Identification of novel enzyme–prodrug combinations for use in cytochrome P450-based gene therapy for cancer
Section snippets
Chemicals
CPA (Cat#C0768), dacarbazine (Cat#D2390), NADPH (Cat#N6505), and troleandomycin (TAO) (Cat#T6514) were purchased from Sigma–Aldrich (St. Louis, MO). IFA was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). MMDX-HCl (PNU-152243) was a gift from Pharmacia & Upjohn (Milan, Italy). Procarbazine (Cat#P6858) and tamoxifen (Cat#T0250) were purchased from LKT Laboratories (St. Paul, MN). Ketoconazole (Cat#30.152.82) was obtained from Research Diagnostics
Evaluation of microsome–prodrug activation using a growth inhibition assay
Evaluation of microsomal CYP–prodrug combinations for potential use in prodrug-activation-based cancer gene therapy was carried out using 9L rat gliosarcoma cells grown in 96-well tissue culture plates. In an initial study, 9L cells were seeded at 0, 250, 450, and 1000 cells/well and allowed to grow for 3, 4, 5, or 6 days, followed by staining with crystal violet. Cell growth was essentially linear over all time points for all cell densities (Fig. 2). The A595 of the stained 1000-cell/well
Discussion
A new generation of treatment strategies is currently being developed to improve the efficacy and reduce the side effects of cancer chemotherapy. One such strategy is cytochrome P450-based prodrug activation gene therapy for cancer treatment [8]. By the delivery of a prodrug-activating CYP gene directly to a tumor, the tumor acquires the capacity for prodrug activation, enabling the localized production of cytotoxic CYP-activated prodrug metabolites. This approach may decrease systemic exposure
Acknowledgements
Supported in part by N.I.H. Grant CA49248 (to D.J.W.). This article is dedicated to Dr. Ronald W. Estabrook in appreciation of the generous support and encouragement that he has extended to young investigators in the field of cytochrome P450 for the past four decades.
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