Action of chlorzoxazone on Ca2+movement and viability in human oral cancer cells

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.

Ca2+; cell viability; chlorzoxazone; endoplasmic reticulum; oral cancer cells.