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.