Effect of clomiphene on Ca2+ movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca2+ indicator. Clomiphene at concentrations between 10-50 muM increased [Ca2+](i) in a concentration-dependent manner. The [Ca+2](i) signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by 41%. Adding 3 mM Ca2+ increased [Ca2+](i), in cells pretreated with clomiphene in Ca2+-free medium, confirming that clomiphene induced Ca2+ entry. In Ca2+-free medium, pretreatment with 50 muM brefeldin A (to permeabilize the Golgi complex), 1 muM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and 2 muM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 muM clomiphene-induced store Ca2+ release. Conversely, pretreatment with 50 IN clomiphene in Ca2+-free medium abolished the [Ca2+](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 [muM clomiphene-induced Ca2+ release was unaltered by inhibiting phospholipase C with 2 muM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 muM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca2+](i) increases in PC3 cells by releasing store Ca2+ from multiple stores in an phospholipase C-independent manner, and by activating Ca2+ influx; and clomiphene was of mild cytotoxicity. (C) 2002 Elsevier Science Inc. All rights reserved.