engleski

Molecular alterations induced in human cervical carcinoma cells by N-fenil-2-(2 piridinil)diazenecarboxamide

The aim of the present study was to investigate the biological effects induced in human cervical carcinoma (HeLa) cells with a new compound N-fenil-2-(2-piridinil)diazenkarboksamid. The cytotoxicity of this compound was determined by MTT method and the type of cell death was investigated by fluorescent microscope. The distribution of cells in the cell cycle phases was examined by flow cytometry. The presence of apoptosis was analysed by flow cytometry (Annexin V-FITC test) and by DNA electrophoresis (determination of internucleosomal cleavage of DNA). The expression of apoptotic proteins and proteins involved in cell cycle regulation was analysed by Western blot method. Results showed that N-fenil-2-(2-piridinil)diazenkarboksamid changed cell’ s and nucleus’ s morphology 3 h after the treatment, but only 24 h after the treatment the cells started to loose membrane integrity. At that time, the number of cells in G1/G0 decreased, while the number of cells in the G2/M phase of the cell cycle increased. However, for the dose that reduced the cell survival to 20 % a peek of sub-G1 cells appeared, that was accompanied by PS externalisation and internucleosomal DNA cleavage. Simultaneously no procaspase-3, procaspase-9 and PARP cleavage, or changes in bax and bcl-2 protein levels was observed, while the levels of proteins involved in cell cycle regulation, survivin and p21, did alter. In conclusion, N-fenil-2-(2-piridinil)diazenkarboksamid induces changes in cell cycle distribution probably by alterations in p21 and survivin expression. Cytotoxicity of this compound is, according to our results, the consequence of induction of caspase-independent cell death.

new anti-cancer drugs; tumor cells

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