Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A:: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia
Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A:: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia
复制标题
DOI：
10.1016/j.bcp.2007.06.014
复制
发表时间：
2007-09-15
影响因子：
5.8
通讯作者：
Patterson, Adam V.
中科院分区：
文献类型：
--
作者：
Guise, Chris P.;Wang, Anderson T.;Patterson, Adam V.
低氧是在许多实体肿瘤中发现的共同特征，因此代表着一个具有相当大潜力的治疗靶点。PR-104是一种目前正在临床试验的缺氧激活前药，是一种在体内转化为相应酒精的水溶性磷酸酯，PR-104A。这种3，5-二硝基苯甲酰胺-2-氮芥末在低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的临床疗效将在一定程度上取决于肿瘤内可实现这种减少的还原酶的表达。在这里，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：苯醌氧化还原酶(NQ01；E.C.1.6.99.2)作为PR-104A还原酶候选基因的作用。在一组八种肿瘤细胞系中，观察到NQ01活性和需氧细胞毒性之间的弱相关性。然而，过表达人NQ01并没有增加PR-104A的细胞毒性或PR-104H/M的形成，表明PR-104A不是NQ01的底物。然而，过表达人细胞色素P450受体确实增加了PR-104A的缺氧细胞毒性，并增加了它对PR-104H和PR-104M的代谢，证明它是PR-104A还原酶。为了评价CypoR在缺氧SIHA细胞中对PR-104A整体激活的作用，采用siRNA转染和反义表达相结合的方法将CYPOR蛋白抑制91%(+/-3%)，这一表型使PR-104A的细胞毒力降低45%(+/-7%)。对所有CYPOR耗竭数据的回归分析发现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苯并多胺抑制。我们的结论是，CYPOR是一种重要的PR-104A还原酶，但在缺氧的SiHA细胞中，其他黄素酶也参与了它的激活。(C)2007 Elsevier Inc.保留所有权利。
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 (NQ01; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQ01 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQ01 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQ01. 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 diphenyhodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells. (C) 2007 Elsevier Inc. All rights reserved.