PRESENTED BY

ANOOP JACOB MATHEW

 Cells of the body begin to grow and reproduce in an uncontrollable manner.

 External Factors (tobacco, chemicals, radiation) and Internal Factors (mutations,

immune conditions).

 One in eight deaths is due to cancer.

 Breast cancer is a type of cancer that forms in tissues of breast – ducts and lobules.

 Breast cancer incidence rates have been rising in developing countries.

 Risk factors include inheritance of genes BRCA1 and BRCA2, family history,

medical procedures etc.

 Estrogen plays an important role in development, progression and

outcome of breast cancer.

 Key Steroidal hormone which regulates physiological processes.

 Estrogen mediates its activity through a receptor protein called Estrogen

Receptor (ER).
 70% of breast cancers are estrogen dependent (ER + ve).
ESTROGEN SIGNALING
 Class of medication that acts on estrogen receptor.

 Agonist action in some tissues, while acting as estrogen antagonist in

others.

 Members of SERM include afimoxifene, arzoxifene, Barzoxifene,

tamoxifen , toremifene, raloxifene.

 Tamoxifen and toremifene are used for breast cancer.

 Safety of SERMs is a big concern.

 Non-steroidal plant compounds that have estrogenic or anti-estrogenic effects.

 Phytoestrogens bind weakly to estrogen receptor.

 Three classses of phytoestrogens: Lignans, isoflavones and coumestans.

 Phytoestrogens provide protective effects against breast cancer.

 Phytoestrogens are inhibitors of enzymes like aromatase, 17β-HSD, estrone

sulphatase and sulphotransferase.

 Antioxidant activity, inhibition of cell signaling pathways.

 Commonly known as the Bodhi or peepal tree.
 Several medical and physiological activities.
 MATERIALS AND METHODS

CELL CULTURE
 Cell line used: MCF-7

 Medium used: Dulbecco’s Modified Eagle Medium (DMEM)

 Materials used: PBS-EDTA, trypsin, Freezing mixture (DMSO in FBS)

Cell culture work involves:

 Thawing and recovery of cells (revival)

 Subculturing

 Freezing of cells
 Ficus religiosa is extracted with different solvents – acetone, chloroform,

hexane and methanol.

 Fractional extraction is carried out using soxhlet apparatus.

 Colorimetric assay for measuring cell viability
 MTT - [3-(4,5- dimethyl thiazol-2-yl)-2,5 diphenyl tetrazolium]

Materials: plant extracts, MTT, Lysis buffer, DMEM.

Procedure:
 Seeding of the cells in a 96 well plate (24 hours incubation)
 Drug addition- Plant extracts in different solvents are added to the wells (48 hours)
 MTT is added to the wells and incubated for 2 hours at 37 0 C
 Lysis buffer is added and incubated for 4 hours.
 Absorbance at 570 nm using ELISA reader
 The distribution of cells in different phases of cell cycle were analysed by
Flow cytometry.
 Materials: DMEM, ice cold PBS, 70% ethanol, 1 x PBS- EDTA, RNase,
Propidium Iodide.

Cell cycle analysis using FACS involves:-

 Seeding of cells in a six well plate (incubated for 24 hours).
 Addition of plant extracts in different solvents (80 μg/ml) to the wells and
incubated for 48 hours.
 Cells were stained using Propidium Iodide (fluorescent stain) for cell cycle
analysis.
 The cells were cultured in DMEM on a glass slide.
 Plant extracts were added and incubated at 370 C for 24 hours.
 Medium was removed and cells fixed by acetic acid/ methanol (1:3) for
10 minutes.
 Cells were air dried and stained by Hoechst stain for 30 minutes at room
temperature.
MTT ASSAY RESULTS
CELL CYCLE ANALYSIS – 24 HOURS

G1 S G2

79.2 6.7 14.0

G1 S G2

Acetone 78.8 7.5 13.6

 Tubes G1 S G2
Tubes G1 S G2
Control 79.2 6.7 14.0
Control 79.2 6.7 14
Hexane 77.9 7.0 14.9
Chloroform 81.0 4.7 14.3

Tubes G1 S G2

Control 79.2 6.7 14.0

Methanol 78.8 5.4 15.7

CELL CYCLE ANALYSIS- 48 hours

G1 S G2

70.9 12.8 16.3

Tubes G1 S G2
Control 70.9 12.8 16.3
Acetone 84.6 7.7 7.7
 G1 S G2 G1 S G2
Control 70.9 12.8 16.3 Control 70.9 12.8 16.3

Chloroform 73.7 14.0 12.3 Hexane 82.6 10.3 7.1

G1 S G2
Control 70.9 12.8 16.3
Methanol 71.8 14.4 13.8
 MICROSCOPIC EVALUATION OF CELL DEATH
CONTROL ACETONE

24 HOURS 48 HOURS 24 HOURS 48 HOURS

CHLOROFORM

24 HOURS 48 HOURS
HEXANE

24 HOURS 48 HOURS

METHANOL

24 HOURS 48 HOURS
 HOECHST STAINED IMAGES

CONTROL ACETONE CHLOROFORM

HEXANE METHANOL
 The present study revealed that different solvent extracts of Ficus
religiosa (L.) inhibit the growth of estrogen receptor positive human
breast cancer cells.
 On 48 hours exposure to these solvent extracts, acetone & chloroform
extract shows significant inhibitory effect on the proliferation of MCF-7
breast cancer cell line in a dose dependant manner.
 FACS analysis revealed that the mechanism behind the inhibition of cell
proliferation by acetone extract and hexane extract is via G1/S phase cell
cycle arrest on exposure for 48 hrs.
 Observations made under the light microscope showed that, after
treatment with acetone and chloroform extract for 48 hours, cell number
had decreased and shape of the cells had changed in comparison with the
control.
 Upon Hoechst staining, acetone and chloroform extract treated cells
shows considerable nuclear condensation with less density.
THANK YOU.....