Baldwin Alex, Huang Zeqi, Jounaidi Youssef, Waxman David J
Division of Cell and Molecular Biology, Department of Biology, Boston University, 5 Cummington St., MA 02215, USA.
Arch Biochem Biophys. 2003 Jan 1;409(1):197-206. doi: 10.1016/s0003-9861(02)00453-8.
Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.
基因导向酶前药疗法可用于增强抗癌前药的治疗活性，这些前药需经肝细胞色素P450（CYP）催化从前药转化为活性药物。本报告描述了一种基于细胞培养的检测方法，用于鉴定能产生旁观者细胞毒性代谢物的CYP基因 - CYP前药组合，这些组合可能对基于CYP的癌症基因治疗有用。在一项为期四天的9L胶质肉瘤细胞生长抑制检测中，评估了一组包含不同药物诱导型肝CYP亚群的大鼠肝微粒体对前药的激活作用。对于CYP前药环磷酰胺、异环磷酰胺和甲氧基吗啉基阿霉素（MMDX），观察到9L细胞毒性有强烈的NADPH和肝微粒体依赖性增加，但另外三种CYP前药丙卡巴肼、达卡巴嗪和他莫昔芬则没有这种现象。地塞米松诱导的大鼠肝微粒体使MMDX的激活增强了约250倍（MMDX的IC(50)约为0.1nM），这表明地塞米松诱导型CYP3A酶有助于激活这种新型蒽环类抗肿瘤药物。在使用未诱导的雄性和雌性大鼠肝微粒体的研究中，以及通过使用CYP3A选择性抑制剂三乙酰竹桃霉素和酮康唑，验证了这种对CYP3A的依赖性。这些发现突出了使用细胞培养检测方法来鉴定新型CYP前药 - CYP基因组合的优势，这些组合的特点是产生可渗透细胞的细胞毒性代谢物，并且可能被纳入基于CYP的癌症治疗基因疗法中。
