In vitro and in vivo models for cytochrome P450-based anticancer gene therapy.

摘要

Cyclophosphamide (CPA) is a widely used anticancer prodrug that is bioactivated in a liver cytochrome P450-catalyzed 4-hydroxylation reaction. The therapeutic efficacy of CPA is limited by host toxicity resulting from the systemic distribution of activated drug metabolites produced in the liver. Tumor cell expression of aldehyde dehydrogenase (ALDH), an enzyme that deactivates 4-hydroxylated CPA, decreases CPA's antitumor activity. The major goal of this thesis was to develop in vitro cell and in vivo tumor models to study the impact of expression of a human CPA-metabolizing P450 enzyme, CYP2B6, on antitumor activity and the role of ALDH in drug resistance associated with CPA treatment. Immunodeficient scid mice implanted subcutaneously with 9L gliosarcomas expressing CYP2B6 and NADPH-P450 reductase were shown to serve as a useful model for investigating the effect of P450 expression on tumor sensitivity to CPA. Using this in vivo model, the impact of P450 reductase expression and the effect of CPA delivery by intratumoral injection or continuous drug infusion compared to intraperitoneal injection could be evaluated. Other parameters investigated using this animal model include the bystander cytotoxicity to P450-deficient tumor cells that is associated with CYP2B6 expression, and the use of an alternate CPA-metabolizing P450 gene, CYP2C18. In addition, the anticancer drug, cisplatin, which is not subject to P450 metabolism, was investigated and found to be equally active toward P450-expressing and P450-deficient tumors.; Human tumor cell lines with varying ALDH levels were studied in combination with chemical inhibitors of ALDH to evaluate the impact of ALDH on CPA sensitivity in the context of tumor cell P450 expression. The cooxidation of CPA by prostaglandin H synthase (PGHS) was also studied to ascertain whether PGHS confers cytotoxicity to CPA in cultured tumor cells. While CPA was found to serve as a cosubstrate of PGHS and to undergo 4-hydroxylation, PGHS metabolized CPA at a rate too low to make a significant contribution to CPA metabolism and antitumor activity.