Cytochrome P-450 2C9 sensitizes human prostate tumor cells to cyclophosphamide via a bystander effect.

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Antimicrob Agents Chemother. 2000 Oct;44(10):2659-63.
Cytochrome P-450 2C9 sensitizes human prostate tumor cells to cyclophosphamide via a bystander effect.
Zhou D, Lu Y, Steiner MS, Dalton JT.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, Tennessee 38163, USA.
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