Potentiation of cytochrome P450/cyclophosphamide-based cancer gene therapy by coexpression of the P450 reductase gene.

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.