Plumb J A, Gerritsen M, Workman P
CRC Department of Medical Oncology, University of Glasgow, Bearsden, UK.
Br J Cancer. 1994 Dec;70(6):1136-43. doi: 10.1038/bjc.1994.461.
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.
已证明对吲哚醌EO9的需氧敏感性与双电子还原酶DT-黄递酶的细胞水平相关。然而，关于单电子和双电子还原酶在EO9缺氧细胞毒性中的相对作用，人们了解甚少。我们已对一组23种人类肿瘤细胞系的生物还原酶活性和对EO9的需氧敏感性进行了表征。然后选择了8种细胞系来比较需氧和缺氧敏感性。DT-黄递酶的活性范围很广（>10,000倍），而单电子还原细胞色素b5和细胞色素P450还原酶的活性通常较低，分别仅显示15倍和25倍的范围。EO9的需氧细胞毒性显然与DT-黄递酶的细胞水平相关（r = 0.87），水平越高敏感性越高，但与单电子还原酶的水平无关。相比之下，对卡莫司汀、顺铂或生物还原剂SR 4233（替拉扎明）的敏感性与这些还原酶中任何一种的活性之间均无关系。在缺氧条件下，DT-黄递酶活性低的细胞系对EO9的敏感性显著增加，而DT-黄递酶活性高的细胞系敏感性仅略有增加。这通过细胞DT-黄递酶活性与对EO9的需氧敏感性与缺氧敏感性之比之间的明显相关性（r = 0.98）得以体现。然而，我们现在首次证明了DT-黄递酶的细胞活性与对EO9的缺氧敏感性之间呈负相关（r = 0.93），即敏感性随DT-黄递酶活性的增加而降低。此外，当细胞在双香豆素存在下接触药物时，这种相关性消失了，这支持了DT-黄递酶参与这种关系。这些观察结果对DT-黄递酶在EO9代谢活化中先前直接的作用提出了质疑。虽然DT-黄递酶与空气中毒性增加有关，但它似乎在缺氧条件下降低了EO9的细胞毒性。这表明要么EO9代谢的单电子还原产物半醌比双电子还原产物对苯二酚毒性更大，要么对苯二酚没有细胞毒性，需氧毒性是由于对苯二酚回氧化时半醌的短暂出现所致。
