The Mechanism of NPC-14686-Induced [Ca^(2+)]i Rises and Non-Ca^(2+)-Triggered Cell Death in MG63 Human Osteosarcoma Cells

Abstract

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.

Effect of Methoxsalen on Ca^(2+) Homeostasis and Viability in Human Osteosarcoma Cells

“…The [Ca 2+ ] i was measured as previously described (6,24,25). Confluent cells grown on 6 cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 10 6 /ml.…”

“…For calibration of [Ca 2+ ] i , after completion of the experiments, Triton X-100 (0.1%) and CaCl 2 (5 mM) were added to the cuvette to obtain the maximal fura-2 fluorescence (6,16,24,25). The Ca 2+ chelator EGTA (10 mM) was subsequently added to chelate Ca 2+ in the cuvette to obtain the minimal fura-2 fluorescence.…”

“…Viability was explored as previously described (6,24,25). The assessment of cell viability was based on the rational that cells can cleave tetrazolium salts by dehydrogenases.…”

Amitriptyline modulated Ca2+ signaling and induced Ca2+-independent cell viability in human osteosarcoma cells

“…The [Ca 2+ ] i was measured as previously described. 13 –15 Confluent cells grown on 6 cm dishes were trypsinized and suspended in medium at a concentration of 10 6 cells per milliliter. Cell viability was assayed by trypan blue exclusion (adding 0.2% trypan blue to 0.1 ml cell suspension).…”

“…11,12 In order to explore the effect of amitriptyline on [Ca 2+ ] i in human osteosarcoma cells, the MG63 cell line was used because it induced significant [Ca 2+ ] i elevation when stimulated by many chemicals. It has been reported that in this cell line, [Ca 2+ ] i rises can be triggered in response to the addition of various chemicals such as NPC-14686, 13 sertraline, 14 and 3,3′-diindolylmethane. 15…”