Chen L, Yu L J, Waxman D J
Department of Biology, Boston University, Massachusetts 02215, USA.
Cancer Res. 1997 Nov 1;57(21):4830-7.
Intratumoral expression of cytochrome P450 2B1 sensitizes tumor cells to the cytotoxic action of the alkylating agent prodrug cyclophosphamide (CPA) and provides a novel strategy for cancer gene therapy that may enhance the selectivity and the effectiveness of this class of antitumor drugs [L. Chen and D. J. Waxman, Cancer Res., 55: 581-589, 1995]. P450-catalyzed drug metabolism is obligatorily dependent on electron input from the flavoenzyme NADPH-P450 reductase (RED), which is widely expressed in many cell types, including tumor cells. Here, we investigate the potential utility of combining RED gene transfer with CPA-based P450 gene therapy. Rat 9L gliosarcoma cells stably expressing either basal or elevated (up to 10-fold increase) levels of RED, in the presence or absence of P450 2B1, were selected and characterized. RED overexpression substantially increased the sensitivity of these cells to CPA, but only when combined with P450 2B1 expression. An enhanced cytotoxic response was also obtained when recombinant adenovirus encoding P450 2B1 was used to deliver the P450 gene to RED-overexpressing tumor cells. CPA cytotoxicity was substantially decreased by the RED inhibitor diphenyleneiodonium chloride or by the P450 inhibitor metyrapone, providing evidence of its dependence on the catalytic contributions of both protein components of the P450 metabolic pathway. Conditioned media from P450 2B1-expressing and RED-overexpressing tumor cells treated with CPA exhibited increased formation of the primary 4-hydroxy metabolite and greater cell contact-independent bystander cytotoxic potential compared to tumor cells containing P450 2B1 and basal levels of RED. Evaluation of the impact of P450/RED combination gene therapy using a s.c. solid tumor model/tumor excision assay revealed a dramatic 50-100-fold increase in tumor cell kill in vivo over that provided by liver drug activation alone. These findings establish the importance of endogenous RED levels as a determinant of the sensitivity of tumor cells to CPA/P450 gene therapy and demonstrate the striking therapeutic effectiveness of an anticancer prodrug activation strategy based on the combination of a cytochrome P450 gene with the gene encoding RED.
细胞色素P450 2B1在肿瘤内的表达使肿瘤细胞对烷化剂前药环磷酰胺（CPA）的细胞毒性作用敏感，并为癌症基因治疗提供了一种新策略，该策略可能会提高这类抗肿瘤药物的选择性和有效性[L. Chen和D. J. Waxman，《癌症研究》，55: 581 - 589，1995]。P450催化的药物代谢必然依赖于黄素酶NADPH - P450还原酶（RED）输入的电子，RED在包括肿瘤细胞在内的许多细胞类型中广泛表达。在此，我们研究将RED基因转移与基于CPA的P450基因治疗相结合的潜在效用。选择并鉴定了在有或无P450 2B1的情况下稳定表达基础水平或升高水平（高达10倍增加）RED的大鼠9L胶质肉瘤细胞。RED的过表达显著增加了这些细胞对CPA的敏感性，但仅在与P450 2B1表达相结合时才会如此。当使用编码P450 2B1的重组腺病毒将P450基因传递给RED过表达的肿瘤细胞时，也获得了增强的细胞毒性反应。RED抑制剂二苯基碘鎓氯化物或P450抑制剂美替拉酮可显著降低CPA的细胞毒性，这证明了其对P450代谢途径两种蛋白质成分催化作用的依赖性。与含有P450 2B1和基础水平RED的肿瘤细胞相比，用CPA处理的表达P450 2B1和RED过表达的肿瘤细胞的条件培养基显示出初级4 - 羟基代谢物的形成增加，并且具有更大的细胞非接触依赖性旁观者细胞毒性潜力。使用皮下实体瘤模型/肿瘤切除试验评估P450/RED联合基因治疗的影响，结果显示体内肿瘤细胞杀伤比单独的肝脏药物激活增加了50 - 100倍。这些发现确立了内源性RED水平作为肿瘤细胞对CPA/P450基因治疗敏感性决定因素的重要性，并证明了基于细胞色素P450基因与编码RED的基因相结合的抗癌前药激活策略具有显著的治疗效果。
