The Ca 2+ Channel Antagonists Mibefradil and Pimozide Inhibit Cell Growth via Different Cytotoxic Mechanisms GABRIEL E. BERTOLESI, CHANJUAN SHI, LINDSY ELBAUM, CHRISTINE JOLLIMORE, GABRIELA ROZENBERG, STEVEN BARNES, and MELANIE E. M. KELLY Laboratory of Retina and Optic Nerve Research, Departments of Ophthalmology (G.E.B., C.S., C.J., S.B., M.E.M.K.), Pharmacology (G.E.B., C.S., L.E., C.J., M.E.M.K.), and Physiology & Biophysics (G.E.B., G.R., S.B.), Dalhousie University, Halifax, Nova Scotia, Canada Received February 15, 2002; accepted April 25, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT We show that mitogenic cells expressing T-type Ca 2+ 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 reti- noblastoma 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 IC 50 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 (IC 50 = 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 antiprolif- erative activity of the drugs together is synergistic; 2) an in- crease 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 mibefradil- treated 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 differ- ent cytotoxic pathways and that in the case of pimozide, it is unlikely that this effect is mediated solely by T-channel inhibi- tion. Mibefradil and pimozide share several common biological properties, which include Ca 2+ channel blockage and, in some cell types, inhibition of cell growth. Mibefradil, a ben- zimidazolyl-substituted tetraline derivative similar in struc- ture to verapamil (Clozel et al., 1990), is distinguished from other Ca 2+ channel antagonists because it preferentially blocks low-voltage–activated T-type Ca 2+ channels (T-chan- nels) with 10 to 20 times more selectivity than high-voltage– activated (HVA) L-type Ca 2+ channels (Mishra and Herms- meyer, 1994; see Lacinova ´ et al., 2000 for a review). Mibefradil seems to bind to a unique receptor site that over- laps with verapamil and indolizine sulfone sites and is also able to interfere with the binding of Ca 2+ channel antago- nists, 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 antihy- pertensive agent but was withdrawn from the market less than a year after its release due to potentially life-threaten- ing interactions when mibefradil and -blockers were taken in combination with, or acutely replaced by, dihydropyridine Ca 2+ channel blockers (Mullins et al., 1998). The diphenylbutylpiperidine antipsychotic drug pimozide has also been shown to be a potent inhibitor of T-type Ca 2+ channels but with less selectivity than mibefradil (Galizzi et al., 1986). In pituitary and heart cells, pimozide inhibits L-type Ca 2+ channels (Enyeart et al., 1990), whereas in adrenal glomerulosa and spermatogenic cells, it blocks T- channels and Ca 2+ influx (Enyeart et al., 1993; Arnoult and Florman, 1998). The actions of pimozide and mibefradil are not restricted to Ca 2+ 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 in- hibit 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 Sco- tia. G.E.B. was supported by the Reynolds Fellowship in Pharmacology and a CaRE-Nova Scotia Trainee Award. ABBREVIATIONS: T-channel, T-type Ca 2+ channel; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; PI, propidium iodide; HVA, high-voltage–activated; T-current, T-type Ca 2+ 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