Cummings J, Allan L, Willmott N, Riley R, Workman P, Smyth J F
Imperial Cancer Research Fund, Western General Hospital, Edinburgh, U.K.
Biochem Pharmacol. 1992 Dec 1;44(11):2175-83. doi: 10.1016/0006-2952(92)90344-i.
We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.
我们之前报道过，Sp 107大鼠乳腺癌中存在的酶可在体内催化阿霉素醌还原（QR）生成7-脱氧糖苷配基代谢物[威尔莫特和卡明斯，《生物化学与药物学》36: 521 - 526，1987]。为了深入了解QR在阿霉素抗肿瘤作用机制中的作用，我们在这项研究中试图鉴定负责该反应的酶。NAD(P)H:（醌受体）氧化还原酶（DT - 黄递酶）是肿瘤中的主要醌还原酶，占微粒体和胞质溶胶中测得的所有活性的约70%（微粒体活性，28.4±4.6 nmol/分钟/毫克；胞质溶胶活性，94.3±11.9 nmol/分钟/毫克）。通过蛋白质印迹分析证实了它的存在。在微粒体中可检测到低水平的NADH细胞色素b5还原酶（15.6±6.3 nmol/分钟/毫克）和NADPH细胞色素P450还原酶（14.5±4.0 nmol/分钟/毫克）。通过蛋白质印迹分析证实了后者的存在。用治疗剂量的阿霉素（48小时）预处理肿瘤，使所研究的所有还原酶的活性水平至少降低了2倍（P < 0.01，学生t检验）。已证明阿霉素不是纯化的大鼠沃克256肿瘤DT - 黄递酶以NADH或NADPH为辅因子时的底物，即使使用高达20,000单位的酶/孵育，但证实它是纯化的大鼠肝细胞色素P450还原酶的底物。纯化的细胞色素P450还原酶形成7 - 脱氧糖苷配基代谢物绝对需要NADPH作为辅因子，受到分子氧和双香豆素的抑制（IC50约为50 microM），并受特异性还原酶抗血清调节。阿霉素还原脱糖基化为7 - 脱氧糖苷配基定位于Sp 107肿瘤的微粒体部分，在胞质溶胶（以NADH、NADPH和次黄嘌呤为辅因子）和线粒体（以NADH和NADPH为辅因子）中活性可忽略不计。肿瘤微粒体酶绝对需要NADPH作为辅因子，受到氧和双香豆素的抑制，并受细胞色素P450还原酶抗血清调节。这些数据有力地表明，NADPH细胞色素P450还原酶是负责催化Sp 107肿瘤中阿霉素QR的主要酶。
