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

10.4077/CJP.2014.BAB178

Jau-Min Chien；Chiang-Ting Chou；Wei-Zhe Liang；Daih-Huang Kuo；Chun-Chi Kuo；Chin-Man Ho；Pochuen Shieh；Chung-Ren Jan

apoptosis ； Ca^(2+) ； human osteosarcoma cells ； NPC-14686

The Chinese Journal of Physiology

57卷3期（2014 / 06 / 30）

158 - 166

英文

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 Ca^(2+) concentrations ([Ca^(2+)]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca^(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca^(2+)]_i. NPC-14686 at concentrations of 100-500 μM induced a [Ca^(2+)]_i rise in a concentrationdependent manner. The response was reduced by 80% by removing Ca^(2+). NPC-14686 induced Mn2+ influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca^(2+) entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca^(2+)]_i rise. At 20-50 μM, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca^(2+) 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 [Ca^(2+)]_i rise by inducing phospholipase C-dependent Ca^(2+) release from the endoplasmic reticulum and Ca^(2+) entry via protein kinase C-sensitive store-operated Ca^(2+) channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways.

1. Chang, K.H.,Tan, H.P.,Kuo, C.C.,Kuo, D.H.,Shieh, P.,Chen, F.A.,Jan, C.R.(2010).Effect of nortriptyline on Ca2+ handling in SIRC rabbit corneal epithelial cells.Chinese J. Physiol.,53,178-184.
   連結：
2. Chen, W.C.,Cheng, H.H.,Huang, C.J.,Lu, Y.C.,Chen, I.S.,Liu, S.I.,Hsu, S.S.,Chang, H.T.,Huang, J.K.,Chen, J.S.,Jan, C.R.(2007).The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells.Chinese J. Physiol.,50,34-40.
   連結：
3. Cheng, J.S.,Lo, Y.K.,Yeh, J.H.,Cheng, H.H.,Liu, C.P.,Chen, W.C.,Jan, C.R.(2003).Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells.Chinese J. Physiol.,46,117-122.
   連結：
4. Fang, Y.C.,Chou, C.T.,Pan, C.C.,Hsieh, Y.D.,Liang, W.Z.,Chao, D.,Tsai, J.Y.,Liao, W.C.,Kuo, D.H.,Shieh, P.,Kuo, C.C.,Jan, C.R.,Shaw, C.F.(2011).Paroxetine-induced Ca2+ movement and death in OC2 human oral cancer cells.Chinese J. Physiol.,54,310-317.
   連結：
5. Bootman, M.D.,Berridge, M.J.,Roderick, H.L.(2002).Calcium signalling: more messengers, more channels, more complexity.Curr. Biol.,12,R563-R565.
6. Burch, R.M.,Weitzberg, M.,Blok, N.,Muhlhauser, R.,Martin, D.,Farmer, S.G.,Bator, J.M.,Connor, J.R.,Green, M.,Ko, C.(1991).N-(fluorenyl-9-methoxycarbonyl) amino acids, a class of antiinflammatory agents with a different mechanism of action.Proc. Natl. Acad. Sci. USA,88,355-359.
7. Bynagari-Settipalli, Y.S.,Lakhani, P.,Jin, J.,Bhavaraju, K.,Rico, M.C.,Kim, S.,Woulfe, D.,Kunapuli, S.P.(2012).Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets.Arterioscler. Thromb. Vasc. Biol.,32,1211-1219.
8. Chang, H.T.,Hsu, S.S.,Chou, C.T.,Cheng, J.S.,Wang, J.L.,Lin, K.L.,Fang, Y.C.,Chen, W.C.,Chien, J.M.,Lu, T.,Pan, C.C.,Cheng, H.H.,Huang, J.K.,Kuo, C.C.,Chai, K.L.,Jan, C.R.(2011).Effect of thymol on Ca2+ homeostasis and viability in MG63 human osteosarcoma cells.Pharmacology,88,201-212.
9. Clapham, D.E.(1995).Intracellular calcium - Replenishing the stores.Nature,375,634-635.
10. Grynkiewicz, G.,Poenie, M.,Tsien, R.Y.(1985).A new generation of Ca2+ indicators with greatly improved fluorescence properties.J. Biol. Chem.,260,3440-3450.
11. Huang, J.K.,Liu, C.S.,Chen, W.C.,Liu, S.I.,Hsu, S.S.,Chang, H.T.,Chou, C.T.,Hsieh, C.H.,Chang, C.H.,Jan, C.R.(2006).NPC-14686 (Fmoc-L-homophenylalanine)-induced Ca2+ increases and death in human prostate cancer cells.Life Sci.,78,964-969.
12. Ishikawa, J.,Ohga, K.,Yoshino, T.,Takezawa, R.,Ichikawa, A.,Kubota, H.,Yamada, T.(2009).A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes.J. Immunol.,170,4441-4449.
13. Jan, C.R.,Kuo, S.Y.,Cheng, J.S.,Lo, Y.K.,Liu, C.P.,Chen, W.C.(2003).Effect of NPC-14686 (Fmoc-L-homophenylalanine) on intracellular Ca2+ levels in human hepatoma cells.Life Sci.,72,2571-2580.
14. Jan, C.R.,Wang, J.L.,Chou, K.J.,Cheng, J.S.,Lee, K.C.,Tseng, L.L.,Wang, S.P.,Tang, K.Y.,Huang, J.K.(2000).NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca2+ concentrations in MDCK renal tubular cells.Int. J. Immunopharmacol.,22,915-921.
15. Jiang, N.,Zhang, Z.M.,Liu, L.,Zhang, C.,Zhang, Y.L.,Zhang, Z.C.(2006).Effects of Ca2+ channel blockers on store-operated Ca2+ channel currents of Kupffer cells after hepatic ischemia/reperfusion injury in rats.World J. Gastroenterol.,12,4694-4698.
16. Kuriyama, T.,Tokinaga, Y.,Tange, K.,Kimoto, Y.,Ogawa, K.(2012).Propofol attenuates angiotensin II-induced vasoconstriction by inhibiting Ca2+-dependent and PKC-mediated Ca2+ sensitization mechanisms.J. Anesth.,26,682-688.
17. Liu, S.I.,Lin, K.L.,Lu, T.,Lu, Y.C.,Hsu, S.S.,Tsai, J.Y.,Liao, W.C.,Huang, F.D.,Chi, C.C.,Liang, W.Z.,Tseng, L.L.,Chiang, A.J.,Jan, C.R.(2013).M-3M3FBS-induced Ca2+ movement and apoptosis in HA59T human hepatoma cells.Chinese J. Physiol.,56,26-35.
18. Lu, Y.C.,Chen, I.S.,Chou, C.T.,Huang, J.K.,Chang, H.T.,Tsai, J.Y.,Hsu, S.S.,Liao, W.C.,Wang, J.L.,Lin, K.L.,Liu, S.I.,Kuo, C.C.,Ho, C.M.,Jan, C.R.(2012).3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells.Basic Clin. Pharmacol. Toxicol.,110,314-321.
19. Merritt, J.E.,Jacob, R.,Hallam, T.J.(1989).Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils.J. Biol. Chem.,264,1522-1527.
20. Ohta, Y.,Kobayashi, T.,Ishiguro, I.(1999).Participation of xanthine-xanthine oxidase system and neutrophils in development of acute gastric mucosal lesions in rats with a single treatment of compound 48/80, a mast cell degranulator.Dig. Dis. Sci.,44,1865-1874.
21. Quinn, T.,Molloy, M.,Smyth, A.,Baird, A.W.(2004).Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro.Life Sci.,74,1659-1669.
22. Shideman, C.R.,Reinardy, J.L.,Thayer, S.A.(2009).γ-Secretase activity modulates store-operated Ca2+ entry into rat sensory neurons.Neurosci. Lett.,451,124-128.
23. Thompson, A.K.,Mostafapour, S.P.,Denlinger, L.C.,Bleasdale, J.E.,Fisher, S.K.(1991).The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells: a role for Gp in receptor compartmentation.J. Biol. Chem.,266,23856-23862.
24. Tsien, R.Y.(1980).New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures.Biochemistry,19,2396-2404.