anticancer activity of withania somnifera on h ep 2 cell
TRANSCRIPT
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Anticancer activity of Withania somnifera
on HEp-2 cell line
Submitted by Samayaditya Singh B.Tech (Bio.tech) MCSCET
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INTRODUCTION
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Withania somnifera (Ashwagandha)
Ashwagandha is a potent adaptogen
and Immunomodulator
It has anti-agening and anti-
cancerous property.
Chemicals present in leaves are
Steroidal Lactone, withanolides,
withaferin A etc.(P.K.Sharma et
al.2010)
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Cont…Chemical components of Ashwagandha Leaves
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Cont… HEp-2 Larynx carcinoma cell line
• Cells of this line contain HeLa marker chromosomes.
• This line was originally derived from an epidermoid
carcinoma, but recently found line was derived by HeLa
contamination.
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Objective
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1. To maintain the Hep-2 cell line for anticancer cell activity.
2. To estimate the cytotoxic effect of Withania somnifera on
Hep-2 cell line.
3. To determine the DNA damaging Potential of Withania
somnifera in Hep-2 cell line.
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Plan of Work
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Culture the Hep-2 Larynx carcinoma cell line and maintain there viability
Check viability of cultured cells
Check cell viability after treatment
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Passaging
Trypan blue exclusion test
Preparation of treatments for cell Viability Assay
MTT AssayNRU Assay
Check Apoptosis rate by DNA Fragmentation process
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METHADOLOGY
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Passaging
DISCARD PREVIOUS
CULTURE MEDIUM
ADD
INCOMPLETE
MEDIA
ADD TRYPSIN-EDTA
SOLUTION AND WAIT FOR
4-5 MINUTES
BY SCRAPER GENTLY
DETACHED REMAIN
ADHERED CELLS
AFTER ASPIRATING PELLETS
ADD APPROPRIATE ALIQUOTS OF
CELL SUSPENSION TO NEW
FLASKS. INCUBATE AT 37°C
DISCARD SUPERNATANT
AND ADD COMPLETE
MEDIA ON PELLETS
TRANSFER CELLS
SUSPENSION IN FALCON
TUBE AND CENTRIFUGE
TUBE FOR 5 MIN
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Trypan Blue exclusion test
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Mix them homogenously
Blue color cell are deadColorless cell viable
Hemocytometer
Take cell suspension and Trypan blue in 1:1 ratio
Load mixture on Hemocytometer
Microscope (on 10X)
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MTT Cell Proliferation Assay(Mosmann, T., 1983) Used to measure rate of reduction in cell viability.
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Extract of LeavesAdd MTT reagent
Incubate for 20hr
Incubate for 2hr
Cell suspension
Add DMSO
Take absorbance
Micro plate reader
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NRU( Neutral Red Uptake Assay) (Winckler, 1974) Live cells incorporate neutral red into their lysosomes.
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Incubate for 20hr
Incubate for 2hr
Take absorbance
Micro plate reader
Add Glacial acetic acid
Add NRU reagent
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DNA Fragmentation Assay (Andrew Wyllie,1980)
DNA fragmentation is Hall Mark of Apoptosis in cells.
Add Lysis Buffer
Phenol:Chloroform:Isoamyl alcohal
Centrifuge
DNA
PROTEIN
INCUBATE FOR 24hr
INCUBATE FOR 2hr
Add RNase and Proteinase KLysates
ISOPROPANOL AND SODIUM ACETATE
Centrifuge
ETHANOL(70%)
Centrifuge
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Cont…q
FORM LOADING SAMPLE
FORM GEL BED WITH 6-WELLS
LOAD SAMPLE
GEL DOC System
Electrophoresis system
Dry the sample in air Nanospectro
photometre
Quantitative Analysis of DNA on
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RESULT
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– Under microscope no dead cells are found, test show 50%
viability of cell line.
Viable cell
Trypan blue exclusion test
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MTT Assay
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NRU Assay
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DNA Fragmentation Assay
L C T-1 T-2 T-3 T-4
Where,
L- Ladder Well; T-1, T-2, T-3, T-4- Treatment Well; C- Control Well
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CONCLUSION
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Trypan Blue Exclusion test
– Average number of cells in large Square 8.5 cells.
– Total number of cells in 1ml is 1.7X105 cells.MTT Assay– Cells show significant death in T-1 treatment, at conc.
1000µg/ml.NRU Assay– Cells show significant death in T-1 Treatment, at concentration
1000µg/ml.DNA fragmentation Assay• DNA fragmentation assay shows apoptosis at 1000µg/ml concentration of ethanolic extract of Ashwagandha.
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AcknowledgementI would like to express my sincere gratitude towards Prof P.K. Seth,
CEO of Biotech Park, Lucknow, who gives me a opportunity to do my training in the field of Molecular Biology Under Aakaar Biotech.
And a heartiest thanks to my mentor Mr. Ashish Yadav and Dr. Swarnita Dixit, for there guidance in my project work and also for maintaining morale.
Also thanks to Dr. Umashankar Sir, who share it’s pecious knowledge and give more knowledge about Biotechnology.
I would also like to thanks Ms. Namita Tripathi, Mr. Pradeep Singh Chauhan and Mr. Anand Kumar Saroi, for there help in improving lab skills and encouragement.
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THANK YOU