Distinct Roles of Cytochrome P450 Reductase in Mitomycin c Redox Cycling and Cytotoxicity

Abstract

Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, Chinese hamster ovary (CHO) cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H(2)O(2); this activity was greater in CHO-OR cells (V(max) = 1.2 +/- 0.1 nmol H(2)O(2)/min/mg protein in CHO-WT cells versus 32.4 +/- 3.9 nmol H(2)O(2)/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC(50) = 72 +/- 20 nmol/L for CHO-WT and 75 +/- 23 nmol/L for CHO-OR cells), as was its ability to induce G(2)-M and S phase arrest. Additionally, in nine different tumor cell lines, although a strong correlation was observed between MMC-induced H(2)O(2) generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in the cytotoxicity of this chemotherapeutic agent.