Phospholipase A2-independent Ca entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

摘要

Meiittin, a peptide from bee venom, is thought to be a phospholipase A2 activator and Ca2"1 influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca2+ concentration ([Ca24]j) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca2+] and killed cells in MG63 human osteosarcoma cells. [Ca2+]: changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 uM increased [Ca~1] in a concentration-dependent manner. The Ca2+ signal was abolished by removing extracellular Ca2+. Melittin-induced Ca2+ entry was confirmed by Mn2+ quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca2+-insensitive. The melittin-induced Ca~+ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A2 with AACOCF3 and aristolochic acid; but was substantially inhibited by blocking L-type Ca2T channels. At concentrations of 0.5 uM and 1 uM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 muM melittin was completely reversed by pre-chelating cytosolic Ca2+ with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca2+] increase by causing Ca21 entry through L-type Ca~~ channels in a manner independent of protein kinase-C and phospholipase A2 activity; and this [Ca~2] increase subsequently caused apoptosis. 500Apoptosis; BAPTA; Ca2+; Fura-2; Melittin; MG63 cells; Osteosarcoma cells