Liao Wei-Chuan, Chou Chiang-Ting, Liang Wei-Zhe, Hao Lyh-Jyh, Kuo Chun-Chi, Lin Ko-Long, Wang Jue-Long, Jan Chung-Ren
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, Republic of China.
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi 61363, Taiwan, Republic of China.
Chin J Physiol. 2018 Dec 31;61(6):341-348. doi: 10.4077/CJP.2018.BAH635.
Niflumic acid, a drug used for joint and muscular pain, affected Ca²⁺ signaling in different models. However, the effect of niflumic acid on Ca²⁺ homeostasis and Ca²⁺-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 [Ca²⁺]i rises. Niflumic acid killed cells at 200-500 μM in a concentration-dependent fashion. Chelating cytosolic Ca²⁺ 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 [Ca²⁺]i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Niflumic acid also induced Ca²⁺-independent cell death.
尼氟灭酸是一种用于缓解关节和肌肉疼痛的药物，在不同模型中会影响钙离子信号传导。然而，尼氟灭酸对人骨肉瘤细胞中钙离子稳态及与钙离子相关的生理功能的影响尚不清楚。本研究检测了尼氟灭酸对MG63人骨肉瘤细胞胞质游离钙离子浓度（[Ca²⁺]i）的影响。使用荧光钙离子敏感染料fura-2监测悬浮细胞中的细胞内钙离子浓度。通过使用4-[3-[4-碘苯基]-2-4(4-硝基苯基)-2H-5-四氮唑]-1,3-苯二磺酸盐]水溶性四氮唑-1（WST-1）检测细胞活力。在MG63细胞中，浓度为250 - 750μM的尼氟灭酸可浓度依赖性地引起[Ca²⁺]i升高。尼氟灭酸引发的钙离子内流通过Mn²⁺诱导的fura-2荧光淬灭得以证实。这种内流受到硝苯地平、益康唑、SKF96365、蛋白激酶C（PKC）激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）的抑制，但不受PKC抑制剂GF109203X的影响。在无钙离子培养基中，用内质网钙离子泵抑制剂毒胡萝卜素（TG）处理可抑制尼氟灭酸引发的[Ca²⁺]i升高。相反，用尼氟灭酸处理可消除TG引发的[Ca²⁺]i升高。用U73122抑制磷脂酶C（PLC）也可部分降低尼氟灭酸引发的[Ca²⁺]i升高。尼氟灭酸在200 - 500μM时以浓度依赖性方式杀死细胞。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸/AM（BAPTA/AM）螯合胞质钙离子并不能逆转尼氟灭酸诱导的细胞毒性。总体而言，我们的数据表明，在MG63细胞中，尼氟灭酸通过引发PLC依赖性的内质网钙离子释放以及通过PKC敏感的钙库操纵性钙离子内流来诱导[Ca²⁺]i升高。尼氟灭酸还可诱导非钙离子依赖性细胞死亡。
