Abstract
BackgroundCytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37).MethodsTo further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase.ResultsThe expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 "factory" cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing.ConclusionsThe introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products.
Highlights
Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism
Characterization of Adeno-2B6/p35 Adeno-2B6/p35 was prepared by introducing the pancaspase inhibitor p35 into Adeno-2B6, which expresses P450 enzyme cytochrome P450 2B6 (CYP2B6) and its redox partner P450 reductase [24] (Figure 1A)
Levels of CYP2B6 protein 48 and 72 hr post infection (Figure 1B, top). p35 protein was only detected in cells infected with Adeno-2B6/p35 (Figure 1B, bottom). quantitative real-time PCR (qPCR) analysis confirmed similar expression levels of CYP2B6 in cells infected with each virus, whereas p35 transcripts were only detectable in the Adeno-2B6/p35-infected cells
Summary
Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Gene-directed enzyme prodrug therapy (GDEPT) for cancer is designed to increase the chemotherapeutic sensitivity of tumor cells by introduction of a gene coding for a prodrug activation enzyme. GDEPT provides the potential to improve cancer therapy by sensitizing tumor tissue to a chemotherapeutic prodrug This strategy may allow for a reduction in drug dosages needed to achieve therapeutic activity, thereby decreasing systemic side effects towards critical host tissues, such as bone marrow, kidney and heart [6,7].
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