Exploration of niflumic acid’s action on Ca ²⁺ movement and cell viability in human osteosarcoma cells

Niflumic acid, a drug used for joint and muscular pain, affected Ca ²⁺ signaling in different models. However, the effect of niflumic acid on Ca ²⁺ homeostasis and Ca ²⁺ -related physiology in human osteosarcoma cells is unknown. This study examined the effect of niflumic acid on cytosolic free Ca2+ concentrations ([Ca2+]i) in MG63 human osteosarcoma cells. Intracellular Ca2+ concentrations in suspended cells were monitored by using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1). In MG63 cells, niflumic acid at concentrations of 250-750 µM evoked [Ca2+]i rises concentration-dependently. Niflumic acid-evoked Ca2+ entry was confirmed by Mn2+-induced quenching of fura-2 fluorescence. This entry was inhibited by nifedipine, econazole, SKF96365, the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was not affected by the PKC inhibitor GF109203X. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) inhibited niflumic acid-evoked [Ca2+]i rises. Conversely, treatment with niflumic acid abolished TG-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also partly reduced niflumic acid-evoked [Ca ²⁺ ] _i rises. Niflumic acid killed cells at 200-500 μM in a concentration-dependent fashion. Chelating cytosolic Ca ²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/ AM (BAPTA/AM) did not reverse niflumic acid-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, niflumic acid induced [Ca ²⁺ ] _i rises by evoking PLC-dependent Ca ²⁺ release from the endoplasmic reticulum, and Ca ²⁺ entry via PKC-sensitive store-operated Ca ²⁺ entry. Niflumic acid also induced Ca ²⁺ -independent cell death.