The mechanism of NPC-14686-induced [Ca ²⁺ ] _i rises and non-Ca ²⁺ -triggered cell death in MG63 human osteosarcoma cells

NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca ²⁺ concentrations ([Ca ²⁺ ] _i ) and viability in MG63 human osteosarcoma cells was explored. The Ca ²⁺ -sensitive fluorescent dye fura-2 was applied to measure [Ca ²⁺ ] _i . NPC-14686 at concentrations of 100-500 μM induced a [Ca ²⁺ ] _i rise in a concentrationdependent manner. The response was reduced by 80% by removing Ca ²⁺ . NPC-14686 induced Mn ²⁺ influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca ²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca ²⁺ ] _i rise. At 20-50 μM, NPC-14686 decreased cell viability, which 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/propidium iodide staining data suggest that NPC-14686 (30-50 μM) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca ²⁺ ] _i rise by inducing phospholipase C-dependent Ca ²⁺ release from the endoplasmic reticulum and Ca ²⁺ entry via protein kinase C-sensitive store-operated Ca ²⁺ channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways.