Natural Polyphenol "Resveratrol" present predominantly in the grape seed plays an very important role in the treatment of devastating disorder Cancer (diseases involving abnormal and uncontrolled growth of cell with the potential to spread to other parts of the body)

1. Dr. Sudharani, N.
Assistant Professor
(Food Science and Nutrition)
College of Horticulture
Mudigere, Karnataka-57713210/12/2016

2. “LET FOOD BE THE MEDICINE AND MEDICINE BE THE FOOD”
- Hippocrates
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3. Topic division
Introduction
Reviews
Summary
Conclusion
Referenc
es
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4. Cancer as a Global Health Problem
• >9.7 million cases are detected / year
• 6.7 million people are dieing
• 20.4 million people are living with cancer
• 2020 :15 million people will die from cancer
WHO , 2014
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5. Nair et al. 2015
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Site of Cancer Incident cases(%)
Males
Lung 10.90
Oral cavity 9.30
Stomach 5.90
Females
Cervix 21.30
Breast 21.00
Ovary 5.60
Table 1: Common cancers in India, 2015

6. Lung
Breast
Colon/Rectum
Stomach
Liver
Prostate
Cervix uteri
Oesophagus
Bladder
Non-Hodgkin
Lymphoma
Leukaemia
Oral cavity
Pancreas
Kidney
Ovary
1000 800 600 400 200 0 200 400 600 8001000
Men
Women
(Thousands)
Incidence
Mortality
337
293
105
0370
241
318
446
234
165
166
471
233
133
111
76
33
121
68
113
86
47
97
101
101
34
71
192
114
810
902
558
405
255
499
398
384
204
543
279
260
227
99
93
167
144
109
81
170
116
112
57
119
5.3 million cases
3.5 million deaths
4.7 million cases
2.7 million deaths
The Global Burden of Cancer, 2012
WHO, 2014
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7. Cancer is an uncontrolled growth
of abnormal cell with the potential to
spread to other parts of the body.
They form a subset of Neoplasms.
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Singh et al., 2014

8. • Division – uncontrolled cell division
Oncogenes, Tumour suppressor genes – p53
• Growth – formation of a lump (Hypertrophy)
Pressure on nerves, blocking organs, stopping
normal function, alters nerve signals
• Mutation – Sudden changes of a Cells
Invasion, Angiogenesis
• Spread – ability to move within the body and survive in another
parts of the body. Metastasis
Characteristic of Carcinogenic cells
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9. Tumor
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10. Malignant Vs Benign tumours
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11. Invasion of malignant versus benign
tumours
Microscopic Appearance of Cancer Cells
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12. Six hallmarks of Cancer
(1) Stimulate own growth
(2) Resist inhibitory signals
(3) Resist their programmed cell
death
(4) Multiply indefinitely
(5) Stimulate the growth of blood
vessels to supply nutrients to
tumors
(6) Invade local tissue and
spread to distant sites
Tissue invasion and
Metastasis
Sustained angiogenesiss
Limitless replicative
potential
No Apoptosis
Insensitivity to anti
growth signal
Self sufficiency in
growth signals
Jayaprakasha et al., 2012
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13.  Angiogenesis
 Metastasis
 Apoptosis
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14. Series of Mutation
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15. Types of Cancer
Often prefixed by the specific cell
Adenomas
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16. Cancerous growth
Normal cell growth
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17. Signs and symptoms
Local symptoms:
due to the its ulceration.
 Eg: cough, pain , swelling
etc..
Systemic symptoms:
due to metastatic spread.
 Eg: Unintentional weight
loss, fever, excessive fatigue and
changes of skin.
Depends on the cancer's type and location
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18. Carcinogenesis: Some factors to consider…
Genetic Inheritance (5% )
Radiation (10%)
Infections(16%)
Environmental factors (69%)
WHO , 2014 1810/12/2016

19. Early detection & screening.
Prevention
Treatment
Chemo therapy/ radiation therapy.
Hormone therapy.
Natural Polyphenols
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20. 2010/12/2016

21. Grape seed extract
 GSE contains OPC’s.
 OPC s are flavonoid like polyphenolic compounds.
 Grape seeds contains a number of antioxidants, mainly
Resveratrol.
Scientific name: Vitis vinifera
Family: Vitaceae
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22. Resverotrol
(C14H12O3)
Resveratrol (3,5,4′-trihydroxy-trans-stilbene)
Natural phenol and phytoalexin.
 Produced in several plants.
Occurrence
• Resveratrol was originally isolated by the roots of
Japanese knotweed in 1963.
22
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23. Grapes (skin &
seeds)
Blueberry
Raspberry
Mulberry
Cran berry
Senna
The amount of Resverotrol varies with the cultivar, geographic origin and exposure to infection
Biosynthesis
&
Biotransformation
23
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24. Beverage Resveratrol (mg/l)
White wine 0.05-1.80
Rose wine 0.43-3.52
Red wine 1.92-12.59
Red wine 1.98-07.13
Red grape juice 1.14-8.69
Total Resveratrol Content of
Grape wines and juice
Sl.No Food source Resveratrol
(μg/g )
1. Red wine 0.2 and 5.8
2. Muscadine grapes >40
3. Grape seed
extract
50-65
6. Mulberries 5.0
Total Resveratrol Content of
Selected Foods
Clement et al., 2012
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25. 2510/12/2016
Bagchi et al., 2012

26. How does Resveratrol Affect Cancer
Inhibits initiation, promotion, and progression stages
• Initiates cell cycle arrest
– Increases transcription factor p53
• Promotes cell apopstosis
– Activation of other apopstotic pathways
• Inhibits angiogenesis
– Feeds tumor
• Inhibits inflammation
– Cellular proliferation & angiogenesis
• Destroys estrogen metabolites &
– Obstructing their reaction with DNA
Mechanism of GSE on Cancer
Bagchi et al., 2012
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27. Products of GSE
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28. 2810/12/2016

29. Grape seed extract induces apoptotic death of human prostate carcinoma
DU145 cells via caspases activation accompanied by dissipation of mitochondrial
membrane potential and cytochrome c release
To investigate the molecular mechanisms of GSE-caused
apoptotic death of human Prostate cancer cell.
Agarwal et al., 2012
Carcinogenesis,
29
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30. • Human prostate carcinoma cell line DU145 and
GSE were obtained from lab
• Cancer cells induced with GSE treatment at various
doses (50–200 μg/ml) for 12–72 h (Adult male
rats=50-65g)
• After 72 h = Apoptotic death & caspase activity.
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31. (A) dose-dependent at 48 h
(B) 150 μg/ml GSE
(C) 200 μg/ml GSE
Fig.1: Dose and time dependent Apoptotic death in DU145 cells by GSE
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17%
7%
3%

32. Fig. 2. Effect of GSE on the cleavage patterns of caspase 3, 7 and 9 and PARP in DU145 cells.
B: Analysis of Caspase 3 activity
A: Immunoblot analysis to identify the
products of caspase 3, 7 & 9.
12
Caspase activation Mechanism
> 3 mechanism
a. Direct Mitochondrial damage
b. Cytochrome C release from Mitochondria c. Caspases activation
Caspases (Cysteine Protease) & PARP (Poly ADP- ribose Polymerase)cleavage
= Biomarkers of Apoptosis.
Mitochondria is a key regulator of Apoptosis by releasing Cytochrome C into cytosol
Cyt C + Caspase to form Apoptome gene leads to further biochemical & morphological
changes = Apoptosis
As GSE , the cleavge of caspase 3 & 9activity = PARP cleavage
Caspase 3 is a central Caspase in the Cascade
Caspase 3= Cell death
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33. Conclusion
GSE causes mitochondrial damage to
cancerous cells, leads to cytochrome c release in
cytosol and activation of caspases resulting in PARP
enzyme which leads significant apoptotic death of
human prostate cancer cells.
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34. A Novel Grape Seed Extract in Vivo enhances
Acetaminophen-Induced Unprogrammed Cell Death in Liver Cancer.
To examine the hepatoprotective ability of short-term and long-term exposures of
a grape seed proanthocyanidin extract (GSPE) in liver Cancer cells.
Ray et al., 2013
Archvs of Biochm
& Biophys,
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35. Methodology
Liver for Histopathological diagnosis
Alanine Amino Transferase activity (ALT)
Animals were Sacrificed and Serum was analyzed
Hepatotoxic doses of AAP (400 and 500 mg/kg for 3 or 7 days.
Male mice (30±40 g) = Nontoxic doses of GSPE (50mg/kg) bw for 3 or 7
days.
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36. Figure 1: AAP induced Liver injury and its reversal by GSPE by serum Alanine amino
transferase activity (ALT).
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37. Figure 2: AAP-induced genomic DNA fragmentation and its reversal by GSPE pre-
exposure. 37
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28%
44%
28%
16%

38. Figure 3: Magnified view of normal hepatocytes undergoing terminal stages of
apoptosis
Due to high toxicity of AAP- indicator of
oxidative tissue damage.
AAP DNA repair enzyme PARP
GSE PARP activity & expression of cancer
cells
Apoptosis
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AAP= Acetaminophen

39. Conclusion
• AAP metabolism triggered production of ROS, leading to oxidative
stress and damages DNA by decreasing DNA repair gene.
• But GSE increases DNA repair enzyme PARP, which increases
apoptotic death linked to down regulation and modification of cancer
cell expression.
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40. Anticancer properties of GSE resveratrol on
chemically induced Hepatocellular carcinoma in
rats: Inhibition of metastasis and angiogenesis
Investigate the therapeutic effect of resveratrol (natural grape
seed extract phytoalexin) on diethyl nitroseamine (DENA) induced
hepatocellular carcinogenesis in rats.
Halim, et al.,2015
J of Chem.
Pharm. Res.,
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41. • GSE Resverotrol & Experimental animals procured from (NRC, Egypt)
• Adult Male, 120-150g
• Acclamatization of rats at 24'C for 72 hr and fed with standard lab diet
• Group 1: (Control) Plain Chow diet
• Group2: (Low dose of resverotrol - 300mg/kg B. w)
• Group3: (High dose of resverotrol - 450mg/kg B. w)
• Group4: DENA - 200mg + CCL4 - 2ml
• Group5: G2 +G4+G2 (Pre)
• Group6: G3+G4+G3 (pre)
• Group7: G4+G2 (Post)
• Group8: G4+G3 (Post)
•Animals were sacrificed for further analysis
DENA=Di Ethyl Nitroseamine CCl4=carbon tetrachloride 4110/12/2016

42. • MMP’s (Matrix metallo Proteinase) and
Heparnase enzymes = markers of Metastasis.
• VEGF:(Vascular endothelial growth
factor)Marker of Angiogenesis.
• Sirtuins: (SIRT) : NAD dependent enzyme
catalyses Histones & others proteins and acts
as regulator for cell proliferation and prevents
cancer.
• As GSE = SIRT1
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43. Group VEGF
(Pg/ml)
Heparanase
(U/ml)
Elastase
(U/ml)
Control 198 2.40 0.25
High Resveratrol 189 2.00 0.21
Low Resveratrol 190 2.30 0.23
DENA 435 6.12 1.01
Low Res Pre treated 300 4.60 0.62
High Res Pre-treated 285 4.00 0.51
Low Res Post-treated 223 2.82 0.33
High Res Post-treated 252 2.97 0.39
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Significant at < 0.05%

44. Table 2: Effect of Resveratrol on MMP-2, MMP-9 and
SIRT 1 in all tested groups
Group MMP-2 (ng/ml) MMP-9 (ng/ml) SIRT1 (pg/ml)
Control 496 265 2.50
High Resveratrol 467 275 9.08
Low Resveratrol 486 280 6.73
DENA 1467 836 8.68
Low RES Pre treated 600 480 11.00
High Res pre-treated 542 320 11.09
Low Res post-treated 551 462 12.08
High Res Post-treated 492 307 12.63
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Significant at < 0.05%

45. GSE = MMP’s & VEGF expression
(Inhibits carcinogenic effect)
VEGF- marker of Angiogenesis
MMP- marker of Metastasis
Promotes tumor by
stimulating TGF
SIRT1
Prevents cell damage
Arrest cell cycle
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46. Conclusion:
Resveratrol may be considered as antimetastatic
agent on DENA-induced hepato carcinogenesis in
rats by decreasing MMP, VEGF and increasing
SIRT1 by arresting cancer cell cycle.
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47. Genotoxic and Histopathological aspects of treatment
with grape seed extract on colon cancer induced with
cyclophosphamide in mice
To evaluate the natural protective efficacy of
grape seed extract (GSE) against CP-induced
genotoxicity of colon cancer cells in mice.
Ela and
Omara, 2014
Cell Biology
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48. Methodology
56 mice = 4 groups (14 mice in each group)
G1: Cyclo phosphamide induced (40mg/kg/day)
G2: Control
G3: 75mg/kg/day GSE liquid (Low dose)
G4: 150mg/kg/day GSE liquid (High dose)
After 1, 7, 14 & 30th day- Histopathological observations
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49. Figure1: Percentage of Carcinogenic cells in Control, Cp induced & GSE treated Mice
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50. Figure : % of Apoptosis in Carcinogenic cells of Control, Cp induced & GSE treated Mice
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51. Induction of GSE stimulates other pro-inflammatory
Cytokines & reduce COX-2 expression
In Cancer cells Expression of COX-2 is more
COX-2 is Pg synthase enzyme
It catalyses Pg production pathway
COX2 = Suppress the Pg synthase and decrease the inflammation
Finally by arresting cell multiplication COX 2 prevent the Metastasis

52. Conclusion
Grape seeds extract down regulated the
COX-2 expression by its antioxidant defense
mechanism to reduce the colon injury.
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53. Resveratrol Affects Protein Kinase C Activity and
Promotes Apoptosis in Human Colon Carcinoma Cells
Investigate the effects of GSE resveratrol on
protein kinase C (PKC) activity and apoptosis in
human colon carcinoma cells.
Fang et al., 2012
Asn. Pa. J of Cancer Prevention,
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54. Methodology
HT 29 Colon cancer cells = diff con’s
of GSE Resverotrol i.e 50, 100, 200,
300 and 400µM for 24, 48 and 72 hr.
PKCα and ERK ½ signalling
pathway were determined by
western blotting method.
The inhibitory effect of resveratrol
on HT29 cells was studied.
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55. Figure 1: Effect of GSE Resveratrol on HT-29 Cell death
%CellDeath
Resveratrol (µM)
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95%
82%
47%

56. Figure 2: Resveratrol Induced Significant Apoptosis after 72 hr
CellApoptotic%
Resveratrol (µM)
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Only after 72 hr
48%
55%

57. Figure 3: Western blot of PKC and ERK Expression in HT-29 cells
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Protein kinase C & ERK1/2 Signaling pathway
PKC = Metastasis
Induced GSE =PKC phosphorylation = Apoptosis
In PKC = ERK1/2 phosphorylation
In PKC pathway= MAPK’s triggers Apoptosis & enhance Chemosensitivity
GSE prevents further Invasion by arresting cell multiplication
Mechanism
ERK= Extra cellular signal
regulated kinase
MAPK= Mitogen activated
protein kinase

58. Conclusion
GSE resveratrol activates the PKC-
ERK1/2 signal pathways and it stimulates
the production of MAPK’s, in turn triggers
the HT29 colon cancer cells Apoptosis.
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59. Inhibitoryroleof GSEoncancer cell metabolismbydownregulating
PyruvateKinaseM2via inhibitionof MammaliantargetofRapamycin
To know the effect of GSE on PKM2
expression on cancer cell metabolism.
Iqbal & Rameshwar, 2012
J. Oncology
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60. Methodology
Procured HeLa, HepG2 & MCF-7 cells (NCCC,
Pune).
Incubated to 24hr in control (DMSO)
Incubated with GSE (50µg/g) to 48 hr
Cells subjected for the proliferation study
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Dimethyl Sulfoxide

61. Figure 1. PKM2 expression on Resveratrol induction by Western
blot
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62. Figure 2. Effect of Resveratrol on the expression of mTOR signaling
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63. Figure 3. Resveratrol inhibits Glycolysis (glucose uptake and lactate
production characteristic of cancer metabolism).

64. Rapamycin (mToR) pathway
Pyruvate kinase M2(PKM) pathway
Marker of
Cell
proliferation
Biosynthesi
TGF
Activity is
very low
L, R, M1 & M2
Hyper
activation of
mTOR
Mutation
Controls cancer
metabolism by
regulating key
enzymes
GF signals
mediated by
mTOR
Phosphophenol
pyruvate - pyruvate
Increase in PKM
Accumulation of
glycolytic intermediates
for PPP
Cell
proliferation
Cancer

65. Conclusion
Resveratrol acts as a promising anti-
cancer agent in hindering pro-cancerous
metabolism through PKM2 and Rapamycin
down regulation pathway.
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66. Summary
 Cancer is an uncontrolled growth of abnormal cell with the
potential to spread to other parts of the body. They form a
subset of Neoplasms.
 Although there are several treatment methods to reduce the
incidence of cancer their is very much scope for the use of
natural Anti-cancer polyphenols.
 In this context Grape seed Extract may provide a potential anti-
tumor effect by several mechanisms i.e.
 As a potent natural antioxidant it reduces the DNA
fragmentation and prevents the cell damage.
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67.  Release cytochrome c in cytosol and activates caspases
resulting in PARP cleavage leads to apoptotic death of human
prostate cancer cells.
 Increases DNA repair enzyme PARP and down regulates the
expression of Liver cancer cell.
 Down regulates the COX-2 expression by its antioxidant
defense mechanism to reduce the colon injury.
 By the activation of PKC- ERK1/2 signal pathways GSE
resveratrol induces colon cancer cell Apoptosis.
 Resveratrol as a promising anti-cancer agent hinders pro-
cancerous metabolism through PKM2 down regulation.
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68. 6810/12/2016

69. References
AGARWAL, C, SINGH, R.P. AND AGARWAL, R., 2012, GSE induces
Apoptotic death of Human Prostate Carcinoma cells via Caspases
activation and Cytochrome ‘C’ release. Carcinogenesis. 23(11):1869-
1876.
BAGCHI, D., BAGCHI, M., STOHS, S., DAS, D.K., RAY, S.D. AND
JOSHI, S.S., 2012, Grape Seed Proanthocyanidin Extract: Importance in
Human health and disease prevention. Toxicology. 148: 187-197.
CLEMENT, M.V., HIRPERA, J., CHAWDHARY, S.A. AND PERVAIZ, 2012,
Cell proliferation properties of Carcinogenic cells, The American So of
Hematology, 92: 996-1002.
ELA, EI. AND OMARA, E.A., 2014,Genotoxic and Histopathological aspects
of treatment with Grape Seed Extract on CPA induced mice. Cell Biology.
2(3):18-27.
FANG, J.Y., LI, Z.H., LI,Q., HUANY, W.S., 2013, Resveratrol affects Protein
Kinase ‘C’activity and promotes Apoptosis in Human Colon Carcinoma
Cells. Asian Pacific J. Cancer prevention. 13 (12): 6017-6022. 6910/12/2016

70. HALIM, A.H., FYIAD, A.A., ALI, M.A. AND SOLIMAN, S.M.,2015, J. of
Chemical Pharmaceutical Research, 7 (4):913-921.
JAYAPRAKASHA, G.K., SELVI, T. AND SAKARIAH, K.K., 2012, J. Food
Research International, 36: 117-122.
NAIR, M.K., VARGHESE, C. AND SWAMINATHAN, R., 2015, Current
scenario, intervention strategies and projections for 2015, Regional cancer
centre, Thiruvananthapuram: 219-225.
SINGH, R.P., TYAGI, A.K., DHANALAKSHMI, S., AGARWAL, R. AND
AGARWAL, A., 2014, GSE inhibits advanced Human Prostate cancer growth,
Int. J. Cancer, 108: 733-740.
WHO, 2014, World cancer report.
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71. Thank You 7110/12/2016