Guise Chris P, Wang Anderson T, Theil Anke, Bridewell David J, Wilson William R, Patterson Adam V
Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
Biochem Pharmacol. 2007 Sep 15;74(6):810-20. doi: 10.1016/j.bcp.2007.06.014. Epub 2007 Jun 17.
Hypoxia is a common trait found in many solid tumours and thus represents a therapeutic target with considerable potential. PR-104, a hypoxia-activated prodrug currently in clinical trial, is a water-soluble phosphate ester which is converted in vivo to the corresponding alcohol, PR-104A. This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) in hypoxic cells. The clinical effectiveness of PR-104 will depend in part on the expression of reductases within tumours that can effect this reduction. Here, we evaluate the roles of NADPH:cytochrome P450 oxidoreductase (CYPOR; E.C.1.6.2.4) and NAD(P)H:quinone oxidoreductase (NQO1; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQO1 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQO1 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQO1. Overexpression of human CYPOR did, however, increase the hypoxic cytotoxicity of PR-104A, and its metabolism to PR-104H and PR-104M, demonstrating it to be a PR-104A reductase. To assess the contribution of CYPOR to overall activation of PR-104A in hypoxic SiHa cells, a combination of siRNA transfection and antisense expression were used to suppress CYPOR protein by 91% (+/-3%), a phenotype which conferred 45% (+/-7%) decrease in cytotoxic potency of PR-104A. Regression analysis of all CYPOR depletion data was found to correlate with cytoprotection and metabolism (p<0.001). Residual PR-104A reductase activity could be inhibited by the flavoprotein inhibitor diphenyliodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells.
缺氧是许多实体瘤中常见的特征，因此是一个具有相当潜力的治疗靶点。PR - 104是一种目前正在进行临床试验的缺氧激活前药，是一种水溶性磷酸酯，在体内可转化为相应的醇PR - 104A。这种3,5 - 二硝基苯甲酰胺 - 2 - 氮芥在缺氧细胞中通过还原为相应的5 - 羟胺（PR - 104H）和5 - 胺（PR - 104M）而被激活。PR - 104的临床疗效部分取决于肿瘤内能够实现这种还原的还原酶的表达。在此，我们评估了NADPH：细胞色素P450氧化还原酶（CYPOR；E.C.1.6.2.4）和NAD(P)H：醌氧化还原酶（NQO1；E.C.1.6.99.2）作为候选PR - 104A还原酶的作用。在一组8种肿瘤细胞系中观察到NQO1活性与需氧细胞毒性之间存在弱相关性。然而，人NQO1的过表达并未增加PR - 104A的细胞毒性或PR - 104H/M的形成，表明PR - 104A不是NQO1的底物。然而，人CYPOR的过表达确实增加了PR - 104A的缺氧细胞毒性及其向PR - 104H和PR - 104M的代谢，证明它是一种PR - 104A还原酶。为了评估CYPOR对缺氧SiHa细胞中PR - 104A整体激活的贡献，采用siRNA转染和反义表达相结合的方法将CYPOR蛋白抑制91%（±3%），这种表型使PR - 104A的细胞毒性效力降低了45%（±7%）。发现所有CYPOR缺失数据的回归分析与细胞保护和代谢相关（p<0.001）。残留的PR - 104A还原酶活性可被黄素蛋白抑制剂二苯基碘鎓抑制。我们得出结论，CYPOR是一种重要的PR - 104A还原酶，但其他黄素酶也有助于其在缺氧SiHa细胞中的激活。
