title: Cytochrome P-450 2C9 sensitizes human prostate tumor cells to cyclophosphamide via a bystander effect.
abstract: The goal of the present study was to examine the ability of cytochrome P450-2C9 (CYP2C9) to activate cyclophosphamide (CPA) and elicit tumor cell death. A CYP2C9-deficient human lymphoblastoid cell line (AHH-1 cells) and a derivative cell line (H2C9 cells) stably transfected with a cDNA encoding CYP2C9 were used. The catalytic activity present in cell lines was examined by measuring the conversion of diclofenac, a CYP2C9-specific substrate, to its 4'-hydroxy metabolite by high-pressure liquid chromatography. Initial rate plots were constructed and the maximal rate of formation (V(max)) and the Michaelis-Menten constant (K(m)) for diclofenac metabolism were determined. Cytotoxicity was studied by exposing the cells to 0.01 to 4 mM CPA in the presence or absence of sulfaphenazole, a CYP2C9-specific inhibitor. Cell survival was quantitated by determination of the level of tritiated thymidine incorporation. H2C9 cells quickly metabolized diclofenac, indicating the presence of high levels of CYP2C9. Kinetic experiments demonstrated a V(max) and K(m) of 0.62+/-0.012 pmol/min/10(6) cells and 6.16+/-0.62 microM, respectively, for diclofenac metabolism. Diclofenac 4'-hydroxylase activity was undetectable in AHH-1 cells. H2C9 cells were more sensitive to the cytotoxic effects of CPA (50% inhibitory concentration [IC(50)], 0.80+/-0.03 mM) than AHH-1 cells (IC(50), 4.07+/-0.35 mM). The cytotoxicity (IC(50), 1.99+/-0.14 mM) of CPA to H2C9 cells was blocked by sulfaphenazole, demonstrating that the chemosensitivity of these cells is a consequence of intracellular prodrug activation. H2C9 cells mediated a bystander killing effect for CYP2C9-negative PPC-1 cells, reducing the IC(50) of CPA from about 14 to 3.62+/-0.73 mM in PPC-1 cells when they were cocultured with H2C9 cells. These results suggest that the enzyme-prodrug system of CYP2C9 and CPA may be an effective combination for gene-directed enzyme prodrug therapy. Ongoing studies are examining the utility of this system for use in prostate cancer cells.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, Tennessee 38163, USA.
authors.forename: D
authors.initials: D
authors.lastname: Zhou
authors.forename: Y
authors.initials: Y
authors.lastname: Lu
authors.forename: M S
authors.initials: MS
authors.lastname: Steiner
authors.forename: J T
authors.initials: JT
authors.lastname: Dalton
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D003520:Cyclophosphamide / Q000378:metabolism / Q000494:pharmacology*; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004008:Diclofenac / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D011355:Prodrugs; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D018906:Antineoplastic Agents, Alkylating; D011355:Prodrugs; D004008:Diclofenac; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; C108036:4'-hydroxydiclofenac