Role of NADPH cytochrome P450 reductase in activation of RH1.

Purpose: RH1 is a new bioreductive agent that is an excellent substrate for the two-electron reducing enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). RH1 may be an effective NQO1-directed antitumor agent for treatment of cancer cells having elevated NQO1 activity. As some studies have indicated that RH1 may also be a substrate for the one-electron reducing enzyme, NADPH cytochrome P450 reductase (P450 Red), P450 Red may contribute to the activation of RH1 where NQO1 activities are low and P450 Red activities are high. The mean P450 Red activity in the human tumor cell line panel used by NCI for evaluation of new anticancer agents is 14.8 nmol min(-1) mg prot(-1), while the mean NQO1 activity in these cell lines is 199.5 nmol min(-1) mg prot(-1). Thus, we investigated whether P450 Red could play a role in activating RH1.

Methods: Reduction of RH1 by purified human P450 Red was investigated using electron paramagnetic resonance and spectroscopic assays. The ability of RH1 to produce DNA damage following reduction by P450 Red was studied using gel assays. To determine the role of P450 Red in activation of RH1 in cells, cell growth inhibition studies with inhibitors of P450 Red and NQO1 were carried out in T47D human breast cancer cells and T47D cells transfected with the human P450 Red gene (T47D-P450) that have P450 Red activities of 11.5 and 311.8 nmol min(-1) mg prot(-1), respectively.

Results: Reduction studies using purified P450 Red and NQO1 confirmed that RH1 can be reduced by both enzymes, but redox cycling was slower following reduction by NQO1. RH1 produced DNA strand breaks and crosslinks in isolated DNA after reduction by either P450 Red or NQO1. DPIC, an inhibitor of P450 Red, had no effect on cell growth inhibition by RH1 in T47D cells, and had only a small effect on cell growth inhibition by RH1 in the presence of the NQO1 inhibitor, dicoumarol, in T47D-P450 cells.

Conclusions: These results demonstrated that P450 Red does not contribute to the activation of RH1 in cells with normal P450 Red activity and plays only a minor role in activating this agent in cells with high levels of this enzyme. These studies confirmed that P450 Red could activate RH1 and provided the first direct evidence that RH1 could produce both DNA strand breaks and DNA crosslinks after reduction by P450 Red. However, the results strongly suggest that P450 Red does not play a significant role in activating RH1 in cells with normal P450 Red activity.