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

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.