Effects of Ketoconazole on the Proliferation and Cell Cycle of Human Cancer

M A T E R I A L S AND M E T H O D S The growth-inhibitory effects of ketoconazole, an antifungal agent which inhibits arachid0nic acid iip0xygenases and cyt0chr0me P-450 enzymes, were tested in human colon and breast cancer cell lines. In the serum independent HT29-S-B6 colon cell clone, ketoconazole reduced cell proliferation and [3H]thymidine incorporation in a dose-dependent fashion, with a 50% inhibitory concentration of approximately 2.5 ~M. Flow cytometry showed an accumulation of cells in the Go-Gt phase of the cell cycle and a concomitant decrease of the percentage of cells in S phase. Ketoconazole also inhibited [3H]thymidine incorporation in the hormone-independent breast cancer cells MDA-MB-231 and Evsa-T, with respective 500 inhibitory concentrations of approximately 13 and 2 ~M. The mechanism of action of ketoconazole is unknown. However, another lipoxygenase inhibitor, BW755C, inhibited only weakly [3H]thymidine incorporation and accumulated the cells in S and G2. Conversely, clotrimazole and SKF525A, inhibitors of cytochrome P-450 enzymes, had effects similar to those of ketoconazole on HT29-S-B6 cells whereas metronidazole and secnidazole, other azole derivatives which do not inhibit cytochrome P-450 enzymes, had no effect. The results suggest that cytochrome P-450 enzyme(s) activity(ies) could be implicated in the antiproliferative effects of ketoconazole. I N T R O D U C T I O N Ketoconazole, an antifungal drug (1), blocks steroidogenesis (2) by inhibit ion of the cholesterol side chain cleavage (3) and has been used for this reason in the t reatment of hormonedependent prostate cancer (4, 5). Besides its indirect effect on hormone-dependent cancers, ketoconazole has been reported to inhibit hepatic metastasis from a human pancreatic adenocarc inoma in the nude mouse (6) and to reduce the incidence of pulmonary metastases in a mouse melanoma (7). In addition, ketoconazole has been found to exert a cytotoxic effect in various cancer cell lines [breast, colon, pancreas, prostate, and leukemia[, as indicated by clonogenicity assay in soft agar (8), and to potentiate the an t i tumor effect of interleukin l a on murine RIF tumors [mainly by inhibiting the secretion of cort icosterone (9)]. The mechanism of action of ketoconazole in the hormoneindependent cell lines has not been elucidated. Ketoconazole inhibits the activities of cytochrome P-450 enzymes (2, 10, 11) and of arachidonic acid lipoxygenases (12) and has antiglucocorticoid properties (13); these processes may be indispensable for mitogenic signaling or for cell cycle progression. In the work reported here, we have studied the effect of ketoconazole in three cell lines: (a) the clone HT29-S-B6, derived from the human colon adenocarcinoma cell line HT29, and able to proliferate and to be subcultured in a defined medium without exogenous growth factors (14); and (b) two (estrogen-independent) human breast cancer cell lines (Evsa-T and MDAMB-231). Received 6/8/92; accepted 10/2/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by INSERM. 2 To whom requests for reprints should be addressed. Materials. Ketoconazole was from Sigma Chemical Co., St. Louis, M0; secnidaz01e and metr0nidaz01e were from Rh0ne-e0ulenc, Vitrysur-Seine, France; clotrimazole was from Laboratoires Roger Bellon, Neuilly-sur-Seine, France; SKF525A was from Smith, Kline and French Laboratories, Welwyn Garden, England; BW755C was from Wellcome Research Laboratories, Beckenham, England; and RU 38486 was from RousseI-UCLAF, Romainsville, France. All other materials were of reagent grade. Cell Culture. The subline HT29-S derived from HT29 cells and adapted to serum-free culture (14) was cloned by limiting dilution in the presence of serum. Clone B6, passages 5 to 30, was used throughout the present study. The HT29-S-B6 cells were maintained in Dulbecco's modified Eagle's medium: Ham F-12 nutrient mixture (50/50; Seromed, ATGC, Noisy-le-Grand, France), supplemented with 10 mM glutamine, glucose to a final concentration of 4.5 g/liter, nonessential amino acids, and 15 #g/ml iron-saturated transferrin (Boehringer Mannheim, Mannheim, Germany), at 37~ in a humidified atmosphere of 5% CO2 in air. Stock cultures were passaged each week and the medium was changed every 1-2 days. Evsa-T and MDA-MB-231 cell lines were cultured in Dulbecco's modified Eagle's medium:Ham's F12 medium (50:50) containing 5% fetal calf serum (Boehringer Mannheim). The BALB/c mouse 3T3 A31 fibroblasts were cultured in a-minimal essential medium (Gibco-BRL, Cergy-Pontoise, France) supplemented with 6% fetal calf serum. Evaluation of the Rate of Cell Proliferation: Growth Curves, [3H]Thymidine Incorporation, and Flow Cytometry. HT29-S-B6 cells (5 x 105) were plated in 35-mm Petri dishes. The next day, the medium was changed and effectors were added in a small volume (10-20~1). The incubation medium was renewed every day during the experiments. The same triplicate dishes were used for cell counts, [3H]thymidine incorporation, and flow cytometry. [3H]Thymidine (0.5 #Ci) was allowed to incorporate for 24 h; at the end of incubation, cells were rinsed with 1 ml of medium, detached with 1 ml of trypsin-EDTA, and diluted (1:3) with the culture medium. An aliquot (0.5-1 ml) was used for cell count with a Coulter Counter (Coultronics, Luton, England). Another cell aliquot (0.5 ml) was precipitated with cold trichloroacetic acid (5% final concentration) and the remaining cells were pooled for flow cytometry analysis. The aliquots in trichloroacetic acid were allowed to precipitate overnight at 4~ and then pelleted and dissolved in 0.5 ml of 0.1 M NaOH-0.1% sodium dodecyl sulfate; the associated radioactivity was counted in a beta scintillation spectrometer. Cells sampled for flow cytometry were pelleted, resuspended in I ml of PBS,3 fixed by addition of 2 ml of absolute ethanol, and stored at 4~ until use. On the day of analysis, the cells were pelleted and resuspended to a final concentration of 1-2 x l06 cells/ml in PBS containing 0.2% Triton X-100; then 1 mg/ml RNase (Sigma) and 20 #g/ml propidium iodide were added. After a 15-30-min incubation at room temperature, the suspension was analyzed at a rate of about 500 cells/s in the flow cytometer (Orthocytofluorograph 50H; Ortho Instruments, Ortho Diagnostic Systems, Westwood, MN). Excitation was at 488 nm wavelength from a 3-W argon ion laser operating at 500 mW. The instrument is standardized every day for a mean channel fluorescence with fluorescent microspheres. Aggregates were gated out by using pulse shape analysis (peak versus area fluorescence cytograms). The red fluorescence was detected in linear mode above 550 nm wavelength. All data were collected in list 3 The abbreviations used are: PBS, phosphate-buffered saline; FCS, fetal calf serum; IC5o, 50% inhibitory concentration. 6827 Research. on January 13, 2018. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from