Free-radicals dependent and independent pathways of cddp-mediated cytotoxicity and apoptosis in ovarian tumor-cell lines.

Cis-platinum (CDDP) is a chemotherapeutic drug widely used alone or in combination with other drugs in the treatment of cancer. However, many tumors become resistant to CDDP and the mechanisms of resistance are complex. The present study investigated the role of free radicals in CDDP-mediated cytotoxicity and apoptosis. Four human ovarian cancer cell lines were chosen for the study: two lines, 222 and A2780, are sensitive to CDDP and two lines, AD10 and C30 are resistant to CDDP. The importance of free radical formation was tested by the use of an inhibitor, 2,6 di-tert-butyl-4-methoxy phenol (BHA), that inhibits the production of free radicals and detoxifies free radicals. Cytotoxicity was assessed by the MTT assay and apoptosis assessed by flow cytometric analysis of DNA hypoploidy. Three of the 4 cell lines, 222, AD10, and C30, were completely inhibited by BHA in CDDP-mediated cytotoxicity. The CDDP-sensitive A2780 cell line, however, was not inhibited by BHA, even at high non-toxic concentrations of BHA. The DNA analysis for apoptosis paralleled the findings obtained in the cytotoxicity assay. In order to rule out that the A2780 cell line was not reactive to BHA, VP-16 mediated cytotoxicity was examined. All four cell lines were sensitive to VP-16 and all four were inhibited by BHA. In contrast, there was no detectable inhibition by BHA of actinomycin-D-mediated cytotoxicity in all four lines tested. Overall, the findings demonstrate that either free-radical dependent (CDDP, VP-16) or free radical-independent (CDDP, actinomycin-D) pathways of cytotoxicity and apoptosis are utilized by different drugs. Further, a single agent like CDDP can mediate killing by both a free radical-dependent and by a free-radical independent pathway. Therefore, new agents that are developed to reverse resistance by a particular drug must take into consideration alternative cytotoxic pathways mediated by the same drug.