Effect of antidepressant doxepin on Ca ²⁺ homeostasis and viability in PC3 human prostate cancer cells

The effect of the antidepressant doxepin on cytosolic Ca ²⁺ concentrations ([Ca ²⁺ ] _i ) and viability in PC3 human prostate cancer cells was explored. The Ca ²⁺ -sensitive fluorescent dye fura-2 was applied to measure [Ca ²⁺ ] _i . Doxepin at concentrations of 500-1000 μM induced a [Ca ²⁺ ] _i rise in a concentration-dependent manner. The response was reduced partly by removing Ca ²⁺ . Doxepin-evoked Ca ²⁺ entry was suppressed by Ca ²⁺ entry blockers (nifedipine, econazole, SK&F96365), and protein kinase C (PKC) modulators. In the absence of extracellular Ca ²⁺ , incubation with the endoplasmic reticulum Ca ²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibit doxepin-induced [Ca ²⁺ ] _i rise. Incubation with doxepin nearly inhibited thapsigargin or BHQ-induced [Ca ²⁺ ] _i rise. Inhibition of phospholipase C (PLC) with U73122 failed to alter doxepin-induced [Ca ²⁺ ] _i rise. At concentrations of 200-250 μM, doxepin killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca ²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/PI staining data implied that doxepin (200 and 250 μM) did not induce apoptosis. Collectively, in PC3 cells, doxepin induced a [Ca ²⁺ ] _i rise by evoking PLC-independent Ca ²⁺ release from stores including the endoplasmic reticulum and Ca ²⁺ entry via PKC-sensitive store-operated Ca ²⁺ channels. Doxepin caused cell death that was independent of [Ca ²⁺ ] _i rises.