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.
Jiann
Bang-Ping
BP
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
Lu
Yih-Chau
YC
Chang
Hong-Tai
HT
Huang
Jong-Khing
JK
Jan
Chung-Ren
CR
D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000031:analogs & derivatives* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002996:Clomiphene / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004963:Estrenes / Q000494:pharmacology; D005300:Fertility Agents, Female / Q000494:pharmacology*; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004963:Estrenes; D005300:Fertility Agents, Female; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D002996:Clomiphene; D020126:Brefeldin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D019284:Thapsigargin; D002118:Calcium