Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Melittin, a peptide from bee venom, is thought to be a phospholipase A, activator and Ca2+ influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca2+ concentration ([Ca2+] (i))and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca2+](i) and killed cells in MG63 human osteosarcoma cells. [Ca2+](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 mu M increased [Ca2+](i) 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 Ca2+ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PNIA) and GF 109203X, or inhibition of phospholipase A, with AACOCF3 and aristolochic acid; but was substantially inhibited by blocking L-type Ca2+ channels. At concentrations of 0.5 mu M and 1 mu M, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 mu M melittin was completely reversed by pre-chelating cytosolic Ca2+ with BAPTA. Taken together, these data showed that in MG63 cells, melitfin induced a [Ca2+](i) increase by causing Ca2+ entry through L-type Ca2+ channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca2+](i) increase subsequently caused apoptosis. (c) 2006 Published by Elsevier Inc.