This document summarizes a study on the effect of resveratrol on human breast cancer MCF-7 cell lines. The study found that resveratrol inhibited the viability of MCF-7 cells in a dose-dependent manner, with an IC50 value of 125μM. Treatment with resveratrol induced apoptosis in the cells, as shown by DNA fragmentation, phosphatidylserine externalization, and morphological changes observed under electron microscopy. The apoptosis was further confirmed by the activation of apoptotic proteins like t-Bid and cleavage of α-fodrin. The study concludes that resveratrol shows promise as an anti-cancer agent for its ability to induce apoptosis in breast cancer cells.

1. Submitted
BY
ABHISHEK BANERJEE
ROLL-586-112-020
Under the Suprevision of
Dr. Sandip Kumar
Bandyopadhyay
K.P.C Medical College &
Hospital
EFFECT OF RESVERETROL
ON HUMAN BREAST CANCER
(MCF-7 ) CELL LINE

2. WHAT IS RESVERATROL ?
Resveratrol is a stilbenoid, a
type of poly phenol , and a
phytoalexin produced
naturally by several plants
when under attack by
pathogens such as bacteria
or fungi .
Source :
Resveratrol is found in
the skin of Red grapes ,
Berries etc as phytoalexin .
Japanese not weed
shown in the picture is one
rich source .

3. Natural Sources of Resveratrol

4. [Resveratrol]
Trans- 3, 5, 4’ tri
hydroxy stilbene
SFRR, 2009
STRUCTURE OF RESVERATROL

5. AIMS AND OBJECTIVES
 Anticancer effect of Resveratrol .
 Anti proliferative effect of Resveratrol
along with its path way is being studied .

6. MATERIALS & METHODS
1. Materials:
 MCF-7 cell line.
 Resveratrol
2. Methods:
 Cell Titer Glo luminscent cell viability assay
 Phase Contrast Microscopy
 Electron Microscopy
 Determination of DNA fragmentation by TUNEL Assay
 Study of the nature of cell death by FACS analysis
 Western Blot

7. MCF-7
 MCF-7 is a breast cancer cell line isolated in 1970 from a 69-
year-old Caucasian woman.
 MCF-7 is the acronym of Michigan Cancer Foundation
.MCF-7 (human breast cancer)

8. CELL VIABILITY ASSAY
 CellTiter-Glo® Luminescent Cell Viability
Assay is a homogeneous method to
determine the no. of viable cells in the
culture based on the quantification of ATP
present, which signals the presence of
the metabolically active cells.
 The amount of ATP present is directly
proportional to the no of cells present in
culture.
 The cells (5 x104
in 100 microlitre
medium /well) were plated in 0.02-
0.07%DMSO in media as control in 96
well plates.
 The cells as such or in presence of RESV
(DISSOLVED IN 0.02- 0.07% DMSO in
media) were incubated for 48 hrs .

9. REACTION :

10. CELL VIABILITY ASSAY (Continued)
 At the end of the treatment each
well was treated with a volume
of cell titer Glo® reagent equal
to the volume of cell culture
medium present in each well.
 Then contents are mixed for 2
min on an orbital shaker to
induce cell lysis & the plate is
incubated at room temp for 10
min in dark to stabilize
luminescent signal .
 At the end the cellular
luminiscence was recorded in a
luminometre .
 A significant reduction in cell
viability was observed at 48 Hr
of treatment at their varying
concentrations .The IC50 value of
Resv in MCF-7 cell line is 125um .

11. PHASE CONTRAST MICROSCOPIC OBSERVATION OF
RESVERETROL TREATED CELL
RESVERETROL TREATED
CELL((100 μM)
CONTROL(DMSO 0.05% V/V)

12. OVERVIEW OF APOPTOSIS

13. Detection of apoptosis by double staining method
 .
 comparison with control (0.02% and 0.05%
DMSO) Resv treated unfixed MCF-7 cells
showed Annexin V- Alexa fluor 488-binding
but PI staining was insignificant indicating
the mode of cell death as apoptosis, not
necrosis.
Control
Study of apoptosis by FACS
Resv treated
Early apoptotic
Late apoptoticNecrotic
viable
Late apoptotic
Necrotic
Early apoptoticviable
viable
viable
viable Early apoptotic
viable
viable Early apoptotic
Early apoptotic
Early apoptotic
Late apoptotic
ate apoptotic
Late apoptotic
ate apoptotic
NecroticNecrotic
Necrotic
Necrotic

14. Transmission Electron Microscopic analysis to study
the cellular morphology by the treatment of Resv:-
 After 48 hr treatment with Resveratrol apoptosis induction was evident from the
morphologic alteration as shown in the TEM .
 It clearly indicated morphological changes, degenerated organelles and
fragmented nucleus compared to vehicle control .
 Electron microscopic analysis of MCF-7 cells with Resveratrol treatment after
48 h treatment. 25μM Resv in MCF-7 cells were treated for 48 h and TEM
analysis were carried out . Nucler fragmentation is indicated by arrows.
Control cell
Resv Cell

15. study the DNA fragmentation:
Terminal Transferase dUTP Nick End Labeling
(TUNEL) ASSAY
 It is a method used to detect DNA
fragmentation by labelling the terminal ends
of nucleic acids .
 The assay relies on the presence of nicks or
DNA breaks .
 Nicks or DNA breaks can be identified by
TdT(Terminal Deoxy Nucleotidyl Transferase)
an enzyme that will catalyze template-
independent addition of deoxyribonucleoside
triphosphates to the 3’ hydroxyl ends of
double- or single-stranded DNA generating
DNA strands with exposed 3'-hydroxyl ends
 Non-apoptotic cells do not incorporate much
of the F-dUTP because of absence of exposed
3’-hydroxyl DNA ends.
Control cell
Resv treted cell

16. STUDY OF Apoptotic protein activation due to Resv
treatment BY WESTERN BLOT
Antibody raised in rabbit is used as
primary antibody and Goat anti
rabbit is used as Secondary
antibody .
Role of proteins used :
t-Bid : Promotes leakage by altering
membrane curvature .
Alpha-
fodrin : Maintains normal membrane
structure .
β-actin : Used as loading control .
t-bid
α-fodrin
β-actin

17. CONCLUSION
 From the above experiments it is visualized that
RESVERATROL induced apoptosis in MCF-7 Cell line.The
mode of work(hypothysed) is supported by the cleavage
of alpha fordin indicating apoptotic mode of cell
death.Besides, several researchs show positive result of
Resveratrol over cancer cell. So on the basis of these &
also on the basis of first phase clinical trial
RESVERATROL , now a days has established itself as a
promising drug against the treatment of cancer cells.
 Resveratrol concentration ranging between 25
micromolar to 125 micro molar shows positive
result .this indicates the concentration of Resv that
can be used to as standard for cancerous cell
treatment .