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Lucie HlouškováSupervisor : Aleksandra Kapuscik
28. 07. 2011
Application of cytotoxicity assays on HeLa and HepG2 cells for estimation of health risk for humans as
response to cyanobacterial metabolites.
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Introduction Places of finding Unicellular and colonial species Secondary metabolites of cyanobacteria Biological activity - antifungal, anticancer, immunosupresants, ion channel blockers, enzyme inhibitors, cytotoxic agents…
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Aim of the project
Characterization of toxic effects of cyanobacterial metabolites to various human and murine cancer cell lines
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Material 15 strains (Nostoc, Phormidium, Leptolyngbia,Oscillatoria and Wiollea) The cyanobacterial biomass was lyophilized and used for experiments
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Methods Sample preparation (70% MeOH extraction) Cytotoxicity methyl-tetrazolium test (MTT) performed on cell lines: Hep G2
(human liver carcinoma), HeLa (human cervix carcinoma), Sp2 (murine myeloma), Yac-1 (murine lymphoma) Caspase 3/7 assay – detection of apoptosis
(Hep G2 cell line)
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Our samples for testing…
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Cytotoxicity screening – MTT test
yellow purple crystals
centrifugation crystals dissolved in DMSO absorbance measurement in 590nm
+ 10mg/ml extracts
MTT (methyl-tetrazolium salt)
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MTT resultsCytotoxicity groups:non cytotoxic: >75% viabilitymodetate cytotox: 50-75%strong cytotox: <50%
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Caspase assay results
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Photos
Sp 2 control
Yac – 1 control
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HeLa 77 5h
HepG2 control
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Our work
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Conclusions
HeLa cells are the most resistant cell line to tested extracts, whereas Yac-1 shows the highest sensitivity
For some cyanobacterial strains a good cytotoxicity correlation to all tested cell lines has been proved
Both 77 - Phormidium sp. CALU 52 and 183 - Nostoc sp. CALU 1533 strains, in concentration 2 mg/ml, activated caspase 3 and 7 what correlates also with observed morfology
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Acknowledgements to my supervisor Aleksandra Kapuscik for her help and advice, and to Pavel Hrouzek my colleague Sofia Chysta to all organizers of the Summer School