Abstract
Aerobic sensitivity to indoloquinone EO9 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 EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. 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 EO9 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 EO9 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 EO9. 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 EO9, 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 EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 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.
Highlights
Since the two cell lines are sensitive to a range of other cytotoxic drugs, we suggested that this could indicate that the one-electron reduction product, the semiquinone, is more cytotoxic than the two-electron reduction product, the hydroquinone
While our results support the correlation between levels of DT-diaphorase and sensitivity to E09 in air, we show for the first time that DT-diaphorase appears to protect cells from the toxicity of E09 under hypoxic conditions
We propose that the semiquinone metabolite of E09 may be the cytotoxic entity under both aerobic and hypoxic conditions
Summary
Chemicals cis-Diamminedichloroplatinum(II) (cisplatin), menadione, 3(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytochrome c, dicoumarol, NADH and NADPH were purchased from Sigma (Poole, Dorset, UK). E09 was provided by the New Drug Development Office of the EORTC and tirapazamine (SR 4233, WIN 59075) was kindly donated by Drs M. Tracy and W.W. Lee of SRI International (Menlo Park, CA, USA). BCNU was obtained from the US National Cancer Institute
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