anticancer and antimetastatic activities of yukyung karne ... · marker left, right events % gated...
TRANSCRIPT
Anticancer and antimetastatic activities of
Yukyung Karne- Traditional Tibetan medicine
By
Tenzin Choedon Jamling PhD
International Centre for Genetic Engineering and
Biotechnology
New Delhi
Hanahan and Weinberg, Cell, 144, 2011
Hallmarks of cancer
2
Ovarian cancer is the fifth leading cause of cancer related death
and the leading cause of death from gynecological malignancies.
Difficult to diagnose
Late diagnosis: Stage 3 cancer of the ovary
80% of Ovarian cancer present with omental metastasis
Screening test: CA 125 (50%) accuracy, late marker
Standard treatment : no change in survival rate
Ovarian cancer
Scenario in India
Massive surge in cancer cases in India
As per WHO, 500,000 people die of cancer and
expected to rise to 700,000 by 2021.
3
Surgery , Radiotherapy and chemotherapy to kill the cells that are dividing
uncontrollably.
Chemotherapy : Platinum based drugs - paclitaxel and carboplatin - are the drugs
most widely used. Tumours usually respond to this form of treatment but often
eventually return.
Standard treatment of Ovarian cancer
4
■Anticancer properties of Yukyung Karne–a Traditional Tibetan
Medicine
■ Anti-Metastatic activity of Traditional Tibetan Medicine-Yukyung
Karne
Parts:
Objectives
⁎ In vitro cytotoxic screening of Traditional Tibetan medicine,
Yukyung Karne in ovarian and other cancer cells
⁎ To validate Yukyung karne efficacy of Inducing apoptosis in
ovarian cancer cells- SKOV6
⁎ To study the effect of Yukyung karne on migration and
transformation of ovarian cancer cells
⁎ To evaluate, Yukyung Karne impact on Extracellular matrices and
Epithelial to mesenchymal transition in ovarian cancer cells- SKOV6
6
Screening Anticancer activity of Yukyung
Karne in cancer cells
7
8
Anti cancer drug screening
MTT assay
MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by mitochondrial reductase in metabolically active cells.
Fractions from 4 Traditional Tibetan medicine
1
Screening strategy
Fractions from 2 TTM
2
Fractions from 1 TTM: YK
3
Viability of cells after treatment with Yukyung Karne (YK)
0
20
40
60
80
100
120
Control
Doxorobucin
1
10
100
Cel
l via
bil
ity (
%)
10
Cel
l via
bili
ty (
%)
YK (μg/ml)
0
20
40
60
80
100
120
Control Doxorubicin 1 10 100
HEK293T
A549
HepG2
Hela
IHH
SKOV6
Cel
l via
bil
ity (
%)
Cel
l via
bil
ity (
%)
0
20
40
60
80
100
120
Control Paclitaxel Pac+YK
*
Cell viability using positive reference
*, p < 0.005.
Cel
l via
bil
ity (
%)
11
Control Paclitaxel Paclitaxel + YK
YK 100μg YK10μg YK 1μg YK200μg
Morphological changes of SKOV6 cell line
after Yukyung Karne treatment
12
Yukyung Karne: Induction of apoptosis in
ovarian cancer cells
13
Cell viability assay by crystal violet staining
Control Pac Pac+YK YK
14
0
20
40
60
80
100
120
140
Control Pac Pac+YK YK
Cell viability assay by crystal violet staining C
ell
via
bil
ity (
%)
**
**
**
Mitochondria
Mitochondria: executor of apoptosis
15
Control Pac Pac+ YK YK
Mitotracker -
dsRED
Cytochrome C
-GFP
Merged
Cellular localisation of Cytochrome C using ds red
mitotracker and GFP cytochrome C in SKOV 6 cell lines
**, p < 0.001.
16
Control Pac Pac+YK YK
JC1
(TRITC)
JC1
(FITC)
DAPI
Merged
Analysis of mitochondrial membrane potential using JC1 dye
*, p < 0.05; **, p < 0.001.
17
Control Doxorubicin
TUNEL
(FITC)
DAPI
Merged
YK (μg)
1 10 100
-1
0
1
2
3
4
5
6
7*
*
*
YK
(mg)
TU
NE
L p
osi
tiv
e ce
lls
(no.)
In situ Cell Death (Apoptosis) Detection-TUNEL assay
*, p < 0.001
18
Fragmentation of genomic DNA from SKOV6 cells treated with
different concentrations of YK
Ctrl Pac Pac+YK 1 10 100 200 50 bpM
YK (mg)
19
“The dream of a bacterium is to become two
bacteria.” FranÇois Jacob, 1965
• Replication must be an exact process.
• Errors of under-replication and over-replication.
• Cancer, birth defects, developmental abnormalities.
• The genome must be replicated once and only once per cell cycle.
20
0
10
20
30
40
50
60
70
80
90
100
Control Paclitaxel Pac+YK YK
G2/M
S
G1
Effect of YK on Cell cycle progression in SKOV6
cells by FACS
0 200 400 600 800 1000FL2-A
Data.001
M1
M2
M3
Histogram Statistics
Marker Left, Right Events % Gated % T otal Mean Geo Mean CV Median Peak Ch
All 0, 1023 13576 100.00 67.88 307.51 293.18 31.17 291.00 216
M1 134, 265 5390 39.70 26.95 219.35 217.67 12.06 220.00 216
M2 268, 329 2917 21.49 14.59 297.26 296.72 6.06 297.00 319
M3 329, 450 3820 28.14 19.10 379.05 377.61 8.80 375.00 368
Fi le: Data.001 Log Data Uni ts: Linear Values
Sample ID: Patient ID:
T ube: Unti tled Panel : Untitled Acquisition Tube List
Acquisi tion Date: 01-Jan-70 Gate: G1
Gated Events: 13576 T otal Events: 20000
X Parameter: FL2-A (Linear)
0 200 400 600 800 1000FL2-A
Data.002
M1
M2
M3
Histogram Statistics
Marker Left, Right Events % Gated % T otal Mean Geo Mean CV Median Peak Ch
All 0, 1023 16145 100.00 80.73 292.27 279.29 30.96 276.00 211
M1 134, 265 7400 45.83 37.00 216.59 214.98 12.05 214.00 211
M2 268, 329 3553 22.01 17.77 297.37 296.86 5.90 297.00 282
M3 329, 450 3874 24.00 19.37 373.56 372.26 8.45 368.00 347
Fi le: Data.002 Log Data Uni ts: Linear Values
Sample ID: Patient ID:
T ube: Unti tled Panel : Untitled Acquisition Tube List
Acquisi tion Date: 01-Jan-70 Gate: G1
Gated Events: 16145 T otal Events: 20000
X Parameter: FL2-A (Linear)
0 200 400 600 800 1000FL2-A
Data.003
M1
M2
M3
Histogram Statistics
Marker Left, Right Events % Gated % T otal Mean Geo Mean CV Median Peak Ch
All 0, 1023 5407 100.00 60.48 281.53 266.88 33.91 260.00 195
M1 134, 265 2772 51.27 31.01 209.29 207.32 13.69 205.00 195
M2 268, 329 1081 19.99 12.09 297.18 296.65 6.02 297.00 270
M3 329, 450 1065 19.70 11.91 375.24 373.76 9.03 367.00 332
Fi le: Data.003 Log Data Uni ts: Linear Values
Sample ID: Patient ID:
T ube: Unti tled Panel : Untitled Acquisition Tube List
Acquisi tion Date: 01-Jan-70 Gate: G1
Gated Events: 5407 T otal Events: 8940
X Parameter: FL2-A (Linear)
0 200 400 600 800 1000FL2-A
Data.004
M1
M2
M3
Histogram Statistics
Marker Left, Right Events % Gated % T otal Mean Geo Mean CV Median Peak Ch
All 0, 1023 13881 100.00 69.41 266.70 251.17 36.25 245.00 178
M1 134, 265 7852 56.57 39.26 199.99 197.46 16.09 193.00 178
M2 268, 329 2789 20.09 13.94 297.63 297.11 5.94 297.00 281
M3 329, 450 2136 15.39 10.68 378.31 376.61 9.56 372.00 331
Fi le: Data.004 Log Data Uni ts: Linear Values
Sample ID: Patient ID:
T ube: Unti tled Panel : Untitled Acquisition Tube List
Acquisi tion Date: 01-Jan-70 Gate: G1
Gated Events: 13881 T otal Events: 20000
X Parameter: FL2-A (Linear)
Control Paclitaxel
Paclitaxel + YK YK
21
22
p53
Check point
p53
Tumor suppressor and cell cycle regulator
p21
Cyclin dependent kinase inhibitor
Inhibits activity of cyclin-CDK2 or 4 complexes
Promotes cell cycle arrest (G1 )
Cyclin B
Sequence specific DNA binding protein
Most frequent mutated in cancer Essential for transition from G2 to M phase
Essential for survival and proliferation
Overexpressed in many cancers
pTEN:
A tumor suppressor protein
Act as a phosphatase
Regulates PI3K/AKT survival pathway
Effects of Yukyung Karne on
tumor suppressor and cell cycle regulator
23
α -p53
α -Cyclin B1
α -Mdm2
α-pTEN
α -GAPDH
Control Pac Pac+YK YK
α -PCNA
α -p21
1 0.1 2.7 2.7
1 1.1 0.9 1.2
1 1.3 1.6 1.7
1 1.2 0.8 0.6
1 0.6 0.3 0.4
Control Pac Pac+YK YK
-10
0
10
20
30
40
50
60
Control Pac Pac+YK YK
Pro
life
rati
ng c
ells
(%
)
* * *
*
Brdu
(FITC)
DAPI
Merged
Brdu incorporation-cell proliferation assay
*, p < 0.02, **, p < 0.002.
24
⁎Yukyung Karne had cytotoxic as well as anti-proliferative
properties in ovarian cancer cells
⁎ Yukyung Karne induce cell cycle arrest at G1 phase
⁎ Yukyung Karne induces efflux of cytochrome c and
activates mitochondrial dependent apoptosis in ovarian
cancer cells
⁎ Yukyung Karne restore tumor suppressor p53 and pTEN
expression
⁎ Yukyung Karne and paclitaxel have synergistic effects on
induction of apoptosis
Summary
25
Anti-metastatic activity of Yukyung Karne in
ovarian cancer cells
Part 2
Steps
*Shedding of cells from primary tumor
*Entrance into the vascular system
*Travel to distant site
*Growth
*Angiogenesis
Metastatic Overview
Effect of Yukyung Karne on tumor cell
adhesion to ECM protein Collagen
Control Pac+YK YK Pac
, P<0.05, , P<0.001
Significant role in cancer progression
Most abundant protein
Provides structural integrity and tensile strength
Determines the spread of metastatic cells
28
Control Pac Pac+YK YK
Paclitaxel Control Pac+YK
YK50μg YK200μg YK100μg
Effect of Yukyung Karne on tumor cell invasion
No. of
filt
rate
d c
ells
Cell invasion studies
0
10
20
30
40
50
60
Control Paclitaxel Pac+YK YK50ug YK100ug YK200ug
*
**
**
:, P<0.05, , P<0.001
**
**
29
Control YK
6h
24h
Effect of Yukyung Karne on cancer cell migration
30
No. of
colo
nie
s
Control Pac
Effect of Yukyung Karne on Colony formation and angiogenesis
, P<0.001
Pac+YK YK
31
Control Pac Pac+YK YK
Effect of YK on VEGF secretion from SKOV6 cells
Effect of Yukyung Karne on Epithelial to
mesenchymal transition
32
Epithelial to mesenchymal transition
Progression towards malignancy is accompanied by
Loss of epithelial differentiation
Shift towards mesenchymal phenotype
Characterized by invasive nature of cells
Marked by increase in expression of mesenchymal marker and decrease in
epithelial marker
33
Control Pac Pac+YK YK
E- Cadherin
Effect of Yukyung Karne on Epithelial markers
GAPDH
E-Cadherin:
Mediates cell – cell adhesion
Regulation of cell polarity and maintenance of epithelial
organization
Loss of E-cadherin : predictive of poor survival
34
1.0 1.2 2.3 2.5
N- Cadherin
GAPDH
:, P<0.05
Effect of Yukyung Karne on mesenchymal markers
N-Cadherin
Associated with acquisition of EMT phenotype
Vimentin
Requisite regulator of mesenchymal cell migration Control Pac Pac+YK YK
35
Control Pac Pac+YK YK
1.0 0.5 0.6 0.5
Effect of YK on 5 major steps of metastasis
Inhibits collagen mediated cell adhesion
Inhibits active invasion and migration
Suppresses the growth at secondary sites
Inhibits VEGF secretion for angiogenesis
Effect of YK on ECM and EMT
Repression of matrix metalloproteinases (MMP2/9)
Down regulates β-Catenin, Integrin and pFAK
Upregulates Caveolin
Regulates Epithelial to mesenchymal transition
Yukyung Karne exhibited property of an effective anti-metastatic agent
38
Anti-metastatic activity of Yukyung Karne
International Centre for Genetic Engineering and Biotechnology
Acknowledgements
Dr.Vijay Kumar ( ICGEB) Dr. Dawa Dolma (TMAI)
Dr. Ganeshan Mathan (BDU)
Thank You All!