Effects of MK-886, a leukotriene synthesis inhibitor, on [Ca ²⁺]_i and apoptosis in MG63 human osteosarcoma cells

The effect of MK-886 (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert- butylthioindol-2-yl]-2, 2-dimethylpropanoic acid), a compound widely used to inhibit leukotriene synthesis, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in osteosarcoma cells has not been explored. This study examined whether MK-886 altered [Ca²⁺]_i levels in suspended MG63 human osteosarcoma cells using fura-2. MK-886 at 0.1 μM and above increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK-886 induced Mn²⁺ quenching of fura-2 fluorescence, implicating Ca²⁺ entry. MK-886-induced Ca²⁺ influx was inhibited by store-operated Ca²⁺ entry inhibitors, nifedipine, econazole, and SKF96365; and by the protein kinase C modulators, phorbol 12-myristate 13-acetate (PMA) and GF109203X. In Ca²⁺-free medium, after pretreatment with 5 μM MK-886, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca ²⁺]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished MK-886-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change MK-886-induced [Ca²⁺]_i rises. Collectively, in MG63 osteosarcoma cells, MK-886 induced [Ca²⁺]_i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca ²⁺ influx via protein kinase C-regulated store-operated Ca ²⁺ entry.