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Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy

Cancer Gene Therapy volume 10, pages 571–582 (2003)Cite this article

Abstract

Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity. While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated. The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time. 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time- and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity. This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content. By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment. This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells. At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially. This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPA-induced apoptotic death. These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 gene-directed enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue.

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Abbreviations

P450:

cytochrome P450

CPA:

cyclophosphamide

4-OH-CPA:

4-hydroxy-cyclophosphamide

GDEPT:

gene-directed enzyme prodrug therapy

9L/P450 cells:

9L gliosarcoma cells that stably express human P450 2B6 and human P450 reductase

AUC:

area under the curve

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Acknowledgements

This work was supported in part by NIH grant CA49248 (to DJW).

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  1. Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, 02215, Massachussetts, USA

    Pamela S Schwartz, Chong-Sheng Chen & David J Waxman

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  1. Pamela S Schwartz
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Correspondence to David J Waxman.

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Schwartz, P., Chen, CS. & Waxman, D. Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy. Cancer Gene Ther 10, 571–582 (2003). https://doi.org/10.1038/sj.cgt.7700601

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