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RH1 is a novel diaziridinylbenzoquinone bioreductive agent that has recently entered phase 1 clinical trials. It is an excellent substrate for the obligate two electron-reducing enzyme NQO1 (DT-diaphorase), and studies have demonstrated a relationship between NQO1 expression levels and drug response. It has been proposed that RH1 may be an effective NQO1-directed antitumor agent for the therapy of cells with elevated NQO1 activity. However, bioreductive agents may also be substrates for other reducing enzymes such as the 1-electron reducing enzyme, NADPH-cytochrome P450 reductase (P450 reductase), and therefore the role of this reducing enzyme in activating RH1 was examined. RH1 reduction by purified P450 reductase yielded a very strong electron paramagnetic resonance (EPR) signal, suggesting that RH1 could be reduced by this enzyme. Spectroscopic studies monitoring the disappearance of NADPH when RH1 was incubated with purified P450 reductase or NQO1 confirmed that this agent can be a substrate for either enzyme, and that under aerobic conditions RH1 undergoes redox cycling. To examine whether P450 reductase can play a role in activating RH1 in tumor cells, we used T47D human breast cancer cells and T47D cells transfected with the P450 reductase gene which have 20-fold increased P450 reductase activity. Pretreatment of the transfected T47D cells with the P450 reductase inhibitor, diphenyliodonium chloride (DPIC), did not inhibit RH1 cytotoxicity. In contrast, pretreatment of the transfected T47D cells with the NQO1 inhibitor, dicoumarol, significantly inhibited the of cytotoxicity of RH1, confirming the role of NQO1 as an activator of this agent. However, if the transfected cells were pretreated with both inhibitors there was additional inhibition of cytotoxicity compared with dicoumarol alone, suggesting that in the absence of NQO1 activity, high levels of P450 reductase activity could contribute to RH1 activation and cytotoxicity. A similar study in the parental T47D cells showed that pretreatment with both enzyme inhibitors did not result in greater inhibition of cytotoxicity than pretreatment with dicoumarol alone. These results suggest that although P450 reductase can reduce RH1, NQO1 is the major activator of this agent. and P450 reductase likely does not play a role in RH1 activation at normal cellular levels of this enzyme. (Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the CancerCare Manitoba Foundation)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]