The cell death response to gamma-radiation in MCF-7 cells is enhanced by a neuroleptic drug, pimozide.

Neuroleptic drugs that bind sigma sites were tested for their ability to inhibit growth and radiosensitize MCF-7 human breast cancer cells. Inhibition of growth by approximately 50% occurred in cells exposed to pimozide (0.6 microM), haloperidol (10 microM), and the sigma ligand DTG (1,3-di(2-tolyl)guanidine, 20 microM), but no growth inhibition occurred in cells exposed to clozapine, a neuroleptic drug lacking sigma binding activity, or dextromethorphan, a selective sigma 1 binding ligand. Pimozide (2.5 microM), but not haloperidol (3.6 microM), enhanced the sensitivity of MCF-7 cells to gamma radiation in clonogenic survival assays. Pimozide significantly decreased MCF-7 clonogenic survival following a 5 or 8 Gy dose of gamma radiation, and the dose of radiation required for 1% survival (survival enhancement ratio, SER) was decreased by a factor of 2. Exposure of normal WI-38 human embryonic lung cells to pimozide did not increase their sensitivity to gamma radiation. Pimozide (2.5 microM) activated early apoptotic changes in MCF-7 cells that were detected by the uptake of Hoechst 33342 dye, and 10 microM pimozide activated a complete apoptotic pathway resulting in the death of > 90% of the cells within 24 hours. MCF-7 cells exposed to gamma radiation alone (8 Gy) showed giant cell formation, mitotic arrest, and a limited degree of apoptosis and necrosis. Within 50 hours of treatment with a combination of radiation and pimozide, cell numbers were sharply reduced compared with cultures exposed to either radiation or pimozide alone. We conclude that pimozide augmented the sensitivity of MCF-7 cells to radiation-induced cell killing through a mechanism not shared by haloperidol, but suggest that concentration of pimozide in MCF-7 cells as a result of an enrichment of sigma 2 sites might target the radiosensitization.

Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture.

The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF-7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration-dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3-day or 5-day exposure to drug. The IC50 values for these five drugs, quinidine (25 microM), glibenclamide (50 microM), linogliride (770 microM), 4-aminopyridine (1.6 mM), and tetraethylammonium (5.8 mM) were estimated from their respective concentration-response curves. Four other potassium channel blockers were tested at supra-maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF-7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in G0/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in G0/G1 phase and the threshold concentrations for inhibition of growth and G0/G1 arrest were similar. The G0/G1 arrest produced by quinidine and glibenclamide were reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of approximately 24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP-sensitive potassium channels in these human mammary carcinoma cells, reversibly arrests the cells in the G0/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation.

A survey of human breast cancer sensitivity to growth inhibition by calmodulin antagonists in tissue culture.

We compared the ability of N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), a calmodulin antagonist, to inhibit the growth of seven human breast cancer cell lines in tissue culture, to determine whether drug sensitivity was related to estrogen receptor (ER) status, tamoxifen resistance (tamr), or levels of calmodulin activity. We examined three ER+ (estrogen receptor-positive) cell lines (MCF-7, ZR-75-1B, and T47D), two ER+/tamr lines (LY2 and RR), and two ER- (estrogen receptor-negative) cell lines (MDA-MB-231 and MDA-MB-435). There was no difference in the inhibition of cell growth by W-13 in MCF-7 cells and the two tamr MCF-7 cell derivatives, LY2 and RR. In addition, the sensitivity to W-13 did not appear to be related to ER status. Although the mean Ki of the five ER+ cell lines (31 microM) was somewhat higher than the mean Ki of the two ER- cell lines (23 microM), the two cell lines most sensitive to W-13 were the ER+ T47D cells (Ki 15 microM) and the ER- MDA-MB-435 cells (Ki 10 microM). Calmodulin activity was measured in three representative cell lines, MCF-7, LY2, and MDA-MB-435. Calmodulin levels were higher in the most sensitive cell line (MDA-MB-435, 2.7 ng calmodulin/micrograms protein) than in the two less sensitive cell lines, MCF-7 and LY2 (1.3 and 1.6 ng calmodulin/micrograms protein, respectively). However, the MCF-7, LY2, and MDA-MB-435 cells were equally sensitive to another specific calmodulin antagonist, calmidazolium. We conclude that neither ER status, tamoxifen resistance, nor levels of calmodulin activity predict the sensitivity of human breast cancer cell lines to growth inhibition in tissue culture by calmodulin antagonists.

Tamoxifen-resistant human breast cancer cell growth: inhibition by thioridazine, pimozide and the calmodulin antagonist, W-13.

Estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and ER-positive derivatives of the MCF-7 cell line selected for growth in the presence of antiestrogens (LY2 and RR) were used as in vitro models of tamoxifen-resistant human breast cancer in this study. The sensitivity of the tamoxifen-sensitive (MCF-7) and tamoxifen-resistant human breast cancer cell growth to two noncytotoxic neuroleptic drugs, pimozide and thioridazine, and the anticalmodulin agent, W-13, were compared. Inhibition of cell growth was measured as a decrease in cell number following a 72-h incubation with drug. Growth of the ER-negative cell lines MDA-MB-231 and MDA-MB-435 was inhibited by all three drugs. The average Ki values in these two lines were 6.3 and 3.8 microM for pimozide and 4.1 and 15 microM for thioridazine, respectively. Both ER-negative cell lines were more sensitive than MCF-7 cells to growth inhibition by W-13. MCF-7 cells selected for antiestrogen resistance were sensitive to growth inhibition by W-13 and thioridazine (LY2, average Ki = 10.4 microM; RR, average Ki = 5.2 microM). LY2 and RR cells were resistant to pimozide except when treated with estradiol (Ki = 4.6 and 7.9 microM, respectively). Pimozide, thioridazine and W-13 all exerted different effects on the distribution of human breast cancer cells within the cell cycle, suggesting that each drug may utilize a distinct pathway for inhibition of cell growth. We conclude that all three drugs are potential noncytotoxic alternatives to tamoxifen for the treatment of tamoxifen-resistant human breast cancer.

Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine.

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.