hence contributing to worldwide elevated rates of mortality and

morbidity. Apart from this, a major setback is cancer relapse which has further emphasized the

urgency of better future treatment modalities (Jones et al., 1992; Brandwein-Gensler et al., 2005;

Kademani et al., 2005; Massano et al., 2006; Bell et al., 2007; Montoro et al., 2008 and

Rusthoven et al., 2010). OSCC, the sixth ranked cancer, has worldwide cases of about 4% where

95% of these are of OSCC alone. In addition, the incidence of oral cancer in Indian subcontinent

is escalating at an unprecedented rate where India has 45% and Pakistan has 10% cases of OSCC

(Williams 2000; Siddiqui et al., 2006; Baykara et al., 2013; Albano et al., 2013; Abbasi et al.,

2014 and Jafarian et al., 2015). From 2004 to 2009, over three million newly identified oral and

oropharyngeal neoplasm cases with over 7000 deaths were reported. It is alarming that out of one

million there is a 6.6% and 2.9% morbidity chance in men and women while a 2.9% and 1.4%

mortality risk in the same subjects respectively (Mehrotra and Yadav, 2006 and Sharma et al.,

2010). Since the last thirty years scientists have been unsuccessful to reduce the morbidity and

mortality rates associated with OSCC which is indeed shocking. Early diagnosis promises a

survival rate of 80-90% while a 15-50% rate if diagnosed at highly progressed stages (Scott et

al., 2006 and Markopoulos et al., 2010).

However, in recent years a burgeon of OSCC cases has been reported in individuals less

than 45 years specifically from 18 to 44 years (Warnakulasuriya 2009 and Patel et al., 2011). It

has been observed that younger people especially males are more prone to develop OSCC than

females due to excessive use of tobacco and tobacco associated products (Feller and Lemmer,

2012 and Krishna et al., 2013). A myriad of factors predispose the risk of OSCC including

family history, cannabis, excessive use of tobacco and tobacco products, regular alcohol intake,

narcotics and antioxidant deficient diet. Moreover Hepatitis C, Herpes virus, Candida albicans,

Human immunodeficiency virus, Human papilloma virus and Epstein Barr virus have also been


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considered potential causative factors (Thavarajah et al., 2006; Garavello et al., 2008; Nagao and

Data, 2009; Shah and Gil, 2009; Petti 2009; Jalouli et al., 2010 and Nuovo 2011). In addition

inflammation, immune defects, potentially malignant disorders, defection in DNA repair

mechanisms, genetic polymorphism and mutation of certain genes (CYPIA1, GSTM1, CCND1,

ADH3, Cyclin D and N-acetyl transferase-2) also places a threat of oral cancer progression.

Although the prospective risk factors in different ethnic populations vary largely by far tobacco

and smokeless tobacco (betel quid, areca nut, gutka, maras powder) poses the greatest risk in

development of oral cancer. Tobacco smoking accounts for 75% of the reported cases, while

both smokers and alcoholics have a six fold risk of oral neoplasm than non-smokers and non-

alcoholics whereas those individuals practicing both habits have a fifteen fold risk of OSCC than

healthy individuals. By and large tobacco and alcohol in the West, gutka and betel quid in

Taiwanese, Maras powder in Turks, betel quid in Asians and in Indians areca nut, tobacco, and

betel quid along with alcohol consumption account for the onset of OSCC (Ogden 2005;

Subapriya et al., 2007; Su et al., 2007; Scully and Bagan, 2009; Attar et al., 2010de

FreitasCordeiro-silva et al., 2011; Marichalar-Mendia et al., 2011; Zygogianni et al., 2011;

Tanaka et al., 2011; Markopoulos 2012; Kurtul and Gokpinar, 2012).

Conventionally imaging techniques followed by blood, serum and biopsy samples are

used for OSCC detection but in the recent years the distinguished role of saliva as a diagnostic

tool has been widely accepted (Arellano-Garcia et al., 2008). Saliva without a doubt has an

advantage over serum in that it is easy to collect, noninvasive, safe, cost effective and in direct

contact with the cancerous site. It also provides an excellent source of genomic, proteomic and

oxidative stress biomarkers which are exploited for diagnostic, predictive and post therapy

monitoring purposes (Krishna Prasad et al., 2013; Cheng et al., 2014 and Bano et al., 2015).

Usually surgery followed by radiotherapy is the prime treatment in OSCC stages I and II

whereas for stage III and IV a combination of surgery, radiotherapy and chemotherapy is

utilized. Likewise immunotherapy, gene therapy, altered fractionated radiotherapy; cervical

lymph adenectomy, selective neck dissection, concomitant chemo-radiotherapy (CT-RT) and

targeted molecular therapy are also novel prospective treatments (Markopoulos, 2012). Recently

over-expressed matrix metalloproteinases (MMPs) have been found to be the culprits behind

various carcinomas including OSCC. The utilization of MMP-targeted synthetic drugs, Matrix

metalloproteinase inhibitors (MMPIs), specifically decreases the activity of various matrix


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metalloproteases. On the basis of drug type MMP inhibitors are classified as peptidomimetic,

non-peptidomimetic, chemically modified tetracyclines, reform proenzyme structures, analogues

of inorganic pyrophosphates, bisbiguanide antiseptics, non-steroidal inhibitors of aromatose,

synthetic peptides based on PSP94, cyclophosphinamides, cyclophosphinamide based

hydroxamic acids, caffeoyl pyrrolidine derivatives, sulfonamide hydroxamates and

nonhydroxamates. Nanomedicine in combination with chemotherapeutics has no doubt also

shown promising results in terms of lesser side effects and tumour resistance. Recently

antioxidant therapy has gained popularity in treating OSCC (Rossi et al., 2010; Vilen et al.,

2013; Liboiron and Mayer, 2014; Oliviera et al., 2014).

Globally, a notable reason behind the drastic boom in OSSC cases is that the masses are

not fully cognizant of the hazardous effects of tobacco products and alcohol. In order to get rid of

this social taboo, a substantial number of awareness programs should be arranged to enlighten

the public about the debilitating effects of tobacco chewing, tobacco smoking, alcohol

consumption and excessive use of tobacco related products. The government can contribute by

taking strict steps on multiple levels to ban the sale and use of these noxious products. Likewise

emphasis on preventive measures such as opting for early screening, avoiding unnecessary

irradiation and consuming diet rich in iron and antioxidants can promise an oral cancer free life

(Kurtul and Gokpinar, 2012; Bansal et al., 2012 and Bhuvaneswari, 2014). Moreover the need

for effective MMP-2 and MMP-9 inhibitors is also in demand in order to stop OSCC in its track.

In addition by understanding the underlying disease mechanism, we can get a vantage point of

the potential predictive markers and novel treatment targets. Another aspect is to hinder

associated pathways leading to OSCC progression. Since the last decade, the search for novel

markers and effective treatments has spurred with the growing advancement in diagnostic tools.

In order to achieve improved outcomes and to minimize death rates, the need of the hour is to

detect oral neoplasms as earliest as possible and devise effective targeted treatments accordingly

(Bagan et al., 2010; Bello et al., 2010 and Prasad and McCullough, 2013). For this purpose, the

search for novel biomarkers is highly in demand and work on synthetic matrix metalloproteinase

inhibitors (MMPIs) has accelerated to a new level.

1.1 AIMS AND OBJECTIVES

The present study was to determine role of prognostic variables and their potential role in

the development of oral squamous cell carcinoma (OSCC).

CHAPTER TWO REVIEW OF LITERATURE

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REVIEW OF LITERATURE


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2.0 REVIEW OF LITERATURE

To date, there are increasing number of appreciable data regarding promising, potential

salivary biomarkers at hand provided by (Khanna 2008, Markopoulos et al., 2010 and Tsuda and

Ohba, 2012; Krishna Prasad et al., 2013; Warnakulasuriya 2014; Bano et al., 2015). The current

research throws light on the combined role of MMPs specifically gelatinases, inflammatory and

oxidative stress markers involved in the pathogenesis of oral squamous cell carcinoma. Lastly,

the curative effect of antioxidants has also been conversed.

2.1 MATRIX METALLOPROTEINASES (MMPS) IN OSCC

These zinc dependent endopeptidases are produced according to the cell's need by wound

cells as well as inflammatory cells and are known to play essential roles in both normal and

diseased state. Under normal conditions, MMPs are secreted in very low amounts and they are

involved in tissue remodeling processes like ossification, placenta development, and embryonic

development and wound healing. Nevertheless in diseased states defected regulation leads to

their elevated level which contributes to rheumatoid arthritis, cardiovascular diseases, dermal

photoaging, autoimmune blistering disorders of skin, osteoarthritis, tumor invasion, metastasis

and periodontitis etc. As zymogens, these metalloendopeptidases either may be attached to

membranes or secreted in the extracellular matrix where proteolysis, other MMPs and serine

proteases activate them in order to act on different substrates ranging from matrix substrates to

bioactive entities. MMPs are strictly regulated by cytokines, stress and growth factors however

they can be rendered inactive through natural inhibitors including tissue inhibitors of

metalloproteinases (TIMPS) and α-2-macroglobulin or by synthetic matrix metalloproteinase

inhibitors (MMPIs). So far about 25 MMPs and four TIMPs (TIMP 1, TIMP 2, TIMP 3 and

TIMP 4) have been reported in vertebrates (Howard et al., 1991; O'Connell et al., 1994; Butler et

al., 1999 and Liu et al., 1997; Murray 2001; Sternlichit and Werb, 2001 and Hadler-Olsen et al.,

2011;).

On the basis of substrate specificity, sequence similarity and domain organization

metalloproteinases are categorized as: collagenases, gelatinases, stromelysins, matrilysins and

membrane type MMPs (MT-MMPs). Generally all MMPs consist of a signal peptide, a pro-

peptide having highly conserved Cysteine residues, a zinc containing catalytic domain and a

hemopexin like domain which is attached to the catalytic domain by a linker. But the striking

feature that separates gelatinases from other MMPs is the presence of a fibronectin type II like


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domain (Egeblad and Werb, 2002; Nagase et al., 2006; Butler and Overall, 2009 and Klein and

Bischoff, 2011;).

One of the characteristic marks in cancer invasion is the deterioration of extracellular

matrix (ECM) which is efficiently done by matrix metalloproteinases (MMPs). Myriad MMPs

(MMP 1, 2, 3, 7, 9, 11, 13 etc.) take part in OSCC disease progression but the crucial role of

gelatinases has been widely established (Hong et al., 2000; Eglebad and Werb, 2002 and Lee et

al., 2008 and Vilen et al., 2013). Additionally studies show that sophisticated methods such as

Immunohistological analysis, In situ hybridization, PCR, ELISA, gelatin zymography, in situ

zymography, Confocal laser scanning microscopy, western blotting, northern blotting, southern

blotting, use of MMPIs or transfected cell lines and knockout animal models have been utilized

to estimate gelatinases successfully (Jin and Lloyd, 1997; Ikebe et al., 1999; Pirila et al., 2001;

Berg et al., 2002; Snoek-van Buerden and Von Den Hoff, 2005; Hotary et al., 2006; Patel et al.,

2007; Vilen et al., 2008; Suojanen et al., 2009; Liu et al., 2009; Singh et al., 2010).

MMP-2, a 72 kDa gelatinase, is physiologically found in both normal and neoplastic

endothelial cells, keratinocytes, fibroblasts and chondrocytes while gelatinase B, a 92 kDa

gelatinase, is normally expressed by macrophages, keratinocytes, monocytes and multiple

neoplastic cells. Both ECM destroyers occur in secreted forms where MMP-9 can be sojourned

by all four types of TIMPs (Westermarck and Kahari, 1999). Predominantly the secreted MMPs

have to be activated in order to perform their respective functions. The pro MMP 9 is naturally

activated extracellularly by unknown activators or by other MMPs which may be MMP 1, 2, 3,

7, 10, 13 and 26. Moreover cathepsin G, dentin matrix protein-1, TAT-2, plasmin, α-

chymotrypsin and trypsin also activate MMP-9 (Fridman et al., 1995; Knauper et al., 1997 and

Nakamura et al., 1998). MMP 3 cleaves pro MMP 9 at Glu59-Met60 as well as Arg106-Phe107

while MMP 26 cleaves at Ala93-Met94 (Ogata et al., 1992 and Zhao et al., 2003). Studies also

show that trypsin-2 activates the same zymogen at Arg87-Phe88 but in very small quantities

(Sorsa et al., 1997). Another activator is a serine protease in nature that has cleavage site at

Lys65-Ser66 (Vilen et al., 2008). Other proteolytic agents include chymotrypsin like domain and

plasmin (Mazzieri et al., 1997 and Han et al., 2002). Whereas gelatin bound and type IV

collagen bound MMP-9 zymogen is activated by allosteric activation, S-nitrosylation or ROS

mediated oxidation (Peppin and Weiss, 1986; Gu et al., 2002 and Bannikov et al., 2002).

Furthermore, in vitro studies indicate that all four types of TIMPs have active role in inhibiting


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92 kDa gelatinase B (Howard et al., 1991; O'Connell et al., 1994; Liu et al., 1997 and Butler et

al., 1999). On the other hand, 72 kD gelatinase A (MMP-2) is activated by certain substances

hydrogen peroxide, MMP 1, 7, 13, 14, 16, 17, 24, 25 along with thrombin and bone sialoprotein.

MMP 2 further activates MMP-9 and -13. The gelatinase-A takes part in tumor invasion and

metastasis by activating other metalloproteinases to enhance its role in disease progression where

it is located in special cells called carcinoma associated fibroblasts (CAFs) in OSCC neoplasm.

Integrins, the adhesion molecule, has been reported to play a vital role in induction of OSCC and

elevates MMP-9 expression directly. Interestingly MMP-2 via the fibronectin-integrin pathway

(αVβ6 pathway) activates MMP-9 (Sutinen et al., 1998 and Impola et al., 2004).

Predominantly gelatinases have a wide range of substrates, the common ones in both

MMP-2 and -9 include collagens IV, V, VII, X, XIV (Welgus et al., 1990; Murphy et al., 1991

and Sires et al., 1995), fibronectin, gelatin, hyaluronidase-treated versican, galectin-3, MBP, a1-

AT, GST-TNF/TNF peptide, IL-1β, Aβ 1-40, aggrecan, elastin, proteoglycan link protein (Mast et

al., 1991; Desrochers et al., 1991; Senior et al., 1991; Fosang et al., 1993; Mitchell et al., 1993;

Nguyen et al., 1993; Gearing et al., 1994; Ochieng et al., 1994; Walsh et al., 1994; Sires et al.,

1994; Perides et al., 1995; Chandler et al., 1995; Fosang et al., 1996; Roher et al., 1994; Ito et

al., 1996; Backstrom et al., 1996). Apart from these laminin-1, osteonectin, laminin-5, APP695,

decorin, FGFR1, MMP-1, IGF-BP5, Ab 10-20, prolysyl oxidase fusion protein, IGF-BP3, MMP-

13, collagen I, collagen XI and MMP-9 are other substrates of neutrophil gelatinase (Miyazaki et

al., 1993; Fowlkes et al., 1994; Crabbe et al., 1994; Fridman et al., 1995; Aimes and Quigley,

1995; Le Page et al., 1995; Thrailkill et al., 1995; Levi et al., 1996; Panchenko et al., 1996;

Knauper et al., 1996; Giannelli et al., 1997; Imai et al., 1997 and Sasaki et al., 1997). On the

other hand, gelatinase B also acts on plasminogen, osteonectin and entactin (Sires et al., 1993;

Sasaki et al., 1993 and Patterson and Sang, 1997).

The normal cellular microenvironment when disturbed has been known to promote

various syndromes. In OSCC neoplasm tumor cells, stromal cells and ECM make up the tumor

microenvironment. Surprisingly, both tumor and stromal cells can largely amend each other’s

effects, facilitating cancer invasion and progression. Mainly tumor associated macrophage

(TAMs), endothelial cells (ECs) and carcinoma associated fibroblasts (CAFs) are pivotal players

in cancer progression (Li et al., 2014). In OSCC cancer cells, specialized carcinoma cells and

inflammatory cells are responsible for the release of MMP-9 and carcinoma associated


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fibroblasts (CAFs) are in charge for the release of MMP-2 (Impola et al., 2004 and Fullar et al.,

2012). TAMs and CAFs cross talk with the cancer cells by autocrine and paracrine signaling and

therefore are of prime significance in OSCC. Usually in wound healing activated fibroblasts

form myofibroblasts, carry out their role, and are marked for death. Nonetheless when the

activated fibroblasts are not degraded, they result in carcinoma associated fibroblasts which

generate MMPs, cytokines and growth factors hence promoting neoplastic progression. Other

imperative stromal resident, macrophages, possess two phenotypes: the proinflammatory M1 and

the anti-inflammatory M2. Furthermore tumor associated macrophage (TAMs) have been said to

belong to the second phenotype. In addition growth factors and chemokines are behind the

accumulation of TAMs at the tumor site while TAMs are known to secrete growth factors,

chemokines and MMPs (Astekar et al., 2013).

The ECM disruptors, MMPs, are found largely throughout the different stages of

neoplasm but gelatinases play a crucial role in these processes (Zucker et al., 1993; Endo et al.,

1997; Torri et al., 1997; Vu et al., 1998; Hong et al., 2000; Lippman and Hong, 2001; Kawata et

al., 2002 and Zwetyenga et al., 2003). Multiple MMPs have been confirmed to play part in

tumor invasion most importantly MMP-1, 2, 7, 9, 13 and 14. The gelatinases are majorly

involved in the onset of OSCC where gelatinase B (MMP 9) is an aggressive multifaceted

endopeptidase which provides its role in tumor invasion, metastasis, inflammation and

angiogenesis by making ECM degradation possible. Apart from this, these endopeptidases make

distinct interactions with cell too. It has been observed that MMP 2 interacts via cellular receptor

integrin αVβ6 while MMP 9 interacts through CD44, integrin pathway or α2 collagen chain. The

activated MMP-9 effectively degrades ECM components giving way to tumor invasion.

Consequently, a great amount of released cytokines and chemokines provides feasible

environment for inflammation. Throughout inflammation stage, oxidative stress makes matters

worse by enhancing inflammation. A great bunch of emanated inflammatory cytokines,

chemokines, matrix proteinases and free radicals expedite angiogenesis. The hypoxic condition

provides increased expression of VEGF, an angiogenic cytokine, along with its receptor in

specific cancer cells, endothelial cells, tumor associated cells.

It has been observed that in OSCC the HIF-1α is upregulated which is the reason behind

elevated levels of gelatinases and hence decreased prognosis (Bergers et al., 2000; Deryugina

and Quigley, 2006 and Chaudhary et al., 2015). MMP-9 has a dual role in angiogenesis where it


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may act as angiogenic or anti-angiogenic depending on the situation (Rundhaug 2003; Liu et al.,

2009 and Reuter et al., 2010). Various growth factors play essential roles in the first step of

tumorigenesis. For instance overexpression of TGF-β1 is responsible for spiked expression of

MMP-9 in OSCC studies. It has been discovered that Snail, the transcription factor, aids in

elevating MMP-9 expressions (Shi and Massague, 2003; Bjorklund and Koivunen, 2005 and Sun

et al., 2008).

Sufficient data supports the versatile role of gelatinases in oral squamous cell carcinoma.

Gelatinase B (MMP-9) has a significant role in OSCC progression and its expression along with

activity has been thoroughly weighed (Jafarian et al., 2015; Jordan et al., 2004 and Van den

Steen et al., 2002). While Guttman et al. stated that no correlation could be established between

primary tumor size and MMP-9 levels in tongue squamous cell carcinoma patients. On the

contrary, 92 kD gelatinase B is reported to be enhanced in oral cancer (Roukolainen et al., 2005).

A study by Katayama et al. showed that elevated MMP 9 expression was in correlation with poor

prognosis and lymph node metastasis. Whereas De Vicente et al. reported that clinical variables

are not associated with enhanced MMP 9 levels; moreover their study also shows that gelatinases

are involved in tumor invasion and their higher expression is seen with lymph node involvement

whereas MMP-9 has vital role in metastasis. In addition, ethanol has been found to positively

affect expression of gelatinases. Further Hong et al. found that increased expression of MMP-9 is

majorly associated with metastasis. Yet Ikebe et al. showed that increased expression of both

gelatinase A and B in OSCC was in correlation with only tumor invasiveness. Likewise Kato et

al. investigated the association of gelatinases with different tumor stages and concluded that

MMP 9 levels were elevated in OSCC but the active form of MMP 9 was less in quantity as

compared to the levels of activated MMP 2, hence MMP 2 has correlation with advanced stages.

Mukherjee et al. showed that gelatinases are highly involved in cancer and pre-cancer states.

Apart from this, Kato et al. found that gelatinolytic activity in OSCC is due to activated 72 kD

gelatinase A. In a different study moderate expression of MMP-2 was seen while MMP-9 levels

were the highest (Mishev et al., 2014). Another investigative study discovered that it is the

increased gelatinases in OSCC patients that leads to their low survival rates (Yorioka et al.,

2002). Although studies show that gelatinases are expressed in OSCC and the role of gelatinase

in tumor invasion has been established but there is still a contradiction whether MMP-2 plays a

role in metastasis or not. As the part played by gelatinases in OSCC development has not been


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completely understood and elucidated, therefore the role of MMP-2 and MMP-9 is of intense

research now a days.

2.2 OXIDATIVE STRESS

Reactive oxygen and nitrogen species are essentially synthesized in the body to accomplish

certain advantageous tasks but if their levels are increased incredibly would be harmful for the

body. The vulnerable biomolecules carbohydrates, lipids, proteins and nucleotides come under

the devastating attack of these unstable species resulting in DNA damage, genetic mutations and

chemical modifications eventually leading to multifarious diseases (Kang 2002; Van Wijk et al.,

2008; Yokoe et al., 2009; Yadav and Ramana, 2013; Ohnishi et al., 2013). The notorious

oxidative stress is a result of exogenous and endogenous factors. Arguably, certain organelles

peroxisomes, mitochondria and enzymes xanthine oxidase, P450 complex and NADPH oxidase

(NOX) family endogenously produce reactive species whereas noxious chemicals, ultraviolet

radiation, smoking, exercise and environmental pollutants are exogenous sources. It is affirmed

that reactive oxygen species (ROS) have implications in initiation, promotion and progression of

cancer including OSCC (Patel et al., 2009 and Zunli et al., 2015).

Mitochondria are the foremost organelle producing reactive oxygen intermediates and

reactive nitrogen species. Initially superoxide (O2·-) is formed which further forms hydroxyl

radical (OH-) and hydrogen peroxide (H2O2) under aerobic and hypoxic conditions. In addition

superoxide reacts with nitric oxide to form peroxynitrite (Jomova et al., 2010). Moreover, the

endoplasmic reticulum (ER), ubiquitous enzymatic NOX family, cytochrome P450, uncoupled

NO synthase, xanthine dehydrogenase (XDH), cyclooxygenase (COX) and xanthine oxidase

(XO) also contributes in ROS production (Pritsos 2000; Nishino et al., 2008; Popov 2012 and

Segal et al., 2012). Tobacco, smokeless tobacco and alcohol are well-known carcinogens capable

of producing highly reactive free radicals and reactive oxygen species (ROS) in biological

systems namely superoxide anion, hydrogen peroxide, organic peroxides, hypochlorous acid and

hydroxyl radicals which in the long run causes oxidative stress (Yu, 1994 and Bagul et al.,

2013). Apart from other organelles, the main source of oxidative stress is mitochondria which

actively generate ROS, reactive nitrogen species (RNS) and lipid peroxidation by products.

Interestingly free radicals can trigger inflammation while chronic inflammation can set off

oxidative stress, hence providing evidence that both inflammation and oxidative stress are

intertwined (Bektas-Kayhan, 2012 and Lugrin et al., 2014). Further, hypoxia mediated


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production of mitochondrial reactive nitrogen species (RNS) produces lipid peroxidation

byproducts 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The reactive species

release inflammatory cytokines and chemokines which activate inflammatory cells hence

proceeding to chronic inflammation.

Inflammatory cells in turn recruit inflammatory cytokines, chemokines and processed

proteins to activate a wide range of transcription factors, hence indirectly initiating oxidative

stress (Hussain et al., 2003 and Poyton et al., 2009). In addition, pro-inflammatory cytokines

(CXCL -1, -4, -8, -9, -11, and 14), proforms of cytokines (pro TNF-α, proIL-1β) and certain cell

adhesion proteins undergo processing and at last contribute to inflammation. All these routes

eventually contribute in the tumor growth, inflammation and angiogenesis. Additionally 8-

hydroxy-2’-deguanosine (8-OHdG), the genetic biomarker pointing towards DNA damage, is an

important signature molecule for predicting neoplasia and oxidative stress (Valavanidis et al.,

2009). Its elevated levels have been reported in OSCC patients (Agha-Hosseini et al., 2012).

Likewise advanced oxidative protein products (AOPPs), a novel biomarker depicting oxidative

stress (OS), was stated to be drastically increased in oral cancer patients (Barut et al., 2012).

2.3 NITRIC OXIDE

Nitric oxide (NO), an endogenous gas with a short lifespan, has crucial role in redox

biology including respiration, vasodilation, immune response, cell migration and apoptosis.

Exceeded and unchecked levels of NO serve as "messenger of death”, hence triggering certain

illnesses. Studies have confirmed remarkably enhanced levels of nitric oxide (Choudhari et al.,

2012). NO, NO2, NO3 are taken as nitrosative stress markers and they were conversed to be

enhanced in the saliva of OSCC patients (Bahar et al., 2007; Rasheed et al., 2007 and Gokul et

al., 2010). While Gokul et al. reported increased nitric oxide levels in saliva, tissue and blood

samples of individuals suffering from OSCC. Korde et al. reported exceeding levels of total

nitric oxide (NO) and malondialdehyde (MDA) which gave evidence of the active role of ROS

and RNS in oral carcinogenesis. Hence prolonged exposure to ROS has not only destructive

effects on cellular structure and function but also instigates somatic mutations thus leading to

cancer (Fang et al., 2009 and Khandrika et al., 2009).


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2.4 LIPID PEROXIDATION

Lipids undergo peroxidation during stress, causing their destruction. The highly unstable

entities, ROS, attack the polyunsaturated fatty acids (PUFA) in lipid bilayer membranes resulting

in the disruption of biomembranes and deposition of lipid peroxides namely lipid hydroperoxides

(LHP) and malondialdehyde (MDA). In mammals, MDA forms DNA adducts hence contributing

to DNA insults. In addition, this reactive aldehyde can possibly cause genotoxicity, cancer and

mutations. In addition association between cancer and lipid peroxidation has been conveyed too

(Baskaran et al., 1999 and Ray and Hussein, 2002). These end products serve as established

indicators of lipid peroxidation; hence they can be utilized as sophisticated biomarkers of cellular

damage. On the contrary, malondialdehyde (MDA) is widely taken as the indicator of lipid

peroxidation (Jacob and Burri, 1996; Beevi et al., 2004 and Ayala et al., 2014).

It is thought that the raised circulating lipid peroxides may attribute to cancer (Gokul et

al., 2010), and sufficient data provides evidence that elevated levels of MDA were found in

OSCC subjects (Manoharan et al., 2005; Patel et al., 2007; Rasheed et al., 2007; Salzman et al.,

2009; Korde et al., 2011; Agha-Hosseini et al., 2012; Barut et al., 2012; Shilpasree et al., 2013

and Ganesan and Kumar, 2014). In addition increased MDA levels in blood and tissue samples

were reported by Gokul et al. while elevated salivary MDA was reported by Rasool et al.,

Metgud and Bajaj; Gupta et al. and Shetty et al. The study by Rasool et al. as well as Ganesan

and Kumar showed that saliva is a better option than blood in terms of evaluating

malondialdehyde. Other studies, reported that MDA levels in OSCC subjects prior to

radiotheraphy were low but greatly enhanced after radiotherapy (Patait et al., 2011). Similarly

levels of thiobarbituric acid reactive substances (TBARS) were also reported to be raised in

individuals suffering from oral cancer (Manoharan et al., 2005; Srivastava et al., 2012 and

Thomas and Sethupathy, 2015). In contrast, the individuals with reduced levels of lipid

peroxidation and TBARS are said to be less liable to oxidative insults owing to their excellent

antioxidant system (Subapriya et al., 2002 and Kolanjiappan et al., 2003).

2.5 CYTOKINES IN OSCC

The immune system protects the body against harm at all cost yet persistent deviation in

homeostasis leads to chronic inflammation. A number of external and internal factors may

prompt inflammation including viruses, allergens, microbes, tobacco, alcohol, autoimmune

disorders, high calorie diet, malicious chemicals, obesity, radiation and chronic maladies


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(Aggarwal et al., 2009 and Schetler et al., 2010). Importantly, inflammation has been known to

give rise to neoplasia, neurodegenerative diseases, bone disorders, heart disease, dental ailments

and chronic pain (Seaman 2006 and Seaman 2007).

OSCC has a close link with immune system and with the advancement in its severity, a

rapid decline in immune function has been known. In addition, chronic inflammation has

potential of causing oxidative stress and excessive exposure to oxidative stress in turn gives rise

to chronic inflammation which ultimately leads to onset of oral squamous cell carcinoma (Osaka

et al., 2000; Bartsch and Nair, 2006 and Nathan and Cunningham-Bussel, 2013). Moreover

increased bacterial exposure sets off inflammation leading to oxidative stress, reduction of

antioxidants and eventually elevated risk of serious infections and dental diseases (Nishida et al.,

2000 and D’Aiuto et al., 2010).

The oral epithelium, naturally, protects itself from external and internal damaging agents.

On the contrary, the copious use of tobacco products not only harms the oral mucosal epithelium

but also poses a great possibility for certain oral infections (Lee et al., 2012 and Feldman and

Anderson, 2013). The exogenous and endogenous molecular patterns (PAMPs and DAMPs) are

recognized by special pattern recognition receptors (PRRs) which comprise of Toll-like receptors

(TLRs) and NOD-like receptors (NLRs). TLRs and NLRs are crucial receptors for the innate

defense mechanisms yet unchecked TLR activation is associated with multifarious ailments

(Reuven et al., 2014). NLRs reside in the cytosol, sense multitude danger signals, have various

subfamilies and are activated by TLRs. The NLRB (NOD-like receptors BIR domain) and NLRC

(NOD-like receptors CARD domain) subfamily forms inflammosomes, later directing activation

of pivotal inflammatory cytokines IL-1α and IL-1β (Martinon et al., 2009 and Kersse et al.,

2011). The nucleotide binding oligomerization domain (NOD)-like receptors consist of NOD1

and NOD2, both of which have been found to be expressed in mouth cavity provided that NOD1

is more prevalent in oral epithelium (Sugawara et al., 2006; Uehara et al., 2007 and Uehara and

Takada, 2008).

To date, 12 TLRs are known in mammals and all TLRs have the capability to recognize

DAMPs and PAMPs. TLRs are only observable in oral cancer as the normal epithelium lacks

them. Toll-like receptors find their wide use in immunity, promoting cancer progression and

secreting cytokines. The TLR-activated neoplastic cells release inflammatory cytokines and

chemokines resulting in activating immune cells in the vicinity, which in turn secrete cytokines


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and chemokines (Heo et al., 1989 and Wang et al., 2006). A recent study has shown that TLR9 is

greatly expressed in OSCC and its role in tumor growth is by means of elevated cyclin-D1,

which has been known to activate TLR9. Also these receptors have role in increasing expression

of gelatinases. Whereas the transcription factors AP-1 and NF-kB are activated by TLRs and

have been identified as the regulator of MMP-2. In addition, the activated Fos and Jun-D genes

lead to elevated AP-1 activity. Interestingly, TLR9 was not found to influence NF-kB (Ruan et

al., 2014). Literature highlights that TLRs varied in diverse OSCC stages. TLR4 was found to be

linked with lymph node metastasis whereas TLR2 expression was linked with both invasion and

lymph node metastasis. Apart from this, TLR7 levels were stated to be tumor grade dependent

while TLR8 levels surged with the exacerbation in tumor grade (Esmaeeli et al., 2015).

It has been reported that those patients had better disease outcomes that had greater

immune cells in the vicinity of tumor (Scully and Bagan, 2007). In addition inflammatory cells

especially tumor-associated macrophages (TAMs) are actively involved in raising reactive

oxygen species (ROS) levels by recruiting certain cytokines (IL-6, IL-1β, TNF-α), chemokine

(NFκB, iNOS, COX-2, HIF, STAT3, Nrf2, NFAT) and metabolites of arachidonic acid. For

instance arachidonic acid derivatives (AADs) are pro inflammatory in nature and they contribute

in ROS-induced inflammation by activating neutrophils. Similarly other pro-inflammatory

substances nitric oxide (NO), tumor necrosis factor (TNF-α), Interleukin-6 (IL-6) and IL-1β are

generated by Lipopolysaccharide (LPS) (Dana et al., 1994; Zallen et al., 1998; Lee et al., 2004;

Lu et al., 2006; Naik and Dixit, 2011 and Ho and Kuo, 2014). Additionally, free radicals are also

known to raise the levels of inflammatory cytokines including tumor necrosis factor alpha (TNF-

α) and tumor growth factor beta (TGF-β) which facilitates chronic inflammation (Goossens et

al., 1995). Similarly tumor necrosis factor (TNF-α) is also a potential predictive inflammatory

biomarker for oral neoplasms and is known to trigger the NFκβ pathway therefore facilitating

tumor progression. Furthermore the role of ROS-induced interleukin 6 (IL-6) and chemokine IL-

8 in the onset of cancer has also been reported (Reuter et al., 2010).

IL-8, a post-inflammatory cytokine, is a useful biomarker for early detection of oral

cancer disorders and OSCC. Brinkmann et al. utilized PCR and ELISA techniques in order to

observe the trends of salivary proteomic markers in Serbian population and their findings

showed raised IL-8. Furthermore ROS and macrophages has also been found to up regulate an

angiogenic cytokine vascular endothelial growth factor (VEGF), TNF-α, IL-6, IL-1 and GM-


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CSF hence accelerating angiogenesis. Likewise macrophages secrete COX-2, MMP-2, MMP-9,

MMP-7, MMP-12 and thymidine phosphorylase hence facilitating migration of endothelial cells.

The role of VEGF in carcinogenesis has been confirmed by the studies (Pandya et al., 2006).

Also, it is found that angiogenic cytokine VEGF is elevated in OSCC (Mukherjee et al., 2010).

Interestingly, MMPs and cytokines have entwined connections where MMP activation and

expression can be controlled by inflammatory cytokines. A study reports that disruption of a

matrix protein, decorin, releases anchored TGF-β from the matrix. Moreover, sheddases and

convertases have also been reported to free receptors, adhesion molecules and cytokines from the

membranes (Arribas et al., 1996; Imai et al., 1997 and Hooper et al., 1997). Nevertheless early

diagnosis of inflammatory growth factors, adhesion molecules and cytokines may provide us a

better outlook of OSCC progression.

2.6 ANTIOXIDANTS IN OSCC

In order to cope with oxidative stress, the body has its own specialized mechanism to

neutralize the adverse effects of ROS and RNS via antioxidants. These scavengers are classified

as enzymatic and non-enzymatic and may be exogenous, endogenous, naturally produced or

taken as supplements. Largely, antioxidants work by preventing and repairing the already caused

damage therefore enhancing immunity and minimizing disease risks. Furthermore the

disturbance in the native production of pro-oxidants and antioxidants provides feasible

environment for the onset of multiple diseases including OSCC (Gonzales et al., 1984 and Pham-

Huy et al., 2008). In oral cancer (OC), the oral epithelial layer is greatly affected by tobacco and

is prone to damage by ROS and RNS. Tobacco users have an escalated level of reactive species

which potentially reduce antioxidants from the system. Likewise increments of ROS and RNS

have been observed in oral pre-cancer as well as cancer. In the course of carcinogenesis, scanty

or dwindling enzymatic and non-enzymatic antioxidants set off oxidative stress hence formation

of intermediates of reactive nitrogen (RNI) and oxygen (ROI). Later, an elevation in oxidative

stress increases the need for essential antioxidant nutrients in the body as in the case of tobacco

abusers. The inhibitory, preventive and reversal characteristics of antioxidants are well

established in multitude diseases, therefore they have a protective effect against oral cancer.

Moreover antioxidant therapy has gained wide speculation in the recent past few years

(Choudhari et al., 2014).


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Oxidative stress may also be triggered by deficiency or inhibition of antioxidant enzymes.

Antioxidants have been found to reverse the harmful effects of nicotine (Figuero et al., 2006).

Surprisingly, topical antioxidants have excellent results on oral, periodontal, skin and gingival

cells (San Miguel et al., 2010). The protective effects of Lactoferin, black raspberries and black

tea polyphenols in dental disorders have been well established. Animal models and human

studies have shown that freeze dried black raspberries caused an appreciable decline in dysplasia

(Stoner et al., 2007 and Letchoumy et al., 2008). The effective roles of flavonoids have been

found to minimize the expression of inflammatory substances in macrophages and monocytes

residing in the gingival connective tissue. Moreover flavonoids such as luteolin, quercitin and

genistein have been reported to regulate NO formation of LPS-stimulated human gingival

fibroblasts. Luteolin, specifically is involved in down regulating MAP kinases hence hindering

inflammation (Gutierrez Venegas et al., 2006). Ferrulic acid, a vital component of raspberries,

has been reported to scavenge free radicals. The anticancer properties of ferrulic acid have been

known (Lesca 1983; Mori et al., 1999; Kampa et al., 2004 and Lee 2005;). Vitamin E, Ferrulic

acid and Vitamin C in combination decreased OS and prevented formation of thymidine dimers

in skin (Srinivasan et al., 2007). The therapeutic role of alpha tocopherol in treating and

preventing periodontal pathology, reducing wound healing time and oxidative stress has been

known (Chapple and Matthews, 2000 and Barbosa et al., 2009). It is suggested that the lipid

soluble vitamin E may diminish hydroxyl radicals (Royack et al., 2000). Studies also show that

reduced levels of serum folate have been linked to periodontal illnesses (Yu et al., 2007).

Antioxidants are potential predictive biomarkers in multiple diseases and they have been

found to be progressively decreased in OSCC (Rasool et al., 2014 and Thomas and Sethupathy,

2015). Rasheed et al. reported depleted antioxidants in head and neck squamous cell carcinoma

(HNSCC) whereas Bahar et al. found diminished antioxidants in oral cancer subjects. Total

antioxidant capacity (TAC), a prognosticator of antioxidant status, has been found to

progressively reduce in numerous malignancies including oral cancer patients (Hedge et al.,

2013). Apart from this, the reduced levels of total antioxidant capacity (TAC) in OSCC patients

were said to be owing to both oxidative and nitrative stress (Korde et al., 2011 and Agha-

Hosseini et al., 2012). Salivary antioxidants albumin, glutathione, vitamin C, uric acid and

antioxidant enzymes have been seen to be diminished in oral illnesses (Bhuvaneswari 2014).


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2.6.1 ENZYMATIC ANTIOXIDANTS

The enzymatic scavengers superoxide dismutase (SOD), catalase (CAT), glutathione

peroxidase (GPx) and glutathione reductase (GRx) are actively involved in eliminating reactive

species where SOD, CAT and GPx are considered the "backbone of antioxidant defense

mechanism" (Manoharan et al., 2005 and Fiaschi et al., 2005). Superoxide dismutase (SOD), a

ubiquitous antioxidant, degrades the unstable superoxide anion into less reactive molecular

oxygen and another ROS, hydrogen peroxide (Pham-Huy et al., 2008). Likewise catalase (CAT)

further neutralizes the hydrogen peroxide forming water. Other scavengers belong to the famous

glutathione system that efficiently removes oxidants from the biological system. The four

selenium containing most abundant antioxidant, Glutathione peroxidase (GPx), acts on organic

hydroperoxides and hydrogen peroxide while glutathione pro-oxidase scavenges on lipid

hydroperoxides (Khanna and Karjodkar, 2006 and Bathi et al., 2009). In addition the salivary

enzyme glutathione-S-transferase (GST) possesses anti-neoplastic properties and prevents DNA

damage (Shiboski et al., 2005).

Literature demonstrated that elevated levels of antioxidants during diseased states are due to

the burden on defense system to remove oxidants from the body, yet decreased levels were

linked to the extent of disease progression (Rehman, 2007). Bagul et al. found elevated levels of

SOD and GPx in OSCC whereas in other studies, SOD, CAT and GPx were reported to be

diminished in the same subjects (Manoharan et al., 2005; Gupta et al., 2009; Srivastava et al.,

2012 and Shilpasree et al., 2013). Other studies, indicated that tumor samples showed minimum

levels of SOD and CAT while GPx, glutathione reductase (GR) and glutathione-S-transferase

(GST) were enhanced in blood samples from the same subjects (Subapriya et al., 2002 and

Fiaschi et al., 2005), this conflicts with the study by Gokul et al. which showed decreased SOD

and CAT in both type of samples. A speculating study concluded that SOD levels fluctuate in

different malignant conditions where oral cancer has the most minimum levels (Gurudath et al.,

2012; Shetty et al., 2013 and Hegde et al., 2013). While enhanced levels of metastatic marker

SOD2 was reported to be linked with lymph node metastasis in OSCC (Ye et al., 2008). In

addition, contrasting results were reported in OSCC patients and individuals undergoing

radiation treatment (Sabitha and Shayamaladevi, 1999; Yang et al., 2002; Suprapaneni and

Raman, 2006; Bogdanovic et al., 2008 and Pejic et al., 2009;). Vurumadla et al. observed that

although both chemotherapy and radiotherapy were responsible for the elevation in oxidative


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stress in OSCC, the latter was found to be more responsible in raising oxidative stress. Moreover

the antioxidant enzymes SOD, GPx and CAT were found to be reduced after radiotherapy.

2.6.2 NON-ENZYMATIC ANTIOXIDANTS

Reduced glutathione (GSH), vitamin A, Vitamin C and Vitamin E are known to play

protective roles in multitude diseases. The low molecular weight, intracellular glutathione is the

most abundant non-protein thiol in the body. It performs multiple functions including regulation,

metabolism and defense against scavengers. It is observed that minimized and depressed levels

of GSH have been attributed to setting off a myriad number of diseases and inflammation

(Anderson, 1998 and Wu et al., 2004). Study by Subapriya et al. and Fiaschi et al. reported

raised GSH levels whereas Srivastava et al. found reduced levels of GSH in blood of OSCC

patients. In addition, glutathione levels were reported to be greatly reduced in different stages of

carcinoma (Hegde et al., 2013). Likewise another study showed that oral cancer greatly

minimized the available glutathione in contrast to potential malignant disorders (Shetty et al.,

2013). In addition, Vitamin E, the strong free radical scavenger, was found to be dangerously

reduced in oral cancer (OC) patients (Raghuwanshi et al., 2012). Additionally Vitamin E and

GSH were reported to be diminished in plasma and red blood cells (Manoharan et al., 2005 and

Metgud and Bajaj, 2014). On the other hand, Vitamin C levels were reported to be increased in

OSCC tumor samples but ascorbic acid and iron levels were found to be decreased in oral

submucous fibrosis (Fiaschi et al., 2005 and Guruprasad et al., 2014).

CHAPTER THREE MATERIALS AND METHODS

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MATERIALS AND METHODS


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3.0 MATERIALS AND METHODS

3.1 SOURCE OF DATA

This study was performed to evaluate the role of inflammatory and biochemical stress

markers in pathogenesis of oral cancer. All the patients were selected and screened at Shoukat

Khanum Hospital, Lahore. Fifity patients with diagnosed oral squamous were included in the

study. Informed consent was taken from them before starting this research. Whereas twenty

healthy patients that neven use tobacoo were taken as controls. The protocols that were used to

perform this research were approved by the Research Ethical Committee of The Institute of

molecular biology and biotechnology, The University of Lahore. Five ml of blood sample was

withdrawen from each participant from their cubital vein that was centrifuged within an hour and

stored at -70˚C under specific wiles.

3.2 INCLUSION CRITERIA

Subjects having history of any tobacco use and were diagnosed oral cancer through

biopsy and pathological experiments were included in this study.

3.3 EXCLUSION CRITERIA

Subjects that were on medication, using alcohol or diagnosed with any significant

systemic disease were excluded from the study.

3.4 CHEMICALS

Chemicals that were used for this study to perform experiment were taken from

sigma/Invitrogen Chemical Co. (St. Louis, Mo, USA).

3.5 ESTIMATION OF CATALASE (CAT)

Method of Aebi (1974) was used to measure the levels of Catalase (CAT) from serum.

Protocol was performed using the spectrophotometer. CAT was measured by taking the

absorbance at 230nm. Reaction was started in the cuvette by adding serum sample 0.01PB and

2mM hydrogen peroxide (H2O2). U/g is the unit used to represent the specific activity of

catalase. The absorbance value was then compared with the known value of catalase by making a

standard curve.


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3.6 ESTIMATION OF MELONDIALDEHYDE (MDA)

Levels of MDA were measured in serum sample through calorimeter by using the method

of Ohkawa et al, (1979). In this protocol following chemicals were added in the test tube

containing 200μl sample

200μl of 8.1% SDS, 1.5 ml of acetic acid (CH3COOH) (20%) and 1.5 ml of TBA (0.8%)

then test tube was kept on heapting for 60 min. then test tube was cooled down and 4 ml of n-

butanol was added and was centrifuged for 10 min at 3000 rpm. After centrifugation upper layer

of was separated with pipette and the absorbance was taken at 532nm on spectrophotometer.

3.7 ESTIMATION OF SUPEROXIDE DISMUTASE (SOD)

Kakkar, (1984) method was used to estimate the level of Superoxide dismutase (SOD) in

serum. This protocol was performed in falcon tube, 100μl of serum sample was added along with

sodium phosphate buffer having pH 8.3, , Nitro blue tetrazolium (0.3ml; 300μM),

phenazinemethosulphate (0.1ml; 186μM) and NADH (0.2ml; 750μM). Then falcon tube was

incubated on 30°C for one and a half minute (90 sec) after incubation glacial acetic acid (0.1ml)

was added then 4.0ml of n-butanol was added and centrifuged at 4000rpm for 10 minutes.

Absorbance was taken of upper layer of butanol at 560 nm.

3.8 DETERMINATION OF AOPPs

Method of Witko-Sarsatet al,(1996) was used to perform the protocol of estimation of

Advanced oxidation of protein products (AOPPPs). Levels of AOPPs were estimated using size

exclusion chromatography.

3.9 AGEs PREPARATION AND DETERMINATION

AGEs that are significant markers of inflammation were determined by the method of

Goldin et al., (2006).

3.10 ESTIMATION OF REDUCED GLUTATHIONE (GSH)

Method of Moron et al, (1979) was used to measure the levels of GSH in serum. This

method is used to estimate both oxidized and reduced form of GSH. The absorbance values were

then compared with known value of GSH by making a standard curve (Tietze, 1969).


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3.11 ESTIMATION OF GLUTATHIONE REDUCTASE (GR)

Glutathione reductase was evaluated by using method of David and Richard (1983).

3.12 DETERMINATION OF GLUTATHIONE PEROXIDASE (GPx)

Aebi and Bergmeyer, (1983) method was used to measures the level of glutathione

peroxidase (GPx) through spectrophotometer by using buffer/enzyme reagent.

3.13 DETERMINATION OF NITRIC OXIDE (NO)

Levels of NO were measured using Griess reagent by following the protocol of Bredt and

Snyder, (1994).

3.14 ESTIMATION OF VITAMIN E

Vitamin E was evaluated in samples by the Emmerie-Engel reaction as reported by

Rosenberg (1992).

3.15 DETERMINATION OF VITAMIN-A

Vitamin-A was determined by the method of spectrophotometer as an ingredient of

pharmacopeial preparation as adopted in 1956 IUPAC USPharmacopeial Forum (Lawrence-

Evans, 2015).

3.16 ESTIMATION OF MMP-2, 9 and 11

Levels of MMPs 2, 9 and 11 were measure through commercially available ELISA kit

(BioVendor).

3.17 ESTIMATION OF IL-1α ELISA

IL-1α levels were determined by using human ELISA kit (BioVendor)

3.18 ESTIMATION OF TNF-α ELISA

ELISA Kit ( Enzo Pharma) was used to measures the levels of Tumor Necrosis Factor-

alpha(TNF-α).

CHAPTER FOUR RESULTS

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RESULTS


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4.0 RESULTS

Fifty diagnosed oral cancer patients and twenty healthy subjects were included in the

study. Significant biochemical markers, inflammatory, stress markers and antioxidants were

measured to determine the effect of these markers in oral cancer proliferation.

4.1 CIRCULATING STRESS BIOMARKERS PROFILE OF ORAL CANCER

PATIENTS VS CONTROL

The results regarding stress biomarkers MDA, isoprostanes 8-OGdG, NO, AGES and

AOPPs are shown in table 1. According to table 1 levels of MDA were increased (1.36±0.38

nmol/ml) significantly in patients suffered from OSCC as compared to healthy controls

(3.87±1.10 nmol/ml). Where as the levels of 8OHdG and Isoprostanes those were the byproducts

of lipid peroxidation were increased significantly (P=0.012 and P=0.014) in subjects (68.99±5.29

and 0.95±0.005 pg/ml) in comparison to controls (0.88±0.001 and 0.04±0.003pg/ml

respectively). Levels of NO were measured to evaluate the levels of Reactive Nitrogen species

(RNS) in the body that induce oxidative stress. Table 1 depicts that levels of NO were increased

(55.66±9.15 Um/L) significantly as compared to healthy controls (19.19±1.31 Um/L). AGEs and

AOPPs were measured to evaluate the degenerative diseases and oxidative stress in the body.

Advanced Glycated End products (AGEs) were generated due to glycation of protein and lipids

and these are significant markers of any degenerative disease in the body. Significantly (P=0.065

and P=0.025) increased levels of AOPPs and AGEs were observed in subjects (2.68±0.42 U/ml

and 133.36±31.65 mmol/L respectively) infected with OSCC as compared to healthy subjects

(0.85±0.04 U/ml and 83.05±6.63 mmol/L respectively).

4.2 INFLAMMATORY BIOMARKERS PROFILE OF ORAL CANCER PATIENTS Vs

CONTROL

The results of proinflammatory cytokines Interleukin-1α (IL-1α) and Tumor Necrosis

Factor-α (TNF-α) were shown in table 1. Results present in table 1 depicts that levels of IL-1α

significantly (P=0.032) increased (6.76±0.73 pg/ml) in Oral Cancer patients as compared to

healthy controls (5.68±0.53 pg/ml). Several interleukin were released in result of inflammation.

TNF-α is a significant cell signaling protein that release in case of inflammation and is a

significant stress marker. Results of present study showed that level of TNF-α significantly

(P=0.032) increased (32.72±3.12 pg/ml) in subjects as compared to healthy controls (29.57±1.22

pg/ml).


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The mean value of IL-2, IL6, IL10, IL11, IL13, IL4 in control is 7.29±1.09, 2.99±0.08,

4.28±1.09, 4.29±1.08, 5.29±1.25 and 2.19±0.028 pg/ml respectively but the value of this

inflammatory markers was increased in diseased patients like 10.26±2.09, 4.26±1.09,

5.29±1.114, 8.28±2.19, 11.29±3.28 and 4.29±0.03 respectively. Hence, pointing towards an

increasing trend among oral cancer patients. A statistically highly significant result was observed

for ILs (p<0.05).

The mean value of MMP-1 and MMP-2 in controls is 42.29±11.29, 32.58±7.18 and that

of diseased patients is 98.26±14.56, 56.30±5.42 respectively. Therefore, a statistically increasing

trend was observed in oral cancer individuals (p-value=0.035 and p-value=0.023). In addition,

the mean value of MMP-13, MMP-9, MMP-11 and MMP-8 in healthy individuals is 53.26±5.26,

51.13±7.78, 45.60±9.67 and 56.29±4.28 but in oral cancer patients this value is increased to

76.29±7.59, 60.46±5.95, 65.46±4.16 and 144.29±15.25 respectively.

4.3 ANTIOXIDANT BIOMARKERS PROFILE OF ORAL CANCER PATIENTS Vs

CONTROL

The antioxidant biomarkers SOD, Catalase, vitamin E, vitamin C, vitamin D, vitamin A,

GPx, GRx and GSK3 beta are shown in table 1. In controls the mean value of SOD and catalase

was observed like 0.62±0.05 U/ml, 3.29±1.08 U/L but in diseased patients showed a mean value

was 0.32±0.05 U/ml and 0.40±0.04 respectively. An overall decreasing trend was observed in

SOD and catalase levels between OC patients and controls and its results was very significant

like (p-value=0.32 and p-value=0.22). Additionally, the mean value of GSH in controls and

patients is 7.56±2.16 mg/dl and 4.26±1.08 mg/dl respectively. An overall decreasing trend was

also observed in GSH levels between diseased patients and controls. The mean value of vitamin

A, C, E and D in controls is 62.08±4.91 nmol/L, 0.65±0.07 nmol/L, 0.19±0.06 nmol/L and

14.17±2.93 ng/ml respectively but in OC patients it values was decreased like 45.64±9.08

nmol/L, 0.42±0.01 nmol/L, 0.11±0.01 nmol/L and 10.11±1.13 ng/ml respectively. According to

the present study all these levels were significantly among healthy controls and patients having

P<0.05. Levels of GPx and GRx were also measured to evaluate the role of antioxidants in

patients infected with OSCC and results from table 1 describes that levels of GPx and GRx were

differs significantly in patients (1.59±0.29 and 7.30±1.19 µmol/L respectively) as compared to

healthy controls (6.50±0.47 and 3.09±1.28 µmol/L respectively). Levels of uric acid, iron and

copper for controls was found to be 4.26±1.06, 121.09±4.29 and 115.29±9.19 whereas in patients


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diagnosed OSCC showed 1.09±0.01, 109.65±8.19 and 151.93±5.88 µg/dl respectively. Results

of present study were significantly different among healthy and controls (p<0.05).

TABLE: 01 LEVEL OF DIFFERENT VARIABLES AND THEIR INTERPLAY IN THE

DEVELOPMENT OF OSCC

VARIABLES CONTROL (n=100) SUBJECT (n=247) P- VALUE (0.05)

MDA nmoles/ml 1.36±0.38 3.87±1.10 0.016

Isoprostanes (pg/ml) 0.88±0.001 68.99±5.29 0.012

8-OHdG (pg/ml) 0.04±0.003 0.95±0.008 0.014

SOD (U/ml) 0.62±0.05 0.32±0.05 0.032

GSH (μmol/L) 7.56±2.16 4.26±1.08 0.012

CAT (U/L) 3.29±1.08 0.40±0.04 0.222

IL-1 (pg/ml) 5.68±0.53 6.76±0.73 0.032

IL-2 (pg/ml) 7.29±1.09 10.26±2.09 0.032

IL-6 (pg/ml) 2.99±0.08 4.26±1.09 0.012

IL-10 (pg/ml) 4.28±1.09 5.29±1.14 0.152

IL-11 (pg/ml) 4.29±1.08 8.28±2.19 0.011

IL-13 (pg/ml) 5.29±1.25 11.29±3.28 0.000

IL-4 (pg/ml) 2.19±0.02 4.29±0.03 0.016

TNF-α (pg/ml) 29.57±1.22 32.72±3.12 0.033

AOPPs (mmol/L) 83.05±6.6 133.36±3.65 0.025

AGEs (AU) 0.85±0.04 2.68±0.42 0.065

MMP-1 (ng/ml) 42.29±11.29 98.26±14.56 0.023

MMP-2 (ng/ml) 32.58±7.18 56.30±5.42 0.035

MMP-8 (ng/ml) 53.26±5.26 76.29±7.59 0.026

MMP-9 (ng/ml) 51.13±7.78 60.46±5.95 0.032

MMP-13 (ng/ml) 56.29±4.28 144.29±15.25 0.054

MMP-11 (ng/ml) 45.60±9.67 65.46±4.16 0.036

Vit-A (nmol/L) 62.08±4.91 45.64±9.08 0.011

Vit-C (nmol/L) 0.65±0.07 0.42±0.011 0.032

Vit-E (nmol/L) 0.19±0.06 0.11±0.018 0.041

Vit-D (ng/ml) 14.17±2.93 10.11±1.13 0.021

NO (μmol/L) 19.19±1.31 55.66±9.15 0.019

GPx (μmol/L) 1.59±0.29 6.50±0.47 0.032

GRx (μmol/L) 7.30±1.19 3.09±1.28 0.032

Uric Acid 4.26±1.06 1.09±0.018 0.032

Iron (µg/dl) 121.09±4.29 109.65±8.19 0.021

Cu(µg/dl) 115.29±9.19 151.93±5.88 0.032

GSK3 beta (GSK3B) ng/ml 75.29±8.26 33.26±4.19 0.000


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FIGURE: 01 EXPRESSION OF DIAGNOSTIC VARIABLES OF MEDICAL IMPORTANCE AND THEIR

INTERPLAY IN THE DEVELOPMENT OF ORAL SQUAMOUS CELL CARCINOMA (OSCC)

0

1

2

3

4

5

6

Control Subject

MD

A (

nm

ole

s/m

l)

P=

0.0

16

0

10

20

30

40

50

60

70

80

Control Subject

Iso

pro

sta

nes

(p

g/m

l)

P=

0.0

12

0

0.2

0.4

0.6

0.8

1

1.2

Control Subject

8-O

hD

G (

pg

/ml)

P=

0.0

14

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control Subject

SO

D (

U/m

l)

P=

0.0

32

0

2

4

6

8

10

12

Control Subject

GS

H (

µm

ol/

L)

P=

0.0

12

0

1

2

3

4

5

Control Subject

CA

T (

U/L

)

P=

0.0

22


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0

1

2

3

4

5

6

7

8

Control Subject

IL-1

(p

g/m

l)

P=

0.0

32

0

2

4

6

8

10

12

Control Subject

IL-2

(p

g/m

l)

P=

0.0

32

0

1

2

3

4

5

6

Control Subject

IL-6

(p

g/m

l)

P=

0.0

12

0

1

2

3

4

5

6

Control Subject

IL-1

0 (

pg

/ml)

P=

0.0

15

0

2

4

6

8

10

12

Control Subject

IL-1

1 (

pg

/ml)

P=

0.0

11

0

2

4

6

8

10

12

14

16

Control Subject

IL-1

3 (

pg

/ml)

P=

0.0

00


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0

1

2

3

4

5

Control Subject

IL-4

(p

g/m

l)

P=

0.0

16

26

27

28

29

30

31

32

33

34

Control Subject

TN

F-α

(pg

/ml)

P=

0.0

33

0

20

40

60

80

100

120

140

160

180

Control Subject

AO

PP

s (m

mo

l/L

)

P=

0.0

25

0

0.5

1

1.5

2

2.5

3

3.5

Control Subject

AG

Es

(AU

)

P=

0.0

65

0

20

40

60

80

100

120

Control Subject

MM

P-1

(n

g/m

l)

P=

0.0

23

0

10

20

30

40

50

60

70

Control Subject

MM

P-2

(n

g/m

l)

P=

0.0

35


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0

10

20

30

40

50

60

70

80

90

Control Subject

MM

P-8

(n

g/m

l)

P=

0.0

26

0

10

20

30

40

50

60

70

Control Subject

MM

P-9

(n

g/m

l)

P=

0.0

32

0

20

40

60

80

100

120

140

160

180

Control Subject

MM

P-1

3 (

ng

/ml)

P=

0.0

45

0

10

20

30

40

50

60

70

80

Control Subject

MM

P-1

1 (

ng

/ml)

P=

0.0

36

0

10

20

30

40

50

60

70

80

Control Subject

Vit

-A (

nm

ol/

L)

P=

0.0

11

0

0.1

0.2

0.3

0.4

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0.6

0.7

0.8

Control Subject

Vit

-C (

nm

ol/

L)

P=

0.0

32


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0

0.05

0.1

0.15

0.2

0.25

Control Subject

Vit

-E (

nm

ol/

L)

P=

0.0

41

0

2

4

6

8

10

12

14

16

18

Control Subject

Vit

-D (

ng

/ml)

P=

0.0

21

0

10

20

30

40

50

60

70

80

90

Control Subject

GS

K3

-β(n

g/m

l)

P=

0.0

00

0

10

20

30

40

50

60

70

Control Subject

NO

(µ

mo

l/L

)

P=

0.0

19

0

1

2

3

4

5

6

7

8

Control Subject

GP

x (

µm

ol/

L)

P=

0.0

32

0

1

2

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5

6

7

8

9

Control Subject

GR

x (

µm

ol/

L)

P=

0.0

32


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0

1

2

3

4

5

6

Control Subject

Uri

c A

cid

(m

g/d

l)

P=

0.0

32

95

100

105

110

115

120

125

130

Control Subject

Iro

n (

µg

/dl)

P=

0.0

21

CHAPTER FIVE DISCUSSION

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DISCUSSION


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5.0 DISCUSSION

Significant biomarkers play a key role in the diagnosis of various diseases. Inflammation

and oxidative stress are the major contributors in OSCC disease. Oxidative stress and

inflammatory markers such as MDA, ILs and MMPs have very high demand because they play

key role in the proliferation and is a prognostic variables in various diseases. Early diagnosis of

OSCC by these inflammatory and stress markers provides a new hopoe to overcome this disease

through different treatment regimes (Patton et al., 2008 and Mehrotra and Gupta, 2011). Oral

squamous cell carcinoma (OSCC), currently a global dilemma, is an aggressive malignant

disorder which is increasing at a threatening pace. It has become a serious lethal problem for

developing countries because of late diagnosis of this disease and have poor survival rate (Yi-

Shing and John, 2011). Oral cancer is a slow process that composed of metastasis and tumor

invasion, this disease results in inflammation and triggering angiogenesis. Carcinogenesis is

owing to the interaction between cancerous cells and cells in vicinity. The interplay of cytokines,

chemokines, interleukins, ROS, angiogenic factors and MMPs sets off OSCC. The oral

epithelium is bombarded with unlimited free radicals by the consistent use of tobacco, tobacco

related products and alcohol. Mitochondria, an ROS generating organelle, through the

mitochondrial respiratory chain and mitophagy, leads to the direct and indirect generation of

intermediates of reactive nitrogen (RNI) and reactive oxygen (ROI) under aerobic and hypoxic

conditions. Moreover it initiates hypoxia induced nitrative stress and fabricates lipid peroxidation

byproducts 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). In addition, inflammatory

cytokines, chemokines and processed proteins activate a wide range of transcription factors,

hence indirectly initiating oxidative stress. In OSCC both oxidative stress and inflammation

induce and trigger one another that result in more and more OSCC as shown in the figure 01.

Lipid peroxidation occurs because of oxidative stress, its markers such as MDA, 4-HNE,

Isoprostanes and 8OHdG describes the damage done to lipid membranes. This highly unstable

product of lipid peroxidation has also been known to form DNA-MDA adducts hence causing

collateral damage to the cellular DNA. A number of diseases have shown the presence of lipid

peroxidation as a key factor in their pathogenesis. Numerous studies on oral cancer individuals

have reported elevated MDA and lipid hydroperoxides (LHP) in blood, serum, saliva and tumor

samples. Present study showed a significantly increased MDA level in serum of oral cancer

patients as compared to healthy controls (Shetty et al., 2014 and Thomas and Sethupathy, 2015).


SAIMA, 2017

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Results of present study describes that oral cancer release free radicals that induce cancer on the

other hand OSCC tumor itself participate in the generation of free radicals that leads to cause

decrease in antioxidants and results in lipid peroxidation. Results of present studies are similar

with the studies of Kurtul and Gokpinar, 2012; Agha-Hosseini et al., 2012; Srivastava et al.,

2012; Shilpasree et al., 2013; Ganesan and Kumar, 2014; Rasool et al., 2014; Metgud and Bajaj,

2014 and Malik et al., 2014). Nitric oxide is a key player in redox biology yet its unexplained

massive production or defective regulation has surfaced as contributors of cancer onset. The

multifaceted signaling molecule has both tumor promoting and tumor suppressive properties but

its function depends largely on when, where and in what quantity it is being produced. Deranged

levels of NO elicit various effects on the body which include direct and indirect effects.

Importantly, NO indirectly facilitates both oxidative and nitrosative stress. Also it facilitates

angiogenesis (Connelly et al., 2005 and Choudhari et al., 2012). Results of present study showed

significantly increased levels in oral squamous cell carcinoma patients as compared to controls

and this strongly suggests the massive production of NO from the reaction of L-arginine and

molecular oxygen and unchecked regulation of nitric oxide synthase (NOS) that facilitates this

reaction. It also points towards the fact that the more nitric oxide available, the more it will react

with superoxide ion or molecular oxygen to form peroxynitrite. Various inflammatory cells also

release NO in advanced stages of cancer and greater iNOS levels have also been reported in OC

patients. In addition, at one point the RNS peroxynitrite degrades into potential harmful products

known to interact with DNA. During long run, NO play key role in the induction nitrostative

stress, lipid peroxidation and NO mediated oxidative stress. Furthermore high concentration of

NO leads to induce high pain because NO induce several signaling pathways that leads to cause

activation of pain receptors. According to the results of present study it shows that NO has

significant relation in promoting tumor in oral cancer. Present study results were in accordance

with the studies of (Beevi et al., 2004; Bahar et al., 2007 Rasheed et al., 2007; Gokul et al., 2010

and Korde et al., 2011).

The novel OS marker AGES and AOPPs have emerged as key players in triggering OC.

Like lipids, cellular proteins are viable to free radical attacks during oxidative stress. Albumin

being the widely present protein is the most affected protein in the body. Primarily the

chlorinated oxidants, hypochlorous acid and chlorinated amines, are the products of

myeloperoxidase (MPO) system which cause protein damage resulting in generation of modified


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proteins called AOPPs (Liu et al., 2006). In this study, significantly increased levels of advanced

oxidation of protein products (AOPPs) were found in patients as compared to controls. This

suggests that in patients myeloperoxidase system is relatively contributing to the formation of

huge amounts of hypochlorous acid. Excessive accumulation of this specific ROS causes it to

react with the most abundant protein albumin, oxidizing it to form AOPPs which further triggers

NADPH oxidase that leads to inflammation and oxidative stress. Results of present studies

showed that pateints diagnosed with oral cancer have high level of AOPPs as compared to

AGEs, it proves that AOPPs has major role in releasing inflammatory mediators in OC pateints.

Present results coincided with the study of (Barut et al., 2012).

Advanced glycation end products (AGEs) also provide strong evidence that it is involved

in the oxidative stress of the body. Sugars molecules that are present in the body show various

changes with time, during aging it generates potential compounds that combines with

biochemical compounds of the body and results in oxidative stress and inflammation in the body.

Lippid peroxidation will also release modified sugar through membrane of cell and proliferation

that have much poly unsaturated fatty acids (PUFA) (Kalousova et al., 2005). Levels of AGEs

were significantly high as compared to healthy controls. These results shows that lipid

peroxidation and oxidative stress are the major role player that induce damage to carbohydrates,

lipids and protein, that generates AGEs that results in inflammation. AGEs that are generated in

response of inflammation will activate NOx that leads to the activation of myeloperoxidase

(MPO) system. MPO cause release of OHOO that combines with protein albumin that

synthesize AOPPs. That’s why AGEs and AOPPs are the major factor to induce tumor in OSCC

patients. Other than AGEs and AOPPs, oral cancer can also proliferate due oxidative stress,

nitrative stress, nitrosative stress and lipid peroxidation. Nature has granted the cell with several

enzymatic and non-enzymatic antioxidants such as GPx, GRx, GSH, SOD, Vitamins A, C and E.

Several studies suggests that reduced activation of these antioxidants is the major reason behind

oral cancer.

SOD, a devourer of O2-, plays crucial role in preventing diseased states. This widely

present antioxidant in the body acts to convert highly reactive superoxide to molecular oxygen

and hydrogen peroxide so that the latter may be decomposed into less toxic and reactive

substances i.e water and molecular oxygen (Pham-Huy et al., 2008). Present study showed

significantly reduced levels in oral cancer patients as compared to healthy controls. Decreased


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levels of SOD lead to the release of high levels of endogenous ROS in the body of patient those

results in generation of peroxynitrite by reaction of superoxide with NO. Present results were in

accordance with the studies of Manoharan et al., (2005), , Gupta et al., (2009), Gokul et al.,

(2010), Srivastava et al., (2012), Gurudath et al., (2012), Shilpasree et al., (2013), Shetty et al.,

(2013) Hegde et al., (2013), Vurumadla et al., (2014) and Thomas and Sethupathy (2015).

CAT enzyme involves in the conversion of ROS hydrogen peroxide in to water and

oxygen. In present study levels of CAT reduced singnificantly in subjects infected by oral cancer

as compared to healthy controls. These results show that CAT has negative correlation with

hydrogen peroxide that leads to induce oxidative stress in the body. Beevi et al. suggests that

catalase deficiency is might be due to high production of ROS from molecular oxygen and RNS.

Furthermore, it is to be suggested that free radicals may cause reduction in antioxidants such as

SOD and GPx that leads to decrease levels of catalase in OC patiens. Results of presents study

are resembles with the results of Manoharan et al, (1996); Kolanjiappan et al, (2003); Beevi et

al., (2004); Manoharan et al, (2005); Rasheed et al, (2007); Gokul et al, (2010); Srivastava et al,

(2012) and Thomas and Sethupathy, (2015)

The powerful antioxidant glutathione (GSH) is a resident of cytosol, peroxisomes and

mitochondria. In present study, the levels of GSH were greatly reduced in oral cancer patients

and these results coincided with the results of (Shivashankara and Prabhu, 2011; Srivastava et

al., 2012; Hegde et al., 2013; Shetty et al., 2013; Vurumadla et al., 2014 and Metgud and Bajaj,

2014 and Thomas and Sethupathy, 2015). Present results suggest that most of the GSH levels

may have been utilized to eliminate circulating oxidative stress causing free radicals (hydroxyl,

superoxide ion, hydrogen peroxide, lipid peroxides) or it may have been taken up by the tumor

itself as nutrition. Moreover, compromised state will generated due to decreased levels of GSH

that fails to produce antioxidants alpha-tocopherol and ascorbic acid in the body. Glutathione

peroxidase enzyme is a part of glutathione system. It catalyzes decomposition of hydrogen

peroxide (Khanna and Karjodkar, 2006 and Bathi et al., 2009). In present study levels of GPx

were increased in OSCC patients that suggest that levels of antioxidants such as SOD and CAT

were decreased in OC diagnosed patients. GPx plays a key role in reducing oxidative stress and

reduce hydrogen peroxide to molecule that is less toxic. Furthermore high activity of GPx will

converts GSH into oxidized form that leads to induce oxidative stress. Also it shows that

although the body is under attack of free radicals, it still manages to fight back by fueling its


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antioxidant defense mechanism to counter-act the debilitating effects of free radicals. Present

results were in accordance with the studies of (Bagul et al., 2013).

GRx is the enzyme glutathione reductase that is involved in catalyzing oxidized form of

glutathione (GSSG) to its reduced form (GSH). Present study results showed decreased levels in

oral cancer which contradicts with the study results of (Fiaschi et al., 2005). Present results

revealed that reduced level of SOD, CAT and GSH and high level of GPx that release hydrogen

peroxide. This leads to the initiation of disturbance in the normal GSH to GSSG ration, that

cause increased GSSG levels as compared to GSH that puts pressure on GRx to generate more

GSH levels to overcome oxidative stress. During high ROS levels GRx tries to convert oxidized

GSH into reduced GSH but due to high level of ROS GRx levels gets reduced. Vitamin A,

originally stored in the liver, is considered a good antioxidant and apart from its various

functions, having a key role in normalizing immune system. Normally vitamin-A is synthesized

by macrophages and dendritic cells of mucosa and associated lymph nodes. CD4 T-cells were

converted into T-helper cell due to inflammation that leads to release of many pro inflammatory

cytokines (Green and Mellanby, 1928 and Raverdeau and Mills, 2014). In present study vitamin

A levels were found to be decreased in OC patients and this coincided with the results of

(Ramaswamy et al., 1990). Decreased level of Vit-C depicts that patients are mostly encounterd

with infection. Furthermore less amount of Vitamin-A will release proinflammatory cytokines

that will induce inflammation leading to oxidative stress through NADPH oxidase.

Vitamin E reduces the levels of hydroxyl radicals. Reducede level of vitamin-E also

induce inflammation in the body. Normal function of vitamin E is to minimize wound healing

time, maintaining the structure and function of membranes. Additionally, it plays role in

reversing premalignant lesions. Normal levels of vitamin E in the body prevent onset of disease.

It hinders cancer onset by stopping the growth of cancer cells and sending them to apoptotic

pathways. The release of cytotoxic cytokines causes apoptosis of these cancerous cells, hence

maintaining the equilibrium of normal and neoplastic cells. The results of present study show

reduced levels in oral cancer patients as compared to healthy controls. This implies that the

decreased levels are due to oral cancer, the tumor might have taken up antioxidants as nutrition.

These low levels also led to more accumulation of hydroxyl radicals, which in turn resulted in

more lipid peroxidation hence disturbing the cellular integrity and eventually leading to

enhanced chances of oral cancer development. Present results contradicted with the study of


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40

Kolanjiappan et al. but were in accordance with the respective studies (Manoharan et al., 1996;

Manoharan and Nagini, 1996; ; Halliwell 1996 Subapriya et al., 2002; Kolanjiappan et al., 2003;

Manoharan et al., 2005; and Raghuwanshi et al., 2012).

The members of Toll/interleukin-1 receptor (TIR) family TLR1, TLR2, TLR4, TLR5

and TLR6 are found in plasma membrane and TLR 3, TLR7, TLR8 and TLR 9 reside in

intracellular compartments (Akira et al., 2001 and O'Neill et al., 2013). The cytoplasmic TIR

domain of TLR4 via IRAK4/TRAF6/IRAK1 activates NF-kB and MAPK signalling cascades,

producing inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α (Vogl et al., 2007 and Kawai

and Akira, 2010). TLR9 through MyD88 pathway activates IRF-5, hence producing chemokine

IL-8 (Jozsef et al., 2006). TLR3 through adaptor molecule TRIF (TIR domain-containing

adaptor inducing interferon-β) activates various IRFs and release cytokines IFN alpha and beta.

While the remaining TLRs, through adaptor molecule MyD88 dependent pathway, activate a

myriad of intercellular signaling proteins resulting in activation of numerous transcription factors

hence initiating various signaling cascades. DAMPs and PAMPs, released by pathogens and

oxidative stress, are sensed by impeccable PRRs leading to activation of TLRs and NLRs. The

widely available TLRs give off reactive species at multiple stages containing mitochondrial,

activated NOX and transcription factor (IRF3, AP-1 and NF-κB) mediated liberation of

ROS/RNS. The cytosolic NLR by means of NLR/ASC/casp1 complex triggers release of

proinflammatory cytokines IL-18 and IL-1β. Additionally free radicals can hamper

inflammosome, shackling NLR signalling. Only TLR3 via adaptor molecule TRIF activates

IRF3 and IRF7, eventually generating cytokines. While the remaining MyD88-dependent TLRs

activate myriad intercellular signaling proteins and numerous transcription factors, triggering

various signaling cascades and finally directing the release of inflammatory cytokines,

chemokines, gelatinases or marking the cell for apoptosis.

The crucial pro-inflammatory cytokine 1L-1alpha is detectable in both normal and

dysregulated inflammatory processes. It is one of the earliest cytokines to be released by

inflammatory cells at the slightest sign of inflammation. IL-1α shows both beneficial and

harmful effects in the body normal amount of IL-1α is involved in healing of wounds and tissue

repair but in diseased case this cytokines induce several chronic diseases (Chedid et al., 1994;

Hubner et al., 1996 and Margioris 2009). Present study results showed significantly increased

levels of this cytokine in oral cancer patients, implying that deregulated inflammation is present


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in OSCC. Present results shows that a large amount of cytokines been released by inflammatory

cell such as fibroblast, neutrophils and macrophages that were activated by tobacco, alcohol or

stress. That’s why IL-1α plays a key role in causing tumor progression and metastasis during

OSCC. In present patients enhanced oxidative stress led to activation of this cytokine. Present

study results coincided with the study of Rhodus et al. 2005 and Saheb-Jamee et al. 2008. Tumor

necrosis factor-alpha (TNF-α) is a specific cytokine that can initiate various signaling pathway

and having pro-angiogenic and pro-inflammatory functions. The cells releasing this cytokine are

involved in myriad functions of the cell including immunity, inflammation and defense against

infection. Moreover it has role in anti-inflammatory and immunoregulatory activities. It shows

its response in each stage of carcinogenesis. TNF-α is an angiogenesis regulator and is a

promoter of tumor formation, having tumor necrotic and tumor promoting activities in the body.

This multifunctional cytokine controls inflammatory cell populations and also mediates many

aspects of inflammatory process. Controversial levels of TNF-α have been reported in oral

cancer patients. These results were in accordance with the study of Krishnan et al., Jablonska et

al., Kurokawa et al., Nakano et al., Saheb Jamee et al., Rhodus et al., and Brailo et al.

(Jablonska et al., 1997; Kurokawa et al., 1998; Nakano et al., 1999; Rhodus et al., 2005; Brailo

et al., 2006; Saheb Jamee et al., 2008 and Krishnan et al., 2014). Present results imply that this

cytokine has role in oral cancer prognosis. It is taking part in inflammatory responses and

promoting angiogenesis.

The tumor microenvironment comprises of cancer cells and associated stromal

components. In OSCC, activated MMP-9 released from cancer cells and inflammatory cells

through CD44 degrades ECM. Meanwhile activated MMP-2 released from Carcinoma-

associated fibroblasts (CAFs) via αVβ6 pathway up regulates MMP-9. Both gelatinases break

down ECM, assist tumor invasion and release inflammatory markers. Release of MMPs,

cytokines, chemokines and ROS will activate various transcription factors that leads to the start

of signaling cascade. OSCC progression occurs through a cascade of angiogenesis followed by

EMT that were activated by growth factors. The action of gelatinase B as pro-angiogenic and

anti-angiogenic regulates the crucial step of angiogenesis. MMPs are good biomarkers of oral

squamous cell carcinoma as reported by Wonga et al. (Wonga et al., 2014). The role of various

MMPs as ECM digesters has been known in different diseases. MMP-11, also known as

stromelysin-3, does not cleave the extracellular matrix but it is suspected to have a unique role in


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tumor development and progression. Elevated levels of MMP 11 have been seen in most human

cancers. Importantly, MMP 11 is processed intracellularly and secreted as an active enzyme.

Present results show increased levels of MMP 11 in oral cancer patients; this result contradicts

with the study of Hsin et al. but coincides with the results of (Soni et al., 2003 and Arora et al.,

2005 and Hsin et al., 2014). The increased levels of MMP 11 throw light on the fact that that

carcinoma associated fibroblasts have MMP 11 reserves in them. As CAFs have unique role in

OSCC, therefore these specialized cells are involved in the release of MMP 11 in oral cancer. In

addition, these increased levels leads to conversion from precancerous to malignant form.

Gelatinases are secreted as inactive enzymes which can be effective only after activation.

On the other hand MMP 11 does not need to be activated by certain enzymes. MMP 2 and MMP

9 degrade the extracellular matrix. Present study results show that gelatinases were elevated in

oral cancer patients as compared to controls, this matched with the study of (Ikebe et al., 1999;

Kato et al., 2005 and Lee et al., 2008 and Katayama et al., 2004). Importantly, the levels of

MMP 9 were found to be more enhanced than the levels of MMP 2 in OC patients. This

coincided with the study of (Mishev et al., 2014). Increased level of MMP-9 in OC patients

shows its important role in proliferation of cancer; this is similar with the study of Ravi et al.,

2014. Increased level of MMP shows that rather it get over expressed by cancerous cell or

increase release of inflammatory cytokines that trigger their production from host stromal cells.

Additionally the more secreted levels of gelatinases, the more ECM degradation will occur hence

leading towards tumor invasion and angiogenesis.

The study results show a strong positive correlation of MDA with CAT (r= 0.460**, p=

0.001) and NO (r=0.453**, p=0.001). SOD showed a strong positive correlation with vitamin E

(r=0.283*, p=0.046), MMP 9 (r=0.462**, p=0.00), MMP11 (r=0.356, p=0.011), GPx (r=0.413**,

p=0.003) and GRx (r=0.285, p=0.045) whereas an inverse relation of SOD with GSH (r=-0.320*,

p=0.024) was observed. GSH showed a strong postive correlation with catalase (r=0.536**,

p=0.000) and TNF-α (r=0.297*, p= 0.036) whereas an inverse correlation of GSH exists with

vitamin E (r=-0.281*, p= 0.048) and MMP-9 (r=-0.422**, p= 0.002). Catalase showed a strong

positive correlation with MMP 9 (r=-0.284, p= 0.045). The cytokine IL-1α showed a positive

correlation with MMP 2 (r=0.323*, p=0.022). GPx showed a strong positive correlation with

MMP 9 (r=0.311*, p= 0.028). In addition AGEs showed a strong positive correlation with MMP

2 (r= -0.306*, p= 0.03) and MMP 9 showed a positive correlation with MMP 11 (r= 0.415**, p=


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43

0.003). All these parameters have a direct correlation with each other (p<0.05) except SOD vs.

GSH, GSH vs. Vit. E, GSH vs. MMP 9, Cat vs. MMP 9 and AGEs vs. MMP 2 which has an

inverse correlation (p>0.05). Moreover all values were found to be statistically significant.

Additionally MDA vs. CAT, MDA vs. NO, SOD vs. GPx, SOD vs. MMP 9 and GSH vs. CAT

showed highly significant levels (p<0.01).


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45

FIGURE 07: PATHWAY INVOLVED IN PATHOGENESIS OF (OSCC). The inhibition of apoptosis is the hallmark of oral cancer pathogenesis which is controlled by several mitochondrial

(intrinsic) or by activation of pro-Caspase 8 (extrinsic) pathways. Higher levels of Bcl-2 (anti-apoptotic protein) and lower level of Bax (apoptotic protein) are frequently reported in oral cancer. The

expression of Bcl-2 in OSCC cell lines is affected by minor alterations in the status of pSer9GSK3β. Active GSK3β blocks CREB dependent expression of Bcl-2 (anti-apoptotic protein). The

increase in the expression of Bax protein is regulated by p53 activity resulting in the initiation of apoptosis. But the modulation of several therapeutic molecules like GSK3β and other active

apoptotic proteins increase p53 dependent activation of Bax resulting in the release of cytochrome C from the mitochondrial matrix causing loss of mitochondrial membrane potential and progression

of Caspase 9. Protein kinase A has been identified as the therapeutic target in OSCC because it affects the signaling of many molecules e.g NF-kβ, Cyclin D1, Bcl-2, COX-2 & p21 resulting in the

onset of OSCC. PI3K/Akt pathway has been known to play a significant role in oral squamous cell carcinoma initiation. pSer9GSK3β (oncogenic protein) level in OSCC cell lines is very high and

can be blocked by inhibiting Akt signaling. Protein Kinase C signaling also suggests its role in oral cancer progression as the inhibition of PKC reduces MMP-2, MMP-9 & COX-2. Suppression of

PKC activity promotes Akt signaling which increases apoptosis of epithelial cells. Therefore targeting of PKC has shown promising results in decreasing the invasion and mortality of OSCC cell

lines. Moreover p90RSK has also been studied for its role in epithelial cell motility and invasiveness. Tumor promoting phorbol esters inhibit p70RSK by activating p90RSK (MAPKAP-K1) might

promote oral cancer. GSK3β regulated c-Myc is the master regulator of cell cycle essential for the progression of G0/ G1 to S phase. C-Myc also activates cyclin D1,E1 and A2, CDK4, CDC25A and

E2F-1 and 2.Cyclin D1 is a crucial cell cycle regulator mainly regulated by NK-kβ, β-catanin-TCF/LEF, AP1. But the inactivation of above said oncogenic regulators leads to the stabilization of

Cyclin D1. Oncogenic gains of function of these anti-apoptotic molecules have been established to deregulate cell cycle in OSCC. Normal epithelial cells are connected to each other by E-cadherin

which in turn connects to the actin cytoskeleton. Epithelial mesenchymal transition (EMT) is caused by decreased level of E-cadherin, which is suppressed by snail. MMPs and COX-2 degrade the

intact basement membrane and facilitate the migration of cancer cells. These upregulated MMPs, MDA, IL-2,6 & COX-2 are controlled by various anti-inflammatory biomarkers NF-kβ, AP1, α/ β

integrin and various antioxidants such as Vitamin C ,Vitamin E, GSH, SOD and Catalase, which maintain the balance between proliferation and death resulting in tissue homeostasis in normal

epithelium which might be disturbed in OSCC.


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TABLE 02: PEARSON CORRELATION (TWO-TAILED) IN ORAL SQUAMOUS CELL CARCINOMA (OSCC)

VAR. MDA SOD GSH CAT VIT.E NO IL-1α TNFα GPx GRr AGEs AOPPs VIT- A MMP2 MMP9 MMP11 8-OHdG IL-6 GSK3β Uric Acid

MDA 1

-0.132

0.362

0.236

0.099

0.460**

0.001

0.045

0.758

0.453**

0.001

-0.008

0.959

0.082

0.572

-0.012

0.935

-0.008

0.957

-0.229

0.109

-0.047

0.745

-0.032

0.824

0.019

0.894

-0.164

0.254

-0.084

0.562

0.826

0.00

0.629

0.031

-0.509

0.01

-0.659

0.016

SOD 1 -0.320*

0.024

-0.045

0.755

0.283*

0.046

0.193

0.180

0.020

0.892

-0.106

0.464

0.413**

0.003

0.285*

0.045

0.066

0.648

-0.020

0.889

0.070

0.630

-0.210

0.143

0.462**

0.001

0.356*

0.011

-0.623

0.02

-0.549

0.019

0.629

0.03

0.561

0.031

GSH 1

0.536**

0.000

-0.281*

0.048

-0.263

0.065

0.081

0.578

0.297*

0.036

-0.195

0.176

-0.219

0.127

0.035

0.811

0.028

0.847

-0.017

0.908

0.114

0.430

-0.422**

0.002

-0.189

0.188

0.426

0.03

-0.619

0.043

0.406

0.016

0.465

0.015

CAT 1

0.022

0.880

0.081

0.575

-0.057

0.696

0.263

0.065

-0.101

0.487

-0.116

0.421

0.016

0.914

0.078

0.592

-0.139

0.337

-0.056

0.700

-0.284*

0.045

-0.128

0.377

0.426

0.05

-0.459

0.014

0.506

0.001

0.629

0.001

VIT.E 1 0.217

0.130

-0.109

0.452

-0.072

0.661

0.258

0.070

0.093

0.522

0.024

0.868

-0.242

0.091

-0.136

0.345

-0.162

0.261

-0.022

0.882

-0.019

0.896

0.626

0.06

-0.455

0.06

0.406

0.06

0.265

0.56

NO 1 -0.176

0.222

-0.161

0.264

0.180

0.211

0.030

0.836

-0.093

0.520

-0.133

0.356

-0.151

0.296

-0.140

0.333

0.078

0.591

-0.119

0.409

0.406

0.15

0.629

0.04

0.619

0.04

-0.569

0.015

IL-1 1 -0.003

0.986

-0.026

0.860

-0.253

0.076

0.135

0.350

0.092

0.524

0.179

0.213

0.323*

0.022

0.091

0.531

0.226

0.114

0.826

0.00

0.466

0.056

-0.628

0.016

-0.626

0.056

TNFα 1 -0.221

0.171

0.138

0.394

-0.062

0.703

-0.063

0.701

0.149

0.357

-0.112

0.491

0.232

0.149

0.302

0.058

7.59

0.04

0.629

0.016

-0.826

0.00

-0.765

0.016

GPx 1 0.263

0.065

0.052

0.721

-0.119

0.411

0.223

0.119

-0.169

0.242

0.311*

0.028

0.195

0.174

-0.619

0.03

-0.529

0.264

0.529

0.16

0.658

0.01

GRr 1 -0.057

0.692

0.053

0.715

-0.015

0.918

0.074

0.608

0.195

0.175

0.179

0.214

-0.526

0.01

-0.426

0.01

0.496

0.16

0.465

0.016

AGEs 1 0.127

0.379

0.035

0.808

-0.306*

0.030

0.078

0.589

-0.055

0.704

0.666

0.028

0.426

0.03

-0.495

0.017

-0.165

0.016

AOPPs 1 0.062

0.671

0.083

0.566

0.256

0.072

-0.139

0.336

0.529

0.01

0.526

0.019

-0.165

0.010

-0.426

0.014

VIT.A 1 0.009

0.950

0.025

0.864

0.109

0.450

-0.459

0.05

-0.659

0.016

0.659

0.003

-0.658

0.010

MMP2 1 -0.255

0.074

-0.080

0.582

0.729

0.00

0.806

0.04

-0.659

0.00

-0.659

0.00

MMP9 1 0.415**

0.003

0.816

0.01

0.629

0.016

-0.745

0.019

-0.689

0.018

MMP11 1 0.629

0.011

-0.726

0.016

-0.881

0.001

-0.776

0.000

8-OHdG 1 0.916

0.016

-0.629

0.016

0.666

0.048

IL-6 1 -0.629

0.003

-0.465

0.011

GSK3β 1 0.649

0.04

Uric

Acid

1

CHAPTER SIX SUMMARY

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47

SUMMARY

CHAPTER SIX SUMMARY

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48

6.0 SUMMARY

The most common type of oral cancer is known as OSCC (Oral Squamous Cell

Carcinoma) which is essentially involved in affecting tongue and floor of oral cavity. Apart from

the fact that its detection in early stages remains challenging as it is an asymptomatic

complication whereas, it can be later symptomized by the pain lasting for more than two weeks

and development of lymph node metastasis in later stages. It contributes in worldwide elaborated

rates of mortality and morbidity. In Asian subcontinent especially in countries such as India and

Pakistan the rate of OSCC are significantly higher due to higher rate of people belonging to

socioeconomically statuses and increased number of people habitual of betel quid. Other myriad

factors of OSCC includes family history, cannabis, excessive use of tobacco and tobacco

products, regular alcohol intake, narcotics and antioxidant deficient diet. Moreover Hepatitis C,

Herpes virus, Candida albicans, Human immunodeficiency virus, Human papilloma virus and

Epstein Barr viruses have also been considered potential causative factors. In the recent years

burgeon of OSCC cases has been reported in individuals less than 45 years specifically from 18

to 44 years. It has been observed that younger people especially males are more prone to develop

OSCC than females due to excessive use of tobacco and tobacco associated products. Both

smokers and alcoholics have a six fold risk of oral neoplasm than non-smokers and non-

alcoholics whereas those individuals practicing both habits have a fifteen fold risk of OSCC than

healthy individuals. By and large tobacco and alcohol in the West, gutka and betel quid in

Taiwanese, Maras powder in Turks, betel quid in Asians and in Indians areca nut, tobacco, and

betel quid along with alcohol consumption account for the onset of OSCC.

Conventionally imaging techniques followed by blood, serum and biopsy samples are

used for OSCC detection but in the recent years the distinguished role of saliva as a diagnostic

tool has been widely accepted. Usually surgery followed by radiotherapy is the prime treatment

in OSCC stages I and II whereas for stages III and IV a combination of surgery, radiotherapy and

chemotherapy is utilized. Likewise immunotherapy, gene therapy, altered fractionated

radiotherapy; cervical lymph adenectomy, selective neck dissection, concomitant chemo-

radiotherapy (CT-RT) and targeted molecular therapy are also novel prospective treatments.

Globally, a notable reason behind the drastic boom in OSSC cases is that the masses are not fully

cognizant of the hazardous effects of tobacco products and alcohol. In order to get rid of this

social taboo, a substantial number of awareness programs should be arranged to enlighten the

CHAPTER SIX SUMMARY

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49

public about the debilitating effects of tobacco chewing, tobacco smoking, alcohol consumption

and excessive use of tobacco related products. The government can contribute by taking strict

steps on multiple levels to ban the sale and use of these noxious products. Likewise emphasis on

preventive measures such as opting for early screening, avoiding unnecessary irradiation and

consuming diet rich in iron and antioxidants can promise an oral cancer free life.

These zinc dependent endopeptidases are produced according to the cell's need by wound

cells as well as inflammatory cells and are known to play essential roles in both normal and

diseased state. Under normal conditions, MMPs are secreted in very low amounts and they are

involved in tissue remodeling processes like ossification, placenta development, and embryonic

development and wound healing. On the basis of substrate specificity, sequence similarity and

domain organization metalloproteinases are categorized as: collagenases, gelatinases,

stromelysins, matrilysins and membrane type MMPs (MT-MMPs). Generally all MMPs consist

of a signal peptide, a pro-peptide having highly conserved Cysteine residues, a zinc containing

catalytic domain and a hemopexin like domain which is attached to the catalytic domain by a

linker. But the striking feature that separates gelatinases from other MMPs is the presence of a

fibronectin type II like domain. One of the characteristic marks in cancer invasion is the

deterioration of extracellular matrix (ECM) which is efficiently done by matrix

metalloproteinases (MMPs).

Myriad MMPs (MMP 1, 2, 3, 7, 9, 11, 13 etc.) take part in OSCC disease progression

but the crucial role of gelatinases has been widely established. It has been observed that in OSCC

the HIF-1α is upregulated which is the reason behind elevated levels of gelatinases and hence

decreased prognosis. MMP-9 has a dual role in angiogenesis where it may act as angiogenic or

anti-angiogenic depending on the situation. Various growth factors play essential roles in the first

step of tumorigenesis. For instance overexpression of TGF-β1 is responsible for spiked

expression of MMP-9 in OSCC studies. It has been discovered that Snail, the transcription

factor, aids in elevating MMP-9 expressions. Sufficient data supports the versatile role of

gelatinases in oral squamous cell carcinoma. Gelatinase B (MMP-9) has a significant role in

OSCC progression and its expression along with activity has been thoroughly weighed. MMP-9

has vital role in metastasis. In addition, ethanol has been found to positively affect expression of

gelatinases. It is found that increased expression of MMP-9 is majorly associated with

metastasis. Yet studies show that increased expression of both gelatinase A and B in OSCC was

CHAPTER SIX SUMMARY

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50

in correlation with only tumor invasiveness. Likewise another set of investigation suggests that

the association of gelatinases with different tumor stages and concluded that MMP 9 levels were

elevated in OSCC but the active form of MMP 9 was less in quantity as compared to the levels

of activated MMP 2, hence MMP 2 has correlation with advanced stages. Mukherjee et al.

showed that gelatinases are highly involved in cancer and pre-cancer states. Apart from this,

scientists found that gelatinolytic activity in OSCC is due to activated 72 kD gelatinase A. In a

different study moderate expression of MMP-2 was seen while MMP-9 levels were the highest.

Another investigative study discovered that it is the increased gelatinases in OSCC

patients that leads to their low survival rates. Enhanced amounts of these metalloproteases

pinpoints the fact that greater levels are owing to either the over expression of MMPs by

cancerous cells or the increased inflammatory cytokine expression may have triggered their

production from the host stromal cells. Additionally the more secreted levels of gelatinases, the

more ECM degradation will occur hence leading towards tumor invasion and angiogenesis.

CHAPTER SEVEN CONCLUSION

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51

CONCLUSION

CHAPTER SEVEN CONCLUSION

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52

7.0 CONCLUSION

Oral squamous cell carcinoma (OSCC) is a modern day catastrophe affecting millions of people

around the globe, including Pakistan. It has gained worldwide attention owing to low survival rates, late

detection, poor prognosis, and escalated mortality rates. Aside from tobacco, tobacco products and

alcohol there are myriad potential factors which contribute to etiopathogenesis of OSCC. Oral cancer

affects men more than women as the former are more habitual users of notorious tobacco and tobacco

products. Currently, the search for predictive OSCC biomarkers is in demand to maximize survival rates.

The present study was done to evaluate the oxidant-antioxidant status and inflammatory profile in oral

cancer subjects. Present research work provides strong evidence that oral cancer patients presented

elevated oxidative stress, enhanced inflammation and reduced antioxidant enzymes in their serum as

compared to their healthy counterparts. Therefore the study strongly approves that MMPs, inflammatory

and lipoxidative biomarkers hold promise for their predictive potential in OSCC. Moreover the

implication of antioxidant therapy during early stages, serves as new potential to overcome OSCC. Lastly,

the understanding of potential role of these certain biomarkers calls for their ultimate utilization and

implementation as prognostic and predictive OSCC biomarkers.

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