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Action of chlorzoxazone on Ca2+movement and viability in human oral cancer cells
Ti Lu1, Wei-Zhe Liang2, Lyh-Jyh Hao3, Chun-Chi Kuo4, Pochuen Shieh5, Chiang-Ting Chou6, Chung-Ren Jan7
1 Department of Psychiatry, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
2 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung; Department of Pharmacy, Tajen University, Pingtung, Taiwan
3 Department of Metabolism, Kaohsiung Veterans General Hospital Tainan Branch, Tainan, Taiwan
4 Department of Nursing, Tzu Hui Institute of Technology, Pingtung, Taiwan
5 Department of Pharmacy, Tajen University, Pingtung, Taiwan
6 Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi, Taiwan
7 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
Correspondence Address:
Dr. Chiang-Ting Chou
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi 61363
Taiwan
Dr. Chung-Ren Jan
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362
Taiwan
Dr. Pochuen Shieh
Department of Pharmacy, Tajen University, Pingtung 90741
Taiwan
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/CJP.CJP_20_19
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Chlorzoxazone is a skeletal muscle relaxant. However, the effect of chlorzoxazone on intracellular Ca2+ concentrations ([Ca2+]i) in oral cancer cells is unclear. This study examined whether chlorzoxazone altered Ca2+ signaling and cell viability in OC2 human oral cancer cells. [Ca2+]iin suspended cells was measured using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by water-soluble tetrazolium-1 assay. Chlorzoxazone (250–1000 μM) induced [Ca2+]irises in a concentration-dependent manner. Ca2+ removal reduced the signal by approximately 50%. Mn2+ has been shown to enter cells through similar mechanisms as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths. Chlorzoxazone (1000 μM) induced Mn2+ influx, suggesting that Ca2+ entry occurred. Chlorzoxazone-induced Ca2+ entry was inhibited by 20% by inhibitors of store-operated Ca2+ channels and protein kinase C (PKC) modulators. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) inhibited chlorzoxazone-evoked [Ca2+]irises by 88%. Conversely, treatment with chlorzoxazone-suppressed TG-evoked [Ca2+]irises 75%. Chlorzoxazone induced [Ca2+]irises by exclusively releasing Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C (PLC) with U73122 did not alter chlorzoxazone-induced [Ca2+]irises. PLC activity was not involved in chlorzoxazone-evoked [Ca2+]irises. Chlorzoxazone at 200–700 μM decreased cell viability, which was not reversed by pretreatment with Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl. In sum, in OC2 cells, chlorzoxazone induced [Ca2+]irises by evoking PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Chlorzoxazone also caused Ca2+-independent cell death. Since [Ca2+]irises play a triggering or modulatory role in numerous cellular phenomena, the effect of chlorzoxazone on [Ca2+]iand cell viability should be taken into account in other in vitro studies.
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