Lu Yi-Chau, Chou Chiang-Ting, Liang Wei-Zhe, Kuo Chun-Chi, Hsu Shu-Shong, Wang Jue-Long, Jan Chung-Ren
Department of Orthopedics, 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. 2017 Jun 30;60(3):174-182. doi: 10.4077/CJP.2017.BAF482.
Methoxsalen is a natural compound found in many seed plants. The effect of methoxsalen on Ca²⁺- related physiology in human osteosarcoma is unclear. This study investigated the effect of methoxsalen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in MG63 human osteosarcoma cells. Methoxsalen induced [Ca²⁺]i rises concentration-dependently. Methoxsalen-induced Ca²⁺ entry was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. This Ca²⁺ entry was suppressed by nifedipine, econazole, and SKF96365. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited methoxsalen-evoked [Ca²⁺]i rises by 96%. In contrast, incubation with methoxsalen abolished BHQ-evoked [Ca²⁺]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished methoxsalen-induced [Ca²⁺]i rises. Methoxsalen was cytotoxic at 300-700 μM in a concentration-dependent fashion. Chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) did not prevent methoxsalen-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, methoxsalen induced [Ca²⁺]i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via store-operated Ca²⁺ entry. Methoxsalen also induced Ca²⁺- disassociated cell death.
甲氧沙林是一种存在于多种种子植物中的天然化合物。甲氧沙林对人骨肉瘤细胞中与Ca²⁺相关的生理学的影响尚不清楚。本研究调查了甲氧沙林对MG63人骨肉瘤细胞胞质游离Ca²⁺浓度（[Ca²⁺]i）的影响。甲氧沙林以浓度依赖性方式诱导[Ca²⁺]i升高。Mn²⁺诱导的fura-2荧光淬灭证实了甲氧沙林诱导的Ca²⁺内流。硝苯地平、益康唑和SKF96365可抑制这种Ca²⁺内流。在无Ca²⁺培养基中，与内质网Ca²⁺泵抑制剂2,5-二叔丁基对苯二酚（BHQ）孵育可使甲氧沙林诱发的[Ca²⁺]i升高抑制96%。相反，与甲氧沙林孵育可消除BHQ诱发的[Ca²⁺]i升高。用U73122抑制磷脂酶C（PLC）可消除甲氧沙林诱导的[Ca²⁺]i升高。甲氧沙林在300 - 700 μM时具有浓度依赖性细胞毒性。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸/乙酰氧甲酯（BAPTA/AM）螯合胞质Ca²⁺并不能预防甲氧沙林诱导的细胞毒性。总体而言，我们的数据表明，在MG63细胞中，甲氧沙林通过引发PLC依赖的内质网Ca²⁺释放和通过储存-操纵性Ca²⁺内流使Ca²⁺内流，从而诱导[Ca²⁺]i升高。甲氧沙林还诱导Ca²⁺解离的细胞死亡。
