The Ca Channel Antagonists Mibefradil and Pimozide Inhibit Cell Growth via Different Cytotoxic Mechanisms

We show that mitogenic cells expressing T-type Ca channels (T-channels) are more sensitive to the antiproliferative effects of the drugs pimozide and mibefradil than cells without significant T-channel expression. The growth of Y79 and WERI-Rb1 retinoblastoma cells, as well as MCF7 breast cancer epithelial cells, all of which express T-channel current and mRNA for T-channel subunits, is inhibited by pimozide and mibefradil with IC50 values between 0.6 and 1.5 M. Proliferation of glioma C6 cells, which show little T-channel expression, is less sensitive to these drugs (IC50 8 and 5 M for pimozide and mibefradil, respectively). Neither drug seems to alter cell cycle or the expression of cyclins. Although this strong correlation between T-channel expression and growth inhibition exists, the following results suggest that the drugs inhibit cell growth via different cytotoxic pathways: 1) pimozide and mibefradil have additive effects on T-channel current inhibition, whereas the antiproliferative activity of the drugs together is synergistic; 2) an increase in the number of apoptotic Y79 and MCF7 cells and a decrease in the mRNA for the antiapoptotic gene Bcl-2 is detected only in pimozide-treated cells, whereas in mibefradiltreated cells, the toxicity is primarily necrotic; and 3) growth inhibition by mibefradil is reduced in Y79 cells transfected with T-channel antisense and in differentiated Y79 cells (which have decreased T-channel expression), but growth inhibition by pimozide is affected to a lesser extent. These results suggest that pimozide and mibefradil inhibit cell proliferation via different cytotoxic pathways and that in the case of pimozide, it is unlikely that this effect is mediated solely by T-channel inhibition. Mibefradil and pimozide share several common biological properties, which include Ca channel blockage and, in some cell types, inhibition of cell growth. Mibefradil, a benzimidazolyl-substituted tetraline derivative similar in structure to verapamil (Clozel et al., 1990), is distinguished from other Ca channel antagonists because it preferentially blocks low-voltage–activated T-type Ca channels (T-channels) with 10 to 20 times more selectivity than high-voltage– activated (HVA) L-type Ca channels (Mishra and Hermsmeyer, 1994; see Lacinová et al., 2000 for a review). Mibefradil seems to bind to a unique receptor site that overlaps with verapamil and indolizine sulfone sites and is also able to interfere with the binding of Ca channel antagonists, such as diltiazem, to dihydropyridine receptors without affecting dihydropyridine binding (Rutledge and Triggle, 1995; Bernink et al., 1996; Glasser, 1998). Mibefradil was introduced clinically in 1997 as an antianginal and antihypertensive agent but was withdrawn from the market less than a year after its release due to potentially life-threatening interactions when mibefradil and -blockers were taken in combination with, or acutely replaced by, dihydropyridine Ca channel blockers (Mullins et al., 1998). The diphenylbutylpiperidine antipsychotic drug pimozide has also been shown to be a potent inhibitor of T-type Ca channels but with less selectivity than mibefradil (Galizzi et al., 1986). In pituitary and heart cells, pimozide inhibits L-type Ca channels (Enyeart et al., 1990), whereas in adrenal glomerulosa and spermatogenic cells, it blocks Tchannels and Ca influx (Enyeart et al., 1993; Arnoult and Florman, 1998). The actions of pimozide and mibefradil are not restricted to Ca channels but may also affect other ion channels, including K (Gomora and Enyeart, 1999) and Cl channels (Nilius et al., 1997). Besides their capacity to inhibit T-current, both drugs inhibit the growth of several cell types (Lee and Hait, 1985; This work was supported by operating grants and salary awards provided by the Canadian Institutes of Health Research, Canadian Breast Cancer Foundation-Atlantic Chapter, and Cancer Research and Education, Nova Scotia. G.E.B. was supported by the Reynolds Fellowship in Pharmacology and a CaRE-Nova Scotia Trainee Award. ABBREVIATIONS: T-channel, T-type Ca channel; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PI, propidium iodide; HVA, high-voltage–activated; T-current, T-type Ca channel current; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s). 0026-895X/02/6202-210–219$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 2 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1676/997367 Mol Pharmacol 62:210–219, 2002 Printed in U.S.A. 210 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from Strobl et al., 1990; Schmitt et al., 1995, 1998). However, the functional role of T-channels in cell growth and the pharmacological properties of these compounds as antiproliferative drugs remain unclear. Recently, it was shown that in cells overexpressing cloned T-channels, there was no alteration in the cell-doubling population time (Chemin et al., 2000), although it has been proposed that the inhibition of smooth muscle proliferation and neointima formation by mibefradil is due to its effect on T-channels (Schmitt et al., 1995). Pimozide has been shown to inhibit tumor cell growth of astrocytoma cells, and the apparent mechanism of action may be via inhibition of calmodulin activity (Lee and Hait, 1985). In breast cancer epithelial cells, the antiproliferative activity of pimozide was thought to be associated with its -2–binding capability (Strobl et al., 1998). In addition to actions on Ca and K channels, other studies documenting the antiproliferative activity of mibefradil on endothelial cells have also suggested that the effect of this drug could be associated with actions on Cl channels (Nilius et al., 1997). T-type Ca channels are the predominant Ca channel expressed in mitogenic Y79 cells and the WERI-Rb1 retinoblastoma cell lines that are derived from human retinoblastoma tumors. The cells arise from a primitive neuroectodermal cell and retain the capability to differentiate to neuron or glia-like cells (Kyritsis et al., 1984). Recently, we demonstrated that T-current and mRNA expression of T-channel genes is diminished when Y79 retinoblastoma cells are induced to exit the cell cycle and differentiate (Hirooka et al., 2002). This suggests that T-channels may be important in membrane potential changes and alterations in Ca that occur during proliferation and differentiation of Y79 retinoblastoma cells. In the present study, we examined the growth-inhibitory actions of the Ca channel blockers pimozide and mibefradil in inhibiting cell growth in Y79 and WERI-Rb1 retinoblastoma cells and compared results with those of MCF7 breast cancer epithelial cells and C6 glioma cells, two cell lines in which pimozide has been previously demonstrated to inhibit cell proliferation (Lee and Hait, 1985; Strobl et al., 1990, 1998). Our studies examined whether mibefradil and pimozide share common cytotoxic mechanisms and whether the observed antiproliferative actions of these agents might involve T-channel inhibition. Materials and Methods Cell Culture. Y79 and WERI-Rb1 (human retinoblastoma), MCF7 (human breast cancer epithelial), and rat glioma C6 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) plus 0.02% glutamine supplemented with 10% fetal bovine serum (Invitrogen, Burlington, ON, Canada), without antibiotics. The cells were kept in a humidified 5% CO2-air atmosphere at 37°C, and the medium was changed twice a week. Chemical differentiation of Y79 cells was performed in 96-well plastic culture dishes. The dishes were pretreated with poly-D-lysine (100 g/ml) at room temperature for 6 min, washed once with DMEM, and coated with laminin (10 g/ml) at 37°C for 30 min. The cells were then rinsed with DMEM and incubated with DMEM plus N2 neuronal supplement (insulin, progesterone, transferrin, selenium, and putrescine; Invitrogen) (Albini et al., 1992). This method yielded approximately 70 to 80% differentiated neuronal-like cells. Pimozide (Sigma-Aldrich), mibefradil (Hoffman-La Roche, Nutley, NJ), and nifedipine (ICN Pharmaceuticals Biochemicals Division, Aurora, OH) were dissolved in dimethyl sulfoxide (DMSO) and diluted to final concentrations in culture medium. Control cells were exposed to the same concentrations of DMSO, which never exceeded