m-3M3FBS 是一種新的化合物，被當作是磷酯酶C (PLC)的活化劑。m-3M3FBS
對人類胃癌細胞株 (SCM1) 細胞內鈣離子濃度變化目前並不清楚。本研究主要在
藉著鈣離子敏感的螢光劑 Fura-2 使用來探討m-3M3FBS 是否會改變人類胃癌細
胞株 (SCM1)內鈣離子的濃度。結果顯示， 1-50 μM m-3M3FBS 增加細胞內鈣離
子且有濃度依賴現象，當移除細胞外鈣離子時會部分減弱鈣訊號，這鈣離子流入
細胞內的情形會被會被磷酯酶A2 抑制劑aristolochic acid，鈣池調控鈣離子通道阻
斷劑nifedipine、econazole 和SK&F96365 以及蛋白質激酶C 抑制劑GF109203X 所
抑制，但不受蛋白質激酶C 活化劑 PMA 的影響。在細胞外不含鈣離子的環境中，
先以m-3M3FBS 處理，也大量減少內質網鈣幫浦抑制劑TG 或 BHQ 所造成的鈣
離子濃度升高。相反地，先以TG 或BHQ 處置，部分消除m-3M3FBS 造成的鈣離
子濃度上升。若先以U73122 抑制磷酯酶C 的活性也無法減少m-3M3FBS 所造成
的鈣離子濃度上升。整體來說，在人類胃癌細胞株(SCM1)中，m-3M3FBS 會藉著
內質網鈣離子釋放(非磷酯酶C 相關路徑)與鈣池調控的細胞外鈣流入(透過磷酯酶
A2 與蛋白質激酶C 調控）來升高細胞內鈣離子濃度。

m-3M3FBS is a new compound that has been used as a phospholipase C (PLC)
activator. The effect of m-3M3FBS on cytosolic free Ca2+ concentrations in human
gastric cancer cells (SCM1) is unclear. This study explored whether m-3M3FBS
changed basal [Ca2+]i levels in suspended SCM1 cells by using fura-2 as a
Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 1-50 μM
increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced
partly by removing extracellular Ca2+. This Ca2+ influx was inhibited by phospholiapase
A2 inhibitor aristolochic acid , store-operated Ca2+ channel blockers nifedipine 、
econazole and SK&F96365; and protein kinase C inhibitor GF109203X. Phorbol
12-myristate 13-acetate ([PMA] a protein kinase C activator) had no effect on
m-3M3FBS-induced [Ca2+]i rise. In Ca2+-free medium , pretreatment with m-3M3FBS
abolished thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) - induced [Ca2+]i
rise. Conversely, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors TG
or BHQ partly inhibited m-3M3FBS -induced Ca2+ release. The inhibition of PLC with
U73122 did not alter mMIRC. Collectively, in SCM1 cells, mMIRC by causing PLCindependent
Ca2+ release from the endoplasmic reticulum and Ca2+ influx via
phospholipase A2-protein kinase C-sensitive store-operated Ca2+ channels.

CONTENTS
1. Introduction………………………………………..………………………………...1
1.1 m-3M3FBS……………………………………………………………………..1
1.2 Ca2+ signal………………………………………………………………………3
2. Aim…………………………………………………………………………………..5
3. Materials and methods………………………………………………………………6
3.1 Cell culture………………………………………………………………………..6
3.2 Chemicals………………………………………………………………………....6
3.3 Solutions used in [Ca2+]i measurements…………………………………………..6
3.4 [Ca2+]i measurements……………………………………………………………...7
3.5 Statistics……………………………………………………………………….…..7
4. Results…………………………………………………………………………….…..8
4.1 m-3M3FBS induced [Ca2+]i…………………………………………………………………………………8
4.2 Effect of modulators on mMICR……………………………………………...…9
4.3 Ca2+ stores following mMICR…………………………………...………………9
4.4 Effect of U73122 on mMICR…………………………………………………..10
5. Discussion……………………………………………………………………………11
6. Figure legends………………………………………………………………………..15
6.1 Figure 1 Effect of m-3M3FBS on [Ca2+]i in fura-2-loaded SCM1 cells………..15
6.2 Figure 2 Effect of extracellular Ca2+ removal on m-3M3FBS-induced [Ca2+]i increases.………………………………………………………………………..16
6.3 Figure 3 Effect of Ca2+ channel blockers and phospholipase A2 inhibitor on m-3M3FBS-induced[Ca2+]irise………………………………………………....17
6.4 Figure 4 Intracellular Ca2+ stores of m-3M3FBS-induced Ca2+ release………..18
6.5 Figure 5 Lack of effect of U73122 on m-3M3FBS-induced Ca2+ release.……..19 References……………………………………………………………………………...20

1. Bae YS, Lee TG, Park JC, Hur JH, Kim Y, Heo K, Kwak JY, Suh PG, Ryu SH. Identification of a compound that directly stimulates phospholipase C activity. Mol Pharmacol 63:1043-1050, 2003.
2. Tsai JY, Shieh P, Kuo DH, Chen FA, Kuo CC, Jan CR. Effect of m-3M3FBS on Ca2+ Movement in PC3 Human Prostate Cancer Cells. Chin J Physiol 53:151-159, 2010.
3. Hattori T, Wang PL. Calcium antagonists cause dry mouth by inhibiting resting saliva secretion. Life Sci 81:683-690, 2007.
4. Horowitz LF, Hirdes W, Suh BC, Hilgemann DW, Mackie K, Hille B. Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current. J Gen Physiol 126:243-262, 2005.
5. Zhang B, Ma JX. SERPINA3K prevents oxidative stress induced necrotic cell death by inhibiting calcium overload. PLoS ONE 3:e4077, 2008.
6. Fang YC, Kuo, DH, Shieh P, Chen FA, Kuo CC, Jan CR. Effect of m-3M3FBS on Ca2+ movement in Madin-Darby canine renal tubular cells. Hum Experi Toxicol 28:655-663, 2009.
7. Jajoo S, Mukherjea D, Brewer GJ, Ramkumar V. Pertussis toxin B-oligomer suppresses human immunodeficiency virus-1 Tat-induced neuronal apoptosis through feedback inhibition of phospholipase C-beta by protein kinase C. Neuroscience 151:525-532, 2008.
8. Díaz Añel, AM. Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport. Biochem J 406:157-165, 2007.
9. Nam JH, Lee HS, Nguyen YH, Kang TM, Lee SW, Kim HY, Kim SJ, Earm YE, Kim SJ. Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes. J Physiol 582:977-990, 2007.
10. Rao JN, Liu L, Zou T, Marasa BS, Boneva D, Wang, SR, Malone DL, Turner DJ, Wang JY. Polyamines are required for phospholipase C-gammal expression promoting intestinal epithelial restitution after wounding. Am J Physiol Gastrointest Liver Physiol 292:G335-G343, 2007.
11. Clapp TR, Medler KF, Damak S, Margolskee RF, Kinnamon SC. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25. BMC Biol 4:7, 2006.
12. Lin CH, Liu MC, Lin MS, Lin PL, Chen YH, Chen CT, Chen IM, Tsai MC. Effects of a new isoquinolinone derivative on induction of action potential bursts in central snail neuron. Pharmacology 75:98-110, 2005.
13. Krjukova J, Holmqvist T, Danis AS, Akerman KE, Kukkonen JP. Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation. Br J Pharmacol 143:3-7, 2004.
14. Bootman MD, Berridge MJ, Roderick HL. Calcium signalling: more messengers, more channels, more complexity. Curr Biol 12:R563-R565, 2002.
15. Clapham DE. Intracellular calcium. Replenishing the stores. Nature 375:634-635, 1995.
16. Jung EM, Lee TJ, Park JW, Bae YS, Kim SH, Choi YH, Kwon TK. The novel phospholipase C activator, m-3M3FBS, induces apoptosis in tumor cells through caspase activation, downregulation of XIAP and intracellular calcium signaling. Apoptosis 13:133-145, 2008.
17. Liu SI, Huang CC, Wang BW, Chang PM, Fang YC, Chen WC, Wang JL, Lu YC, Chu ST, Chou CT, Jan C.R. Thimerosal-induced apoptosis in human SCM1 gastric cancer cells: activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation. Toxicol Sci 100:109-117, 2007.
18. Kuo CC, Kuo DH, Huang CJ, Fang YC, Shieh P, Chen FA, Shaw CF, Jan C.R. Nonylphenol-induced apoptotic pathways in SCM1 human gastric cancer cells. Drug Dev Res 71:139-148, 2010.
19. Clapham DE. Intracellular calcium replenishing the stores. Nature 375:634-635, 1995.
20. Clapham DE. Calcium signaling. Cell 80:259-268, 1995.
21. Blaustein MP. Calcium transport and buffering in neurons. TINS 11:438-443, 1998.
22. Berridge MJ. Elementary and global aspects of calcium signaling. J Physiol 499:291-306, 1997.
23. Berridge MJ. Inositol trisphosphate and calcium signaling. Nature 361:315-325, 1993.
24. McPherson PS, Campbell KP. The ryanodine receptor/Ca2+ release channel. J Biol Chem 268:13765-13768, 1993.
25. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 7:1-12, 1986.
26. Park HY, Park JI, Baek DW, Lee SY, Lee MJ, Jin JO, Kim JW, Hong YS, Lee YH, Kwak JY. Modulation of neutrophil apoptosis by beta-amyloid proteins. Int Immunopharmacol 6:1061-1069, 2006.
27. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Aca Sci 87:2466-2470, 1990.
28. van Esch GJ. Toxicology of tert-butylhydroquinone (TBHQ). Food Chem Toxicol 24:1063-1065, 1986.
29. Bootman MD, Berridge MJ, Roderick HL. Calcium signaling: more messengers, more channels, more complexity. Cur Biol 12:563-565, 2002.
30. Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher SK. The aminosteroid U73122 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, 1991.
31. Florenzano F, Viscomi MT, Mercaldo V. P2X2R purinergic receptor subunit mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons. J Com Neurol 498:58-67, 2006.
32. Wang JL, Lin KL, Chen WC, Chou CT, Huang CJ, Liu CS, Hsieh CH, Chang CH, Huang JK, Chang HT, Liu SI, Hsu SS, Jan CR. Effect of celecoxib on Ca2+ fluxes and proliferation in MDCK renal tubular cells. J Recept Signal Transduct Res 25:237-249, 2005.
33. Jan CR, Chou KJ, Lee KC, Wang JL, Tseng LL, Cheng JS, Chen WC. Dual action of palmitoyl trifluoromethyl ketone (PACOCF3) on Ca2+ signaling: activation of extracellular Ca2+ influx and alteration of ATP- and bradykinin-induced Ca2+ responses in Madin Darby canine kidney cells. Arch Toxicol 74:447-451, 2000.
34. Young RC, Schumann R, Zhang P. Nifedipine block of capacitative calcium entry in cultured human uterine smooth-muscle cells. J Soc Gynecol Investig 8:210-215, 2001.
35. Harper JL, Camerini-Otero CS, Li AH, Kim SA, Jacobson KA, Daly JW. Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells. Biochem Pharmacol. 65:329-338, 2003.
36. Quinn T, Molloy M, Smyth A, Baird AW. Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro. Life Sci 74:1659-1669, 2004.
37. Morita K, Sakakibara A, Kitayama S, Kumagai K, Tanne K, Dohi T. Pituitary adenylate cyclase-activating polypeptide induces a sustained increase in intracellular free Ca2+ concentration and catechol amine release by activating Ca2+ influx via receptor-stimulated Ca2+ entry, independent of store-operated Ca2+ channels, and voltage-dependent Ca2+ channels in bovine adrenal medullary chromaffin cells. J Pharmacol Exp Ther 302:972-982, 2002.
38. Ishikawa J, Ohga K, Yoshino T, Takezawa R, Ichikawa A, Kubota H, Yamada T. A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes. J Immunol 170:4441-4449, 2003.
39. Jiang N, Zhang ZM, Liu L, Zhang C, Zhang YL, Zhang ZC. 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, 2006.
40. Shideman CR, Reinardy JL, Thayer SA. Gamma-Secretase activity modulates store-operated Ca2+ entry into rat sensory neurons. Neurosci Lett 451:124-128, 2009.
41. Tedesco E, Rigoni M, Caccin P, Grishin E, Rossetto O, Montecucco C. Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins. Toxicon 54:138-44, 2009.
42. Lupescu A, Bock CT, Lang PA, Aberle S, Kaiser H, Kandolf R, Lang F. Phospholipase A2 activity-dependent stimulation of Ca2+ entry by human parvovirus B19 capsid protein VP1. J Virol 80:11370-11380, 2006.
43. Boittin FX, Gribi F, Serir K, Bény JL. Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta. Am J Physiol Heart Circ Physiol 295:H2466-H2474, 2008.
44. Boittin FX, Petermann O, Hirn C, Mittaud P, Dorchies OM, Roulet E, Ruegg UT. Ca2+ -independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers. J Cell Sci. 119:3733-3742, 2006.
45. Singaravelu K, Lohr C, Deitmer JW. Calcium-independent phospholipase A2 mediates store-operated calcium entry in rat cerebellar granule cells. Cerebellum 7:467-481, 2008.
46. Jung EM, Lee TJ, Park JW, Bae YS, Kim SH, Choi YH, Kwon TK. The novel phospholipase C activator, m-3M3FBS, induces apoptosis in tumor cells through caspase activation, down-regulation of XIAP and intracellular calcium signaling. Apoptosis 13:133-145, 2008.
47. Lee YN, Lee HY, Kim JS, Park C, Choi YH, Lee TG, Ryu SH, Kwak JY, Bae YS. The novel phospholipase C activator, m-3M3FBS, induces monocytic leukemia cell apoptosis. Cancer Lett 222:227-235, 2005.