Submitted BYABHISHEK BANERJEE ROLL-586-112-020Under the Suprevision ofDr. Sandip Kumar BandyopadhyayK.P.C Medical College & Hospital

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 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 .

Natural Sources of Resveratrol

[Resveratrol]Trans- 3, 5, 4’ tri hydroxy stilbene

SFRR, 2009

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

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

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)

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 .

REACTION :

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 .

PHASE CONTRAST MICROSCOPIC OBSERVATION OF RESVERETROL TREATED CELL

RESVERETROL TREATED CELL((100 μM)

CONTROL(DMSO 0.05% V/V)

OVERVIEW OF APOPTOSIS

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

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

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

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

-fodrin

-actin

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 .