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

• Published in: Life sciences (1973) 2007-01, Vol.80 (4), p.364-369
• Main Authors: Chu, Sau-Tung, Cheng, He-Hsiung, Huang, Chun-Jen, Chang, Hong-Chiang, Chi, Chao-Chuan, Su, Hsing-Hao, Hsu, Shu-Shong, Wang, Jue-Long, Chen, I-Shu, Liu, Shiuh-Inn, Lu, Yih-Chau, Huang, Jong-Khing, Ho, Chin-Man, Jan, Chung-Ren
• Format: Article
• Language: English
• Subjects: Apoptosis - drug effects; Calcium - metabolism; Calcium Channels, L-Type - drug effects; Calcium Channels, L-Type - metabolism; Cell Line, Tumor; Cell Survival - drug effects; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Enzyme Inhibitors - pharmacology; Fluorescent Dyes - metabolism; Fura-2 - metabolism; Humans; Indoles - pharmacology; Maleimides - pharmacology; Melitten - pharmacology; Osteoblasts - drug effects; Osteoblasts - metabolism; Osteoblasts - pathology; Osteosarcoma; Phospholipases A - antagonists & inhibitors; Phospholipases A - metabolism; Phospholipases A2; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0024-3205
• DOI: 10.1016/j.lfs.2006.09.024

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 microM 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 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM 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.