anticancer and antimetastatic activities of yukyung karne ... · marker left, right events % gated...

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!