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

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 microM 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 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum CA(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store CA(2+) release. Conversely, pretreatment with 50 microM clomiphene in CA(2+)-free medium abolished the [CA(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced CA(2+)release was unaltered by inhibiting Phospholipase C with 2 microM 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 microM) 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.