DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9

Abstract

Summary Aerobic sensitivity to indoloquinone E09 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one-and two-electron reducing enzymes in the hypoxic cytotoxicity of E09. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to E09. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15-and 25-fold range respectively. The aerobic cytotoxicity of E09 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to E09 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to E09. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to E09, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of E09. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of E09 in hypoxic conditions. This suggests either that the one-electron reduction product of E09 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.

Development and validation of a spectrophotometric assay for measuring the activity of NADH: cytochrome b5 reductase in human tumour cells

“…Methods reported in the literature do not appear to have been validated, i.e. the non-dicoumarol-inhibitable reduction of cytochrome c (Segura-Aguilar et al, 1990) or the NADHdependent reduction of cytochrome c (Plumb et al, 1994), and assume that cytochrome b5 reductase is the only enzyme responsible for the NADH-dependent reduction of cytochrome c. Our data indicate that this assumption is not valid. From Figure 4 it is evident that cytochrome b5 reductase activity does not account for all of the NADH-dependent reduction of cytochrome c in any cell line, as assessed using pHMB as a selective inhibitor.…”

“…For example, the levels of DTdiaphorase have been shown to vary 10 000-fold in a panel of 23 tumour cell lines (Robertson et al, 1994) and also to be elevated in tumour tissue compared with surrounding noncancerous tissue (Riley and Workman, 1992). The aerobic toxicity of the indoloquinone E09 correlates highly with intracellular DT-diaphorase activity (Plumb et al, 1994;Robertson et al, 1994). Therefore, E09 should be targeted at tumours with high levels of DT-diaphorase, and would be expected to be of litle or no therapeutic benefit when used as a single agent to treat tumours with low DT-diaphorase levels.…”

“…the residual activity observed in the presence of dicoumarol) (Segura-Aguilar et al, 1990) or the NADH-dependent reduction of cytochrome c (i.e. in the absence of menadione) (Plumb et al, 1994) are equivalent to cytochrome b5 reductase activity. However, neither of these HM Barham et al S 1189 Figure 2 and discussed in the Results section.…”

Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro

“…The similar DNA damage and sensitivity of each of the human and each of the murine cell lines suggests that they must possess similar levels of activity of the reductase(s) that activates tirapazamine to a cytotoxic species. However, in the clinic, wide variations in reductase activity, as has been reported for human tumour cell lines (Plumb et al, 1994;Robertson et al, 1994), and varying levels of hypoxia , would be expected to result in large differences in sensitivity to tirapazamine. It is of primary importance that in this study, by measuring DNA damage, it was possible to distinguish between cell lines with 2-to 3-fold differences in sensitivity to killing by tirapazamine under hypoxic conditions.…”

“…The response of a tumour to any such bioreductive drug will depend both on the level of activity of the reductase(s) that activate the drug to a cytotoxic species, and the level of tumour hypoxia. Recent studies have shown that reductase activity can vary substantially between different human tumour cell lines (Plumb et al, 1994;Robertson et al, 1994), and measurements made using oxygen electrodes show large variations in oxygen tensions between tumours . It would therefore be highly desirable to be able to predict the response of individual tumours to a bioreductive drug so that patients unlikely to benefit from the drug would not be treated with it, and further, the power of a clinical trial of the drug to produce a significant response would be improved.…”