Action of Chlorzoxazone on Ca^(2+) Movement and Viability in Human Oral Cancer Cells

10.4103/CJP.CJP_20_19

Ti Lu；Wei-Zhe Liang；Lyh-Jyh Hao；Chun-Chi Kuo；Pochuen Shieh；Chiang-Ting Chou；Chung-Ren Jan

Ca^(2+) ； cell viability ； chlorzoxazone ； endoplasmic reticulum ； oral cancer cells

The Chinese Journal of Physiology

62卷3期（2019 / 06 / 30）

123 - 130

英文

Chlorzoxazone is a skeletal muscle relaxant. However, the effect of chlorzoxazone on intracellular Ca^(2+) concentrations ([Ca^(2+)]_i) in oral cancer cells is unclear. This study examined whether chlorzoxazone altered Ca^(2+) signaling and cell viability in OC2 human oral cancer cells. [Ca^(2+)]_i in suspended cells was measured using the fluorescent Ca^(2+)-sensitive dye fura-2. Cell viability was examined by water-soluble tetrazolium-1 assay. Chlorzoxazone (250-1000 μM) induced [Ca^(2+)]_i rises in a concentration-dependent manner. Ca^(2+) removal reduced the signal by approximately 50%. Mn^(2+) has been shown to enter cells through similar mechanisms as Ca^(2+) but quenches fura-2 fluorescence at all excitation wavelengths. Chlorzoxazone (1000 μM) induced Mn^(2+) influx, suggesting that Ca^(2+) entry occurred. Chlorzoxazone-induced Ca^(2+) entry was inhibited by 20% by inhibitors of store-operated Ca^(2+) channels and protein kinase C (PKC) modulators. In Ca^(2+)-free medium, treatment with the endoplasmic reticulum Ca^(2+) pump inhibitor thapsigargin (TG) inhibited chlorzoxazone-evoked [Ca^(2+)]_i rises by 88%. Conversely, treatment with chlorzoxazone-suppressed TG-evoked [Ca^(2+)]_i rises 75%. Chlorzoxazone induced [Ca^(2+)]_i rises by exclusively releasing Ca^(2+) from the endoplasmic reticulum. Inhibition of phospholipase C (PLC) with U73122 did not alter chlorzoxazone-induced [Ca^(2+)]_i rises. PLC activity was not involved in chlorzoxazone-evoked [Ca^(2+)]_i rises. Chlorzoxazone at 200-700 μM decreased cell viability, which was not reversed by pretreatment with Ca^(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl. In sum, in OC2 cells, chlorzoxazone induced [Ca^(2+)]_i rises by evoking PLC-independent Ca^(2+) release from the endoplasmic reticulum and Ca^(2+) entry via PKC-sensitive store-operated Ca^(2+) entry. Chlorzoxazone also caused Ca^(2+)-independent cell death. Since [Ca^(2+)]_i rises play a triggering or modulatory role in numerous cellular phenomena, the effect of chlorzoxazone on [Ca^(2+)]_i and cell viability should be taken into account in other in vitro studies.

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