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(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.
Chu
Sau-Tung
ST
Department of Otolaryngology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
Cheng
He-Hsiung
HH
Huang
Chun-Jen
CJ
Chang
Hong-Chiang
HC
Chi
Chao-Chuan
CC
Su
Hsing-Hao
HH
Hsu
Shu-Shong
SS
Wang
Jue-Long
JL
Chen
I-Shu
IS
Liu
Shiuh-Inn
SI
Lu
Yih-Chau
YC
Huang
Jong-Khing
JK
Ho
Chin-Man
CM
Jan
Chung-Ren
CR
D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008555:Melitten / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012516:Osteosarcoma; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D008301:Maleimides; D008555:Melitten; D004533:Egtazic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2