NSAIDs are used extensively to control pain and inflammation NSAIDs are among the most widely used drugs in the world; each day, it is estimated that 30 million people worldwide benefit from the anti-inflammatory and analgesic effects of these drugs, 1 and some 500 million prescriptions for NSAIDs are written every year. NSAID use is particularly common among elderly patients: it is estimated that 10-20% of the elderly ( 65 years) have a current or recent NSAID prescription. 2 Indeed, 35% of all NSAIDs prescribed are to individuals over 60 years of age. 3 Furthermore, the use of NSAIDs is growing as these drugs become more readily available as ’over-the-counter’ medications, and there is increasing use of low-dose aspirin to prevent thrombotic conditions such as myocardial infarction or stroke. Moreover, the population in many parts of the world is becoming increasingly elderly, with a concomitant increase in the incidence of arthritic diseases and, hence, the need for NSAID therapy.
EFFECT OF RESVERETROL ON HUMAN BREAST CANCER (MCF-7) CELL LINE
Under the Suprevision of
Dr. Sandip Kumar
K.P.C Medical College &
EFFECT OF RESVERETROL
ON HUMAN BREAST CANCER
(MCF-7 ) CELL LINE
WHAT IS RESVERATROL ?
Resveratrol is a stilbenoid, a
type of poly phenol , and a
naturally by several plants
when under attack by
pathogens such as bacteria
or fungi .
Resveratrol is found in
the skin of Red grapes ,
Berries etc as phytoalexin .
Japanese not weed
shown in the picture is one
rich source .
Trans- 3, 5, 4’ tri
STRUCTURE OF RESVERATROL
AIMS AND OBJECTIVES
Anticancer effect of Resveratrol .
Anti proliferative effect of Resveratrol
along with its path way is being studied .
MATERIALS & METHODS
MCF-7 cell line.
Cell Titer Glo luminscent cell viability assay
Phase Contrast Microscopy
Determination of DNA fragmentation by TUNEL Assay
Study of the nature of cell death by FACS analysis
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)
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
The cells (5 x104
in 100 microlitre
medium /well) were plated in 0.02-
0.07%DMSO in media as control in 96
The cells as such or in presence of RESV
(DISSOLVED IN 0.02- 0.07% DMSO in
media) were incubated for 48 hrs .
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
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 .
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
Study of apoptosis by FACS
viable Early apoptotic
viable Early apoptotic
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.
study the DNA fragmentation:
Terminal Transferase dUTP Nick End Labeling
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.
Resv treted cell
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
Role of proteins used :
t-Bid : Promotes leakage by altering
membrane curvature .
fodrin : Maintains normal membrane
β-actin : Used as loading control .
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