Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species⁵

Abstract

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a free radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs.

Introduction

The bioreductive alkylating agent mitomycin C has been used clinically for the treatment of several tumour types. Despite significant antitumour activity, its widespread use is limited as a result of its extensive and unpredictable dose-limiting myelosuppression [1]. An indoloquinone analogue of mitomycin C, EO9 (Fig. 1; [2]), has undergone preclinical and clinical development under the auspices of the EORTC. EO9 was found to have a different spectrum of preclinical antitumour activity to mitomycin C, showed preference for solid tumours over leukaemias, and most importantly was non-myelopsuppressive 3, 4. As a result of these features and information regarding its mechanism of enzymatic activation (see below), EO9 entered Phase I and II clinical trial 5, 6, 7. Although responses were seen in Phase I trials [6], activity was not confirmed in Phase II [7]. Nevertheless, clinical development of EO9 continues, potentially to define a clinical role for this agent in a setting where its short plasma half-life is not so critical, e.g. intravescicular therapy for bladder cancer. In addition, studies continue with the aim of identifying bioreductive agents that will be clinically more effective than EO9 [8].

A characteristic of bioreductive alkylating agents such as EO9 is that they are designed to require reduction for activation to a cytotoxic species. Several enzymes are known to be involved in the reduction of quinone compounds [8]. These include those catalysing one-electron reduction, e.g. NADPH: cytochrome P450 reductase and cytochrome b5 reductase as well as DT-diaphorase, which carries out two-electron reduction. As part of an ‘enzyme-directed approach’ to bioreductive drug development 8, 9, 10, we were interested in elucidating the molecular enzymology and mechanism of action of EO9. We had previously focused on the involvement of the two-electron reducing flavoenzyme DT-diaphorase and provided data to support a role for this enzyme in the activation of EO9 to a cytotoxic species 11, 12, 13, 14, 15.

Initially, extracts of the high DT-diaphorase expressing rat Walker tumour and human HT29 colon carcinoma cells were shown to catalyse reduction of EO9 12, 15. The rate of reduction decreased in the presence of the potent DT-diaphorase inhibitor dicoumarol. Dicoumarol is, however, also known to affect other enzyme systems. Proof of the ability of DT-diaphorase to reduce EO9 was provided by use of purified rat, mouse, and human recombinant DT-diaphorase 11, 16. Activation of EO9 catalysed by DT-diaphorase resulted in generation of a DNA-damaging metabolite(s) able to induce strand breaks and interstrand cross-links in DNA [13]. Studies correlating cellular sensitivity with enzyme expression provided further evidence for the importance of DT-diaphorase in activation of EO9 under aerobic conditions 17, 18, 19, 20, 21 but not in hypoxia [22]. Interestingly, cells expressing low levels of DT-diaphorase were greatly sensitised to EO9 under hypoxic compared with aerobic conditions 19, 23. Also, inclusion of dicoumarol in cytotoxicity assays did not provide complete protection against the cytotoxic effects. These data suggested that other enzymes may also be important in reductive activation of EO9, especially under hypoxia.

Malipaard et al.[24] have shown that xanthine oxidase is able to reduce EO9 to generate a DNA-damaging species. NADPH: cytochrome P450 reductase is known to reduce other bioreductive quinone compounds such as AZQ and mitomycin C 25, 26, 27. Furthermore, recent experiments by Saunders and co-workers [28], involving transfection and overexpression of the NADPH: cytochrome P450 reductase gene, have strongly implicated a role for this enzyme in the cellular reduction of EO9. Here, we provide additional evidence in support of a role for this enzyme in activation of EO9 to free radical and DNA-damaging species. The experiments were carried out under predominantly aerobic conditions and are therefore more directly relevant to cytotoxic effects that EO9 may generate in well-oxygenated normal cells (contributing to toxicity) and in the aerobic population of tumour cells (contributing to therapeutic response). Understanding the enzymology of EO9 metabolism could be useful in the future clinical development of the drug, as well in the development of analogues of EO9 and related indoloquinone bioreductive agents, which continues to be an area of significant activity 8, 28.