Phospholipase A 2-independent Ca 2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Abstract

Melittin, a peptide from bee venom, is thought to be a phospholipase A 2 activator and Ca 2+ influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca 2+ concentration ([Ca 2+] i) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca 2+] i and killed cells in MG63 human osteosarcoma cells. [Ca 2+] i changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 μM increased [Ca 2+] i in a concentration-dependent manner. The Ca 2+ signal was abolished by removing extracellular Ca 2+. Melittin-induced Ca 2+ entry was confirmed by Mn 2+ quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca 2+-insensitive. The melittin-induced Ca 2+ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A 2 with AACOCF 3 and aristolochic acid; but was substantially inhibited by blocking L-type Ca 2+ channels. At concentrations of 0.5 μM and 1 μM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 μM melittin was completely reversed by pre-chelating cytosolic Ca 2+ with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca 2+] i increase by causing Ca 2+ entry through L-type Ca 2+ channels in a manner independent of protein kinase-C and phospholipase A 2 activity; and this [Ca 2+] i increase subsequently caused apoptosis.