Lucie Hloušková Supervisor : Aleksandra Kapuscik 28. 07. 2011 Application of cytotoxicity assays on HeLa and HepG2 cells for estimation of health risk.

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Presentation transcript:

Lucie Hloušková Supervisor : Aleksandra Kapuscik Application of cytotoxicity assays on HeLa and HepG2 cells for estimation of health risk for humans as response to cyanobacterial metabolites.

Introduction  Places of finding  Unicellular and colonial species  Secondary metabolites of cyanobacteria  Biological activity - antifungal, anticancer, immunosupresants, ion channel blockers, enzyme inhibitors, cytotoxic agents…

Aim of the project  Characterization of toxic effects of cyanobacterial metabolites to various human and murine cancer cell lines

Material  15 strains (Nostoc, Phormidium, Leptolyngbia,Oscillatoria and Wiollea)  The cyanobacterial biomass was lyophilized and used for experiments

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)

Our samples for testing…

Cytotoxicity screening – MTT test yellow purple crystals  centrifugation  crystals dissolved in DMSO  absorbance measurement in 590nm + 10mg/ml extracts MTT (methyl-tetrazolium salt)

MTT results Cytotoxicity groups: non cytotoxic: >75% viability modetate cytotox: 50-75% strong cytotox: <50%

Caspase assay results

Photos Sp 2 control Yac – 1 control

HeLa 77 5h HepG2 control

Our work

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 Nostoc sp. CALU 1533 strains, in concentration 2 mg/ml, activated caspase 3 and 7 what correlates also with observed morfology

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