M-3M3FBS-Induced Ca2+ Movement and Apoptosis in HA59T Human Hepatoma Cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+](i)) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca2+ ](i) levels in suspended cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations of 1050 mu M increased [Ca2+](i) in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca2+-free medium, 50 mu M m-3M3FBS pretreatnient inhibited the [Ca2+; rise induced by the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca2+](i) rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca-i(2+]) rise. At concentrations between 10 and 40 [mu M m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. M-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca2+](i) rise by inducing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive store-operated Ca2+ channels. M-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.