The mechanism of NPC-14686-induced [Ca²⁺]i rises and non-Ca²⁺-triggered cell death in MG63 human osteosarcoma cells.
NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]i. NPC-14686 at concentrations of 100-500 μM induced a [Ca²⁺]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca²⁺. NPC-14686 induced Mn²⁺ influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺]i rise. At 20-50 μM, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that NPC-14686 (30-50 μM) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca²⁺]i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways.
Department of Pediatrics, Ping Tung Christian Hospital, Ping Tung 900, Taiwan, Republic of China.
Jau-Min
JM
Chien
Chiang-Ting
CT
Chou
Wei-Zhe
WZ
Liang
Daih-Huang
DH
Kuo
Chun-Chi
CC
Kuo
Chin-Man
CM
Ho
Pochuen
P
Shieh
Chung-Ren
CR
Jan
D000613:Aminobutyrates / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001859:Bone Neoplasms / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D004464:Econazole / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D012516:Osteosarcoma / Q000473:pathology*
D000613:Aminobutyrates; D000935:Antifungal Agents; D002121:Calcium Channel Blockers; D002614:Chelating Agents; D007093:Imidazoles; C417979:NPC 14686; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D004464:Econazole; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D009543:Nifedipine; D002118:Calcium