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, 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 H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/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 (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 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 cytotoxicity of this chemotherapeutic agent.

Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence

“…10B). The quinones mitomycin C and apaziquone were no more cytotoxic to the POR-overexpressing cells than to the parental cells; earlier studies have given conflicting data on whether POR sensitizes other cell lines to these prodrugs under anoxia (16,17,26,63). POR expression also strongly sensitized the cells to the N-oxides under oxic conditions (Fig.…”

The chemical and biological activities of quinones: overview and implications in analytical detection

“…Although the redox properties of the quinones depend largely on their chemical potential, their interactions with proteins at a specific binding site can further modulate the electronic properties and thus their redox potential in situ (Breton and Nabedryk 1996;Jeon 2003). Phytochem Rev (2011) 10:353-370 355 The one-electron reduction of quinones can be catalyzed by a number of enzymes, including microsomal NADPH cytochrome P450 reductase (P450R), microsomal NADH cytochrome b5 reductase (b5R), and mitochondrial NADH ubiquinone oxidoreductase (Holtz et al 2003;Monks and Jones 2002;Wang et al 2010;Yan et al 2008). The semiquinone radical, formed by one electron reduction, gets oxidized under aerobic conditions to the initial quinone with the generation of superoxide anion radicals.…”

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

“…These enzymes require NADPH or NADH as the source of reducing equivalents. NADPH-cytochrome P450 reductase, which contains both FAD and FMN as cofactors, is the best characterized enzyme mediating redox cycling (9,10). It appears that the ability of FAD to accept single electrons from NADPH is critical for redox cycling.…”