Open AccessAction of chlorzoxazone on Ca2+movement and viability in human oral cancer cells
Author(s) -
TungWu Lu,
Wei Liang,
Lyh-Jyh Hao,
Chun-Chi Kuo,
Pochuen Shieh,
Chiang-Ting Chou,
ChungRen Jan
Publication year - 2019
Publication title -
chinese journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.396
H-Index - 31
eISSN - 2666-0059
pISSN - 0304-4920
DOI - 10.4103/cjp.cjp_20_19
Subject(s) - chlorzoxazone , endoplasmic reticulum , viability assay , chemistry , fura 2 , thapsigargin , intracellular , biochemistry , biophysics , cell , biology , enzyme , microsome , cyp2e1 , cytosol
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.