Nordihydroguaiaretic acid-induced Ca 2+ handling and cytotoxicity in human prostate cancer cells

Abstract

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca 2+ levels ([Ca 2+] i) in PC3 human prostate cancer cells was investigated. [Ca 2+] i was measured by using the Ca 2+-sensitive dye fura-2. NDGA increased [Ca 2+] i in a concentration-dependent manner with an EC 50 of 30 μM. The Ca 2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca 2+ partly decreased the NDGA-induced [Ca 2+] i increase, suggesting that the Ca 2+ signal was due to both extracellular Ca 2+ influx and intracellular Ca 2+ release. NDGA-induced Ca 2+ influx was independently confirmed by measuring NDGA-induced Mn 2+-coupled quench of fura-2 fluorescence. The NDGA-induced Ca 2+ influx was not affected by L-type Ca 2+ channel blockers. In Ca 2+-free medium, the NDGA-induced [Ca 2+] i increase was abolished by pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca 2+] i increase. NDGA-induced intracellular Ca 2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20–50 μM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca 2+] i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca 2+] i increase via releasing stored Ca 2+from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca 2+ influx. NDGA also caused cytotoxicity at higher concentrations.