Bioreductive activation of mitomycin C by DT-diaphorase

Abstract

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.

Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

“…However, MMC is a poor substrate for NQO1 and substrate concentration may be the rate-limiting factor for reduction of MMC catalysed by either NQO1 or NQO2. The original observation of MMC reduction catalysed by NQO1 used 5.2 mg/ml (169 nM) purified rat hepatic NQO1 (Siegel et al, 1992) and this is comparable with the 162 nM hrNQO2 or hrNQO1 used in the present study. Despite the nonphysiological conditions used in the cell-free systems described here, NQO2 expression in an isogenic cell model also resulted in more DNA alkylation, as measured by the comet assay, than was seen in an NQO1 and NQO2 null background.…”

“…As a prodrug, the parent compound is relatively nontoxic compared to the highly electrophilic metabolites formed following sequential or simultaneous two electron reduction of the quinone to the hydroquinone (Iyer and Szybalski, 1963). Many enzymes have been implicated in the catalysis of MMC including NQO1 (Siegel et al, 1992) and interest in NQO1-directed cancer chemotherapy arose following the observation that NQO1 is more highly expressed in tumour tissue when compared with matched healthy tissue, for example lung (Schlager and Powis, 1990).…”

“…A correlation between MMC GI 50 and NQO1 activity in the National Cancer Institute 60 cell line panel presented evidence of a more heterogeneous nature that NQO1 has a role in MMC bio-activation (Fitzsimmons et al, 1996). Although MMC has been demonstrated to be a substrate for NQO1, a large amount of enzyme and an acidic pH are needed to see a reaction, indicating that MMC is a poor substrate for NQO1 (Siegel et al, 1992). An added level of complexity is that, when activated, MMC alkylates NQO1, irreversibly inhibiting the enzyme (Siegel et al, 1993).…”

Presence of a heterozygous substitution and its relationship to DT-diaphorase activity

“…NQOI is an inducible homodimenrc enzyme in the active state. NQOI is believed to mediate most cellular quinone reduction since quinone-containing compounds are good substrates for purified human NQO, Siegel et al, 1992;Ross et al, 1993). NQO2 also appears to be an inducible enzyme which is 54% similar to NQOI at the cDNA level (Jaiswal et al, 1990;.…”

DT-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents?

“…DT-diaphorase (DTD, ECI.6.99.2) is considered to be the most important enzyme for the activation of bioreductive drugs such as E09 and diaziquone (AZQ); at low pH it can also reduce mitomycin C (MMC) [Siegel et al, 1990;Walton et al, 1991;Riley and Workman, 1992;Siegel et al, 1992;Workman, 1992]. DTD is present in many mammalian tissues, where it can play a role in the biosynthesis of vitamin K (Wallin et al, 1978;Ernster, 1987), or may act as a detoxifying enzyme, by catalysing a strict two-electron reduction (Benson et al, 1980;Ernster, 1987).…”