Exploration of Niflumic Acid's Action on Ca^(2+) Movement and Cell Viability in Human Osteosarcoma Cells

10.4077/CJP.2018.BAH635

Wei-Chuan Liao；Chiang-Ting Chou；Wei-Zhe Liang；Lyh-Jyh Hao；Chun-Chi Kuo；Ko-Long Lin；Jue-Long Wang；Chung-Ren Jan

Ca^(2+) ； endoplasmic reticulum ； human osteosarcoma cells ； niflumic acid ； viability

The Chinese Journal of Physiology

61卷6期（2018 / 12 / 31）

341 - 348

英文

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 Ca^(2+) concentrations ([Ca^(2+)]_i) in MG63 human osteosarcoma cells. Intracellular Ca^(2+) concentrations in suspended cells were monitored by using the fluorescent Ca^(2+)-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 [Ca^(2+)]_i rises concentration-dependently. Niflumic acidevoked Ca^(2+) entry was confirmed by Mn^(2+)-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 Ca^(2+)- free medium, treatment with the endoplasmic reticulum Ca^(2+) pump inhibitor thapsigargin (TG) inhibited niflumic acid-evoked [Ca^(2+)]_i rises. Conversely, treatment with niflumic acid abolished TG-evoked [Ca^(2+)]_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.

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