Action of chlorzoxazone on Ca 2 + movement and viability in human oral cancer cells.

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.
Competing Interests: None