Cytoprotective activity of amifostine on HeLa cell cultures treated with daunorubicin and evaluation of gene expression by RT-PCR

Abstract

There are reports that amifostine protects normal tissues against chemotherapy and radiotherapy in cancer treatment. In this study, daunorubicin effects on cell kinetics parameters and apoptosis were examined both singly and in combination on HeLa cell culture. Optimum doses of daunorubicin and amifostine (0.1, 1 mu g/mL, respectively) were applied for 24 and 48h. Cell kinetic parameters such as growth rate (WST-1 colorimetric assay), mitotic and apoptotic index were applied to identify cytotoxicity that was induced by daunorubicin. In addition, to evaluate the molecular mechanism of cytoprotectivity we studied DNA degradation in agarose gel electrophoresis and expression of two genes, bcl-2 and bax, known to be important for the regulation of apoptosis. The cytotoxic effect correlated with a decline of growth rate and mitotic index, and a significant increase of apoptotic index, when HeLa cells were treated with daunorubicin alone or in combination with amifostine treatment (p <0.05-0.001). The enhancement of the cytotoxicity by daunorubicin was closely associated with DNA degradation. Investigation of bcl-2/bax expression level by RT-PCR showed that amifostine did not protect tumor cell lines against daunorubicin cytotoxicity.