Lucie Hloušková Supervisor : Ale ks andra Kapuscik
description
Transcript of Lucie Hloušková Supervisor : Ale ks andra Kapuscik
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.
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 resultsCytotoxicity groups:non cytotoxic: >75% viabilitymodetate 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 183 - 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