THE ROLE OF NAD(P)H - QUINONE REDUCTASE (EC 1.6.99.2, DT-DIAPHORASE) IN THE REDUCTIVE BIOACTIVATION OF THE NOVEL INDOLOQUINONE ANTITUMOR AGENT EO9
THE ROLE OF NAD(P)H - QUINONE REDUCTASE (EC 1.6.99.2, DT-DIAPHORASE) IN THE REDUCTIVE BIOACTIVATION OF THE NOVEL INDOLOQUINONE ANTITUMOR AGENT EO9
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DOI：
10.3727/095535491820873164
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发表时间：
1991-01-01
影响因子：
16.2
通讯作者：
WORKMAN, P
中科院分区：
文献类型：
--
作者：
WALTON, MI;SMITH, PJ;WORKMAN, P
EO 9 [3-羟甲基-5-氮丙啶基-1-甲基-2-（H-吲哚-4，7-茚酮）-丙烯醇]是一种新的吲哚醌，结构上与丝裂霉素C相关，丝裂霉素C是一种需要还原生物活化的醌类抗癌药物。NAD（P）H：（醌受体）氧化还原酶（醌还原酶，DT-心肌黄酶，EC 1.6.99.2）是一种专性2-供电子酶，其可以还原多种醌，导致生物活化或生物保护。使用醌还原酶（QR）制剂从大鼠步行者256乳腺癌细胞和人HT 29结肠癌细胞，我们的特点是这种酶在EO 9还原代谢的作用。在最佳条件下，通过在酶、醌底物、NADH和牛白蛋白存在下在550 nm处进行细胞色素c还原来测定QR活性，并通过在550 nm处损失EO 9吸光度来确认。大鼠和人肿瘤细胞酶都催化基准醌甲萘醌的还原，K（m）相似，为1.4-3.1 μ M，但人制剂的V（max）低7 - 8倍。大鼠步行者QR容易降低EO 9。在约15 μ M时，平均K（m）比甲萘醌高约5倍，在约2.5 μ mol细胞色素c还原mg-1蛋白质时，V（max）低6倍。EO 9也被来自HT 29人结肠癌细胞的QR代谢，但效率低于大鼠肿瘤酶。例如，在对两种制剂的比活性的7- 8倍差异进行校正后，在100 μ M底物浓度下，该速率比步行者肿瘤酶的速率低6倍。通过QR抑制剂双香豆素（100 μ M）抑制大鼠和人肿瘤制剂对EO 9的减少。使用高度纯化的大鼠步行者肿瘤酶，显示EO 9被还原为在体外引起pBR 322质粒DNA单链断裂的物质，如通过琼脂糖凝胶迁移率检测的。该活性在10-50 μ M EO 9之间最大。它完全抑制10 μ M双香豆素和不受超氧化物歧化酶高达2000单位mL-1。这些研究表明，大鼠和人QR在体外减少了EO 9，并且对于大鼠酶，这代表了导致DNA单链断裂的生物活化途径。
EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4,7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar K(m) of 1.4-3.1-mu-M, although the V(max) was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean K(m) was about 5-fold higher than for menadione at around 15-mu-M and the V(max) was 6-fold lower at around 2.5-mu-mol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100-mu-M substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations. Reduction of EO9 by both the rat and the human tumor preparations was inhibited by the QR inhibitor dicoumarol (100-mu-M). Using highly purified rat Walker tumor enzyme, EO9 was shown to be reduced to a species causing single strand breaks in pBR 322 plasmid DNA in vitro, as detected by agarose gel mobility. This activity was maximal at between 10-50-mu-M EO9. It was inhibited completely by 10-mu-M dicoumarol and unaffected by superoxide dismutase up to 2000 Units mL-1. These studies show that rat and human QR reduced EO9 in vitro and that for the rat enzyme this represents a bioactivation pathway which causes DNA single strand breaks.