Effect of clomiphene on Ca 2+ movement in human prostate cancer cells

Abstract

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