Unusually marked hypoxic sensitization to indoloquinone E09 and mitomycin C in a human colon‐tumour cell line that lacks DT‐diaphorase activity

Abstract

Studies with purified DT-diaphorase have shown that the enzyme is capable of catalyzing a two-electron reduction of the novel indoloquinone E 0 9 to a DNA-damaging alkylating species. The aim of this study was to determine to what extent DT-diaphorase may be involved in the metabolic activation of E09 and mitomycin C in both aerobic and hypoxic conditions. Two human colon-carcinoma cell lines were used; H l 2 9 has high levels of DT-diaphorase whilst BE lacks this activity because of a point mutation in the NQOl gene. In aerobic conditions the 2 cell lines show similar sensitivities to a number of qtotoxic drugs including cisplatin, doxorubicin and etoposide. They are equally sensitive to the benzotriazine di-N-oxide SR 4233 but H R 9 is more sensitive than BE to mitomycin C and E09. Sensitivity to SR 4233 is increased by about 100-fold for both cell lines in hypoxic conditions. DT-diaphorase-deficient BE cells show markedly increased sensitivity to mitomycin C and particularly E 0 9 in hypoxic conditions, whereas DTdiaphorase-rich H R 9 cells show little hypoxic sensitization to these agents unless exposed in the presence of dicoumarol.These results suggest that DT-diaphorase can reduce E09 and mitomycin C to potent cytotoxic species in aerobic conditions, and this activii predominates over the one-electron-reducing enzymes even in hypoxic conditions. In the absence of DTdiaphorase activity, E 0 9 and mitomycin C are reduced in hypoxic conditions, presumably by one-electron-reducing enzymes, to a similar or greater extent than is achieved with DT-diaphorase.8 1994 Wfq-Liss, Znc.The presence of hypoxic areas in solid tumours suggests a target for the development of selective anti-cancer agents. The main classes of bioreductive agents which show specificity for hypoxic cells are quinone, nitro and N-oxide compounds (Workman, 1992). Hypoxic cell specificity is dependent on preferential metabolic activation by reductase enzymes in hypoxic conditions (Workman, 1992). Mitomycin C is the prototype bioreductive agent which shows clinical activity against some solid tumours but exhibits little selectivity towards hypoxic tumour cells (Stratford and Stephens, 1989; Workman, 1992). It (Oostveen and Speckamp, 1987) is structurally related to mitomycin C (Fig. l), is less myelosuppressive (Hendriks et al., 1993) and is in clinical trial in Europe under the auspices of the EORTC. E09 also requires metabolic activation and has been shown to be a much better substrate for both human and rat DT-diaphorase than mitomycin C (Walton et al., 1991).In hypoxic conditions one-electron reduction of quinone bioreductive agents by enzymes such as cytochrome P450 reductase and xanthine oxidase results in the production of semiquinone free radicals. However, in aerobic conditions toxic oxygen radicals are produced due to back oxidation of these unstable metabolites. In contrast, obligate two-electron reduction by DT-diaphorase (Iyanagi and Yamazaki, 1970) results in production of more stable hydroquinone metabolites under both aerobic an...

Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators

“…1B), there was a ∼10-fold difference in aerobic sensitivity between the most sensitive (RIF-1) and resistant (HT29 and SKOV3) cell lines. The similar pattern for the nitro and amino compounds suggests that the differences in aerobic sensitivity to the nitro prodrugs are not a result of their aerobic activation in a subset of cell lines, unlike the situation with quinone or dinitrobenzamide HAPs, which are activated by variably expressed two-electron reductases (10,12). Consistent with this, the lower hypoxic selectivity of the nitro prodrugs in HT29 relative to UV4 and RIF-1 reflects resistance under hypoxia rather than sensitivity under aerobic conditions [as would occur if, for example, the high activity of DT-diaphorase in HT29 cells (10) resulted in two-electron activation].…”

“…These hypoxia-activated prodrugs (HAP) act as direct oxygen sensors through the ability of O 2 to inhibit the first step in their reductive metabolism, usually by reoxidizing the initial radical intermediate (9). Several HAPs are currently in clinical trial (N-oxides tirapazamine and banoxantrone, nitro compounds PR-104 and TH-302, and quinones apaziquone and RH1), and the approved drug mitomycin C has weak selectivity for hypoxic cells (10).…”

Preclinical Evaluation of the Pharmacodynamic Properties of 2,5-Diaziridinyl-3-Hydroxymethyl-6-Methyl-1,4-Benzoquinone

“…A possibility supported by the fact that cell lines with very low NQO1 but overexpressing P450 reductase do not show enhanced sensitivity to RH1 (49). NQO1 has been reported to be up-regulated under hypoxic conditions (33,34). Consequently, under hypoxia, RH1 is likely to be more stable following any one-electron reduction.…”

“…Whereas numerous in vitro-based studies have shown that NQO1 plays a key role in the mechanism of action of a number bioreductive drugs, such as clinically active mitomycin C (MMC; 14, 24 -28), it is clear that there exits a far from simple relationship between in vivo drug activity and clinical efficacy and NQO1 expression (29 -32) both under hypoxic and aerobic conditions (33,34).…”

In vivo Role of NAD(P)H:Quinone Oxidoreductase 1 in Metabolic Activation of Mitomycin C and Bone Marrow Cytotoxicity

“…The cellular studies using dicumarol as NQO1 enzyme inhibitor indicated that bioactivation by the obligatory two-electron reducing enzyme NQO1 might be especially important for the cytotoxicity of mitomycin C (2,7,8). On the other hand, the cells expressing higher levels of NQO1 failed to show increased sensitivity to mitomycin C compared with cells of similar origin containing normal levels of NQO1 (9,10). The cellular studies have shown a role of NQO2 in metabolic activation of mitomycin C and other drugs leading to cytotoxicity and cell death (11,12).…”