lucie hloušková supervisor : aleksandra kapuscik 28. 07. 2011 application of cytotoxicity assays...

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