FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2093123576> ?p ?o ?g. }

• W2093123576 endingPage "19140" @default.
• W2093123576 startingPage "19133" @default.
• W2093123576 abstract "Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca2+. This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca2+ spike, change in mitochondrial membrane potential (Δψm) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resulted in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca2+ uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced Δψm transition and Bax translocation. This suggests that ROS generation is an event that occurs after the Δψm transition and Bax translocation. Together these data demonstrate that the Ca2+ spike, mitochondrial Bcl-2 presensitization, and subsequent Δψm transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis. Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca2+. This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca2+ spike, change in mitochondrial membrane potential (Δψm) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resulted in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca2+ uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced Δψm transition and Bax translocation. This suggests that ROS generation is an event that occurs after the Δψm transition and Bax translocation. Together these data demonstrate that the Ca2+ spike, mitochondrial Bcl-2 presensitization, and subsequent Δψm transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis. Bcl-2 is overexpressed in many types of tumors, especially relapsed or chemoresistant malignancies (1Reed J.C. Miyashita T. Krajewski S. Takayama S. Aime-Sempe C. Kitada S. Sato T. Wang H.G. Harigai M. Hanada M. Krajewska M. Kochel K. Millan J. Kobayashi H. Cancer Treat. Res. 1996; 84: 31-72Google Scholar, 2Krajewska M. Krajewski S. Epstein J.I. Shabaik A. Sauvageot J. Song K. Kitada S. Reed J.C. Am. J. Pathol. 1996; 148: 1567-1576Google Scholar, 3Borner M.M. Brousset P. Pfanner-Meyer B. Bacchi M. Vonlanthen S. Hotz M.A. Altermatt H.J. Schlaifer D. Reed J.C. Betticher D.C. Br. J. Cancer. 1999; 79: 952-958Google Scholar, 4Villar E. Redondo M. Rodrigo I. Garcia J. Avila E. Matilla A. Tumour Biol. 2001; 22: 137-145Google Scholar, 5Tothova E. Fricova M. Stecova N. Kafkova A. Elbertova A. Neoplasma. 2002; 49: 141-144Google Scholar). It is an important target for anticancer drug development because Bcl-2 plays an essential antiapoptotic role against cell death induced by chemotherapeutic agents, radiation, growth factor withdrawal, or hypoxia (6Reed J.C. Oncogene. 1998; 17: 3225-3236Google Scholar). By interrupting the function of Bcl-2, agents such as peptidic and organic molecules discovered recently have been shown to induce a potent direct and selective killing effect on target cells (7Wang J.L. Zhang Z.J. Choksi S. Shan S. Lu Z. Croce C.M. Alnemri E.S. Korngold R. Huang Z. Cancer Res. 2000; 60: 1498-1502Google Scholar, 8Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Google Scholar, 9Huang Z. Chem. Biol. 2002; 9: 1059-1072Google Scholar). Like most apoptotic stimuli, these peptidic and organic inhibitors of Bcl-2 induce cytochrome c release, caspase-9/-3 activation, and DNA fragmentation. However, the precise molecular mechanism for cytochrome c release triggered by Bcl-2-targeting agents remains obscure. Cytochrome c release is a central step in the initiation of caspase-dependent apoptosis (10Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Google Scholar, 11Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Google Scholar, 12Scorrano L. Korsmeyer S.J. Biochem. Biophys. Res. Commun. 2003; 304: 437-444Google Scholar). It has been demonstrated that overexpression of either mitochondria- or endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; PTP, potential transient pore; ROS, reactive oxygen species; PARP, poly(ADP-ribose) polymerase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; Δψm, change in mitochondrial membrane potential; BH3, Bcl-2 homology 3; H2DCFDA, succinimidyl ester of dichlorodihydrofluorescein diacetate; MEF, mouse embryonic fibroblast; MTS-tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Z-VAD-fmk, carbobenzoxyl-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone; carbobenzoxylvalyl-alanyl- Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; GFP, green fluorescent protein; IAP, inhibitor of apoptosis. 1The abbreviations used are: ER, endoplasmic reticulum; HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; PTP, potential transient pore; ROS, reactive oxygen species; PARP, poly(ADP-ribose) polymerase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; Δψm, change in mitochondrial membrane potential; BH3, Bcl-2 homology 3; H2DCFDA, succinimidyl ester of dichlorodihydrofluorescein diacetate; MEF, mouse embryonic fibroblast; MTS-tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Z-VAD-fmk, carbobenzoxyl-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone; carbobenzoxylvalyl-alanyl- Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; GFP, green fluorescent protein; IAP, inhibitor of apoptosis.-specific Bcl-2 prevents the induction of apoptosis by inhibiting the mitochondrial release of cytochrome c (13Annis M.G. Zamzami N. Zhu W. Penn L.Z. Kroemer G. Leber B. Andrews D.W. Oncogene. 2001; 20: 1939-1952Google Scholar, 14Rudner J. Lepple-Wienhues A. Budach W. Berschauer J. Friedrich B. Wesselborg S. Schulze-Osthoff K. Belka C. J. Cell Sci. 2001; 114: 4161-4172Google Scholar). Although the molecular mechanism governing the cytochrome c release regulated by Bcl-2 has yet to be fully characterized, what does appear to be important is the heterodimerization of Bcl-2 with proapoptotic proteins. Such heterodimerization directly or indirectly inhibits the apoptotic function, especially the homo-oligomerization of multidomain proapoptotic Bax or Bak that corresponds with the release of cytochrome c (15Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Google Scholar, 16Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.C. Science. 1997; 277: 370-372Google Scholar, 17Adams J.M. Cory S. Science. 1998; 281: 1322-1326Google Scholar, 18Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Google Scholar). Heterodimerization of Bcl-2 with proapoptotic proteins or BH3 domain-derived peptides from proapoptotic members such as Bax, Bak, or Bad has been demonstrated by NMR, cell-free protein-protein binding, and yeast two-hybrid studies (19Petros A.M. Medek A. Nettesheim D.G. Kim D.H. Yoon H.S. Swift K. Matayoshi E.D. Oltersdorf T. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3012-3017Google Scholar, 20Reed J.C. Zha H. Aime-Sempe C. Takayama S. Wang H.G. Adv. Exp. Med. Biol. 1996; 406: 99-112Google Scholar, 21Hanada M. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11969Google Scholar). The anti-cytochrome c releasing effect of Bcl-2 can be suppressed by the overexpression of proapoptotic proteins such as Bax (22Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Google Scholar). Conversely the cytochrome c releasing activity of proapoptotic proteins can be limited by the overexpression of Bcl-2 (16Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.C. Science. 1997; 277: 370-372Google Scholar). The ratio rather than the amount of antiapoptotic versus proapoptotic proteins determines whether apoptotic signaling can proceed (15Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Google Scholar, 17Adams J.M. Cory S. Science. 1998; 281: 1322-1326Google Scholar, 23Korsmeyer S.J. Shutter J.R. Veis D.J. Merry D.E. Oltvai Z.N. Semin. Cancer Biol. 1993; 4: 327-332Google Scholar). Normally Bax exists as a soluble monomer in cytosol or is loosely associated with mitochondria. However, upon apoptotic stimulation, Bax translocates to mitochondria where it forms oligomers that are inserted into the outer mitochondrial membrane (24Antonsson B. Montessuit S. Sanchez B. Martinou J.C. J. Biol. Chem. 2001; 276: 11615-11623Google Scholar, 25Valentijn A.J. Metcalfe A.D. Kott J. Streuli C.H. Gilmore A.P. J. Cell Biol. 2003; 162: 599-612Google Scholar). The signals that trigger the translocation of Bax to mitochondria remain largely unknown. In synthetic membranes, Bcl-2 has been shown to directly inhibit the channel or pore forming activity of Bax (16Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.C. Science. 1997; 277: 370-372Google Scholar), which is consistent with the ability of Bcl-2 in preventing Bax oligomerization in outer mitochondrial membranes (22Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Google Scholar, 26Saito M. Korsmeyer S.J. Schlesinger P.H. Nat. Cell Biol. 2000; 2: 553-555Google Scholar). The antiapoptotic function of Bcl-2 is thought to be primarily derived from Bcl-2 presented in the mitochondria (10Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Google Scholar, 27Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Google Scholar). However, growing evidence has indicated that Bcl-2 expressed on ER plays an important role in the inhibition of cytochrome c release and apoptosis (28Rudner J. Jendrossek V. Belka C. Apoptosis. 2002; 7: 441-447Google Scholar). An exclusive ER-targeted form of Bcl-2, Bcl-2/cb5 has been shown to prevent apoptosis in many types of cells induced by c-Myc, tunicamycin, brefeldin A, radiation, or Bax overexpression (13Annis M.G. Zamzami N. Zhu W. Penn L.Z. Kroemer G. Leber B. Andrews D.W. Oncogene. 2001; 20: 1939-1952Google Scholar, 14Rudner J. Lepple-Wienhues A. Budach W. Berschauer J. Friedrich B. Wesselborg S. Schulze-Osthoff K. Belka C. J. Cell Sci. 2001; 114: 4161-4172Google Scholar, 29Lee S.T. Hoeflich K.P. Wasfy G.W. Woodgett J.R. Leber B. Andrews D.W. Hedley D.W. Penn L.Z. Oncogene. 1999; 18: 3520-3528Google Scholar, 30Hacki J. Egger L. Monney L. Conus S. Rosse T. Fellay I. Borner C. Oncogene. 2000; 19: 2286-2295Google Scholar, 31Wang N.S. Unkila M.T. Reineks E.Z. Distelhorst C.W. J. Biol. Chem. 2001; 276: 44117-44128Google Scholar). The protective effect of Bcl-2/cb5 in cells suggests the presence of cross-talk between ER and mitochondria. However, the mediators that connect ER Bcl-2 and mitochondrial cytochrome c release remain to be identified. What does appear to be important is intracellular Ca2+ signaling (32Scorrano L. Oakes S.A. Opferman J.T. Cheng E.H. Sorcinelli M.D. Pozzan T. Korsmeyer S.J. Science. 2003; 300: 135-139Google Scholar, 33Demaurex N. Distelhorst C. Science. 2003; 300: 65-67Google Scholar). It has been shown that Bcl-2 overexpression blocks Ca2+ entry into or release from ER in response to many apoptotic stimuli. For example, overexpression of Bcl-2 decreases the ER Ca2+ load (34Pinton P. Ferrari D. Magalhaes P. Schulze-Osthoff K. Di Virgilio F. Pozzan T. Rizzuto R. J. Cell Biol. 2000; 148: 857-862Google Scholar, 35Lam M. Dubyak G. Chen L. Nunez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Google Scholar) and prevents or delays the depletion of Ca2+ from ER after cells have received a treatment with apoptosis inducers such as thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone, cyclopiazonic acid, or ceramide (36He H. Lam M. McCormick T.S. Distelhorst C.W. J. Cell Biol. 1997; 138: 1219-1228Google Scholar, 37Bian X. Hughes Jr., F.M. Huang Y. Cidlowski J.A. Putney Jr., J.W. Am. J. Physiol. 1997; 272: C1241-C1249Google Scholar, 38Pinton P. Ferrari D. Rapizzi E. Di Virgilio F.D. Pozzan T. Rizzuto R. EMBO J. 2001; 20: 2690-2701Google Scholar). The involvement of Ca2+ signaling in apoptosis has been suggested by a number of recent studies (39Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Google Scholar, 40Pinton P. Ferrari D. Rapizzi E. Di Virgilio F. Pozzan T. Rizzuto R. Biochimie (Paris). 2002; 84: 195-201Google Scholar). With transient increases in cytosolic Ca2+, many intracellular enzymes including phospholipases, proteases, and endonucleases can be activated, while prolonged or unregulated cytosolic Ca2+ elevation can further lead to apoptosis or cell death (41Orrenius S. McCabe Jr., M.J. Nicotera P. Toxicol. Lett. 1992; 64: 357-364Google Scholar, 42Pacher P. Hajnoczky G. EMBO J. 2001; 20: 4107-4121Google Scholar). Although the molecular mechanism underlying apoptosis mediated by Ca2+ in vivo remains to be fully defined, accumulation of mitochondrial Ca2+ or activation of caspase-12 or other Ca2+-dependent intracellular enzymes may be the possible mechanisms (39Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Google Scholar, 43Ermak G. Davies K.J. Mol. Immunol. 2002; 38: 713-721Google Scholar). ER and mitochondria are two crucial organelles in the regulation of intracellular Ca2+ homeostasis in cells. They are physiologically connected (44Hajnoczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Google Scholar, 45Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Google Scholar). After certain stimulations, rapid Ca2+ accumulation in mitochondria always occurs immediately after ER Ca2+ release (46Rapizzi E. Pinton P. Szabadkai G. Wieckowski M.R. Vandecasteele G. Baird G. Tuft R.A. Fogarty K.E. Rizzuto R. J. Cell Biol. 2002; 159: 613-624Google Scholar, 47Pacher P. Thomas A.P. Hajnoczky G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2380-2385Google Scholar). Such a rapid influx of Ca2+ into mitochondria causes mitochondrial PTP to open and subsequent mitochondrial membrane permeability, mitochondrial metabolic alteration, reactive oxygen species (ROS) generation, and cytochrome c release. ROS is another potent promoter of cytochrome c release. In addition to regulating the cytochrome c-mediated caspase-dependent apoptosis, both Ca2+ and ROS are important mediators of caspase-independent apoptosis or necrosis (39Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Google Scholar, 43Ermak G. Davies K.J. Mol. Immunol. 2002; 38: 713-721Google Scholar). Apoptosis, or programmed cell death, is a very important phenomenon in cytotoxicity induced by anticancer treatment. However, chemoresistance occurs when antiapoptotic protein Bcl-2 is overexpressed (48Reed J.C. Hematol. Oncol. Clin. N. Am. 1995; 9: 451-473Google Scholar, 49Inoue S. Salah-Eldin A.E. Omoteyama K. Hum. Cell. 2001; 14: 211-221Google Scholar). Therefore, directly targeting Bcl-2 might overcome such chemoresistance. Based on the evidence that the heterodimerization of Bcl-2 and proapoptotic proteins is mediated by the BH3 domain of the proapoptotic proteins, we recently discovered a novel organic molecule, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (C17H17BrN2O5,), that binds to the proapoptotic protein BH3 domain binding sites on the surface pocket of Bcl-2 (8Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Google Scholar). HA14-1 is a synthetic cell-permeable small molecule with a molecular weight of 409. It specifically competes with Bak BH3 domain-derived peptide in binding Bcl-2 with an IC50 of 9 μm (8Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Google Scholar). HA14-1 alone or in combination with other anticancer agents has been shown to induce apoptosis effectively in many types of cancer cells including Ara-C-resistant HL-60 cells (8Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Google Scholar, 50Yamaguchi H. Paranawithana S.R. Lee M.W. Huang Z. Bhalla K.N. Wang H.G. Cancer Res. 2002; 62: 466-471Google Scholar, 51Milella M. Estrov Z. Kornblau S.M. Carter B.Z. Konopleva M. Tari A. Schober W.D. Harris D. Leysath C.E. Lopez-Berestein G. Huang Z. Andreeff M. Blood. 2002; 99: 3461-3464Google Scholar, 52Pei X.Y. Dai Y. Grant S. Leukemia. 2003; 17: 2036-2045Google Scholar). Cytochrome c release, caspase-9/-3 activation, and DNA fragmentation are evident in HA14-1-induced apoptosis. However, the molecular mechanism upstream of cytochrome c release upon HA14-1 treatment remains unclear. In this study, we investigated and identified several upstream signals in both ER and mitochondria that are critical for cytochrome c release in response to HA14-1. Reagents—HA14-1 was synthesized and purified by our laboratory following our previously published method (53Yu N. Aramini J.M. Germann M.W. Huang Z. Tetrahedron Lett. 2000; 41: 6993-6996Google Scholar). The detailed description of the routes of syntheses and structure has been published previously (8Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Google Scholar). HA14-1 was prepared freshly by dissolving HA14-1 in Me2SO. It was diluted to appropriate concentrations in culture media before use in each experiment. EGTA acetoxymethyl ester, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), Calcium Green, and H2DCFDA (Molecular Probes) were dissolved in Me2SO, and aliquots were stored at -20 °C. RU-360 (Calbiochem), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and vitamin E (Sigma) were dissolved in ethanol, and aliquots were stored at -20 °C. Cell Culture—The HL-60/Bcl-2 and HL-60/neo cell lines were obtained from Dr. Kapil N. Bhalla (University of Miami School of Medicine, Miami, FL). Each cell line (HL-60/Bcl-2 or HL-60/neo) had been stably transfected with either pZip-Bcl-2 or pZip-neo (control), respectively. The Bax-/-/Bak-/- knock-out and wild type MEF cell lines were obtained from Dr. Stanley J. Korsmeyer (Dana-Farber Cancer Institute, Boston, MA). All cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 800 μg/ml Geneticin (G418, Invitrogen). The concentrations of HA14-1 and other agents used and the treatment intervals are indicated in the following “Experimental Procedures” and under “Results.” Cells were maintained in a humidified 5% CO2 atmosphere at 37 °C. Measurement of Cell Viability—To measure the cytotoxic effects of HA14-1 on cell survival, cells were plated at 1 × 105 cells/well in 96-well plates. Cells were treated with either a vehicle control or various concentrations of HA14-1 or HA14-1 plus other agents as indicated under “Results.” At the end of each treatment, cell viability was measured by using a CellTiter 96® AQueous assay kit (Promega). This kit contains MTS-tetrazolium in which viable cells convert MTS-tetrazolium into a formazan colored product with an A of 490 nm. According to the manufacturer's instructions, 20 μl of MTS-tetrazolium solution were added to 100 μl of culture media and incubated for 3-4 h at 37 °C. Afterward the absorbance at 490 nm was measured using a Wallac Victor2 multilabel counter (PerkinElmer Life Sciences). Assays were performed in triplicate for each experiment; mean cell viability was compared with vehicle-treated controls. Each experimental data point was generated from at least three independent experiments. Measurement of Caspase Activity—A CaspGLOW green caspase staining kit (Biovision) was used to measure the caspase-9 activity according to the manufacturer's instructions. Briefly cells (106/ml), after different treatments, were incubated with fluorescein isothiocyanate-conjugated caspase-9 inhibitor LEHD-fmk, which specifically interacts with activated caspase-9. After incubation for 30 min at 37 °C, the fluorescence intensity was determined using the Wallac Victor2 multilabel counter. Increase in the fluorescence intensity was determined by comparing the level of the treated cells to that of vehicle controls. Poly(ADP-ribose) Polymerase (PARP) Cleavage and DNA Fragmentation Assay—After appropriate treatments, cells were harvested by centrifugation, washed with ice-cold phosphate-buffered saline, and placed in 1) lysis buffer containing 0.02 m Tris, 1% Triton X-100, 0.14 m NaCl, 2 mm EDTA, 10 μmol/ml protease inhibitor (Sigma), 1 mm phenylmethylsulfonyl fluoride, and dithiothreitol for PARP cleavage assay and 2) lysis buffer containing 50 mm Tris, pH 8.0, 10 mm EDTA, 0.5% SDS, 0.5% sodium chloride, and 100 μg/ml proteinase K for 3 h at 56 °C and for 1 h with 0.5 mg/ml RNase A at 37 °C for DNA fragmentation assay. The PARP cleavage was determined on Western blot using rabbit polyclonal antibody (1:1,000) (Cell Signaling Technology). Signals were detected in the same way as that described for cytochrome c. For the DNA fragmentation assay, DNA was precipitated with isopropanol and analyzed by 2% agarose gel electrophoresis. After ethidium bromide staining, the presence of DNA in the gels was visualized under UV light. Measurement of Cytochrome c Release—Mitochondrial versus cytosol fractions of cells were prepared using a mitochondrial/cytosol fractionation kit (Biovision). Cells at 1 × 107 with or without different treatments were harvested by centrifugation at 700 × g for 5 min and washed twice with cold phosphate-buffered saline. Afterward the cells were resuspended in a 250 μl of extraction buffer containing protease inhibitor mixture and dithiothreitol (Biovision). After incubation on ice for 30 min, the cells were homogenized using a Kontes Dounce tissue grinder (Fisher) on ice. Homogenizations were centrifuged at 700 × g for 10 min at 4 °C, and the supernatant was collected. Then the collected supernatant was centrifuged again at 10,000 × g for 30 min at 4 °C. The resulting supernatants were harvested and designated as cytosolic fractions, and the pellets were resuspended in an appropriate buffer and designated as mitochondrial fractions. Protein content was determined using a Coomassie Blue assay (Bio-Rad). Similarly mitochondrial versus media fractions were obtained after treatments of purified mitochondria with HA14-1. For all experiments, fresh mitochondria were prepared and used within 4 h. The isolated mitochondria were incubated in a buffer containing 150 mm KCl, 3 mm KH2PO4,5mm succinate, 25 mm NaHCO3, 1 mm MgCl2, 2 μm rotenone, 5 mm Tris-HCl, pH 7.4 and treated with 20 μm HA14-1 alone, with 0.1 μm Ca2+ alone, or with HA14-1 plus 0.1 μm Ca2+ for 30 min at 37 °C, respectively. Cytochrome c release in isolated mitochondria was monitored after treatment with HA14-1 in the presence or absence of CaCl2. The cytochrome c distributed in either fraction, the cytosol/media or the mitochondria, in each experiment was analyzed using Western blotting with anti-cytochrome c monoclonal antibody (1:1,000) (BD Pharmingen) or control antibody against β-actin (1:5,000) (Oncogene). Signals were detected using the horseradish peroxidase-conjugated anti-mouse secondary antibody (1:1,000) and enhanced chemiluminescence substrate kit (Amersham Biosciences). Measurement of Intracellular Calcium Concentration—Cells at 1 × 106/ml were incubated with the calcium indicator dye Calcium Green at 5 μm (Molecular Probes) for 30 min at 37 °C in 5% CO2 followed by a 10-min wash in phosphate-buffered saline. Previous studies have demonstrated that the vast majority of Calcium Green fluorescence is associated with ER as determined by using confocal microscopy with ER-Tracker- or MitoTracker-counterstained cells (54Nutt L.K. Chandra J. Pataer A. Fang B. Roth J.A. Swisher S.G. O'Neil R.G. McConkey D.J. J. Biol. Chem. 2002; 277: 20301-20308Google Scholar). The fluorescence intensity of each indicator was measured using a FluoroMax-2 fluorescence spectrofluorometer (JoBin Yvon-Spex Instruments S.A., Inc.). Fluorescence was excited at 485 nm (F485), and emitted fluorescence was recorded at 550 nm. To obtain the fluorescence maximum and minimum, cells were subsequently incubated with 0.1% Triton X-100 or 10 mm EGTA in the presence of saturating concentrations of extracellular Ca2+. Experiments were performed at 37 °C with constant stirring. The Ca2+ concentrations were calculated by the formula: [Ca2+] = (F - Fmin/Fmax - F) × Kd. Determination of Changes in Mitochondrial Membrane Potential (Δψm)—Quantitative changes in Δψm in cells at the early stage of apoptosis were measured using JC-1. This dye exists as a monomer with emission at 530 nm (green fluorescence) at low concentrations but forms J-aggregates with emission at 590 nm (red fluorescence) at high concentrations. Therefore, the fluorescence of JC-1 can be considered as an indicator of the relative mitochondrial membrane polarization state. Briefly cells were seeded at 1 × 106/ml. After different treatments, the cells were incubated with 10 μm JC-1 at 37 °C for 8 min, washed twice, and resuspended in phosphate-buffered saline. Relative fluorescence intensities were monitored using the FluoroMax-2 fluorescence spectrofluorometer with 490 nm excitation/535 nm emission and 570 nm excitation/610 nm emission. The Δψm was expressed as the ratio of the fluorescence of J-aggregate (570 nm excitation/610 nm emission) to monomer (490 nm excitation/535 nm emission) forms of JC-1. Confocal Microscopic Analysis of Bax Translocation—Cells (5 × 105) were transfected with pEGFP-C3/Bax (obtained from Dr. Richard J. Youle, NINDS, National Institutes of Health, Bethesda, MD) using the cationic lipid LipofectAMINE™ 2000 (Invitrogen) according to the manufacturer's instruction. Each well contained 2 μg of plasmid DNA and 8 μg of LipofectAMINE 2000. After 3 h in a serum-free medium, an equal volume of culture medium was added. Twenty-four hours after transfection, the cells were washed once and cultured in regular media for an additional 24 h. Afterward cells were stained with MitoTracker Red at 20 ng/ml for 30 min, and then the cells were resuspended in fresh medium containing 20 μm HA14-1 and transferred to a sealed mounting chamber. Images were collected using a confocal microscope (Olympus). The fluorescence excitation of GFP and MitoTracker Red were 488 and 568 nm, respectively. Measurement of ROS Generation—The generation of ROS was measured in HL-60 cells at the end of different treatments according to the methods published by others (55Brubacher J.L. Bols N.C. J. Immunol. Methods. 2001; 251: 81-91Google Scholar). Briefly, at the end of each treatment, the culture medium was removed and replaced with 100 μl of fresh culture medium containing 100 μm H2DCFDA, which was intracellularly de-esterified to dichlorodihydrofluorescein the substrate that ROS oxidizes to fluorescent dichlorofluorescein. After a 15-min incubation at 37 °C, the fluorescence intensity was measured using the Wallac Victor2 multilabel counter. Fluorescence was excited at 485 nm, and emitted fluorescence was recorded at 538 nm. Statistical Analysis—The statistical analysis was performed using the Student's paired t test. Two-sided p values of <0.05 were c" @default.
• W2093123576 created "2016-06-24" @default.
• W2093123576 creator A5043422612 @default.
• W2093123576 creator A5049328660 @default.
• W2093123576 creator A5054059790 @default.
• W2093123576 date "2004-04-01" @default.
• W2093123576 modified "2023-10-09" @default.
• W2093123576 title "Critical Upstream Signals of Cytochrome c Release Induced by a Novel Bcl-2 Inhibitor" @default.
• W2093123576 cites W1500271849 @default.
• W2093123576 cites W1522763255 @default.
• W2093123576 cites W1523213544 @default.
• W2093123576 cites W1532194993 @default.
• W2093123576 cites W1557686543 @default.
• W2093123576 cites W1566777539 @default.
• W2093123576 cites W1679452661 @default.
• W2093123576 cites W1933007466 @default.
• W2093123576 cites W1963899781 @default.
• W2093123576 cites W1964617786 @default.
• W2093123576 cites W1979995768 @default.
• W2093123576 cites W1986388339 @default.
• W2093123576 cites W1994611236 @default.
• W2093123576 cites W1999349182 @default.
• W2093123576 cites W2002487087 @default.
• W2093123576 cites W2007313590 @default.
• W2093123576 cites W2008888835 @default.
• W2093123576 cites W2013065884 @default.
• W2093123576 cites W2016974966 @default.
• W2093123576 cites W2017102177 @default.
• W2093123576 cites W2021944232 @default.
• W2093123576 cites W2022593308 @default.
• W2093123576 cites W2025818427 @default.
• W2093123576 cites W2036822121 @default.
• W2093123576 cites W2040968120 @default.
• W2093123576 cites W2045216288 @default.
• W2093123576 cites W2046353863 @default.
• W2093123576 cites W2050297509 @default.
• W2093123576 cites W2054820743 @default.
• W2093123576 cites W2060987758 @default.
• W2093123576 cites W2061947498 @default.
• W2093123576 cites W2064196264 @default.
• W2093123576 cites W2068146558 @default.
• W2093123576 cites W2068800778 @default.
• W2093123576 cites W207305739 @default.
• W2093123576 cites W2073706613 @default.
• W2093123576 cites W2074993010 @default.
• W2093123576 cites W2079273110 @default.
• W2093123576 cites W2086365629 @default.
• W2093123576 cites W2090240729 @default.
• W2093123576 cites W2102584757 @default.
• W2093123576 cites W2108131089 @default.
• W2093123576 cites W2108972313 @default.
• W2093123576 cites W2109938472 @default.
• W2093123576 cites W2111209940 @default.
• W2093123576 cites W2116722945 @default.
• W2093123576 cites W2117049476 @default.
• W2093123576 cites W2124221273 @default.
• W2093123576 cites W2134413058 @default.
• W2093123576 cites W2141000153 @default.
• W2093123576 cites W2152247516 @default.
• W2093123576 cites W2154546683 @default.
• W2093123576 cites W2157805786 @default.
• W2093123576 cites W2161641464 @default.
• W2093123576 cites W2171183626 @default.
• W2093123576 cites W2259568108 @default.
• W2093123576 cites W2279323582 @default.
• W2093123576 cites W2315241706 @default.
• W2093123576 cites W2318252390 @default.
• W2093123576 cites W2410237697 @default.
• W2093123576 cites W65521289 @default.
• W2093123576 cites W89280349 @default.
• W2093123576 doi "https://doi.org/10.1074/jbc.m400295200" @default.
• W2093123576 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14966123" @default.
• W2093123576 hasPublicationYear "2004" @default.
• W2093123576 type Work @default.
• W2093123576 sameAs 2093123576 @default.
• W2093123576 citedByCount "94" @default.
• W2093123576 countsByYear W20931235762012 @default.
• W2093123576 countsByYear W20931235762013 @default.
• W2093123576 countsByYear W20931235762014 @default.
• W2093123576 countsByYear W20931235762015 @default.
• W2093123576 countsByYear W20931235762016 @default.
• W2093123576 countsByYear W20931235762017 @default.
• W2093123576 countsByYear W20931235762018 @default.
• W2093123576 countsByYear W20931235762019 @default.
• W2093123576 countsByYear W20931235762020 @default.
• W2093123576 countsByYear W20931235762021 @default.
• W2093123576 crossrefType "journal-article" @default.
• W2093123576 hasAuthorship W2093123576A5043422612 @default.
• W2093123576 hasAuthorship W2093123576A5049328660 @default.
• W2093123576 hasAuthorship W2093123576A5054059790 @default.
• W2093123576 hasBestOaLocation W20931235761 @default.
• W2093123576 hasConcept C12554922 @default.
• W2093123576 hasConcept C185592680 @default.
• W2093123576 hasConcept C190283241 @default.
• W2093123576 hasConcept C191172861 @default.
• W2093123576 hasConcept C29311851 @default.
• W2093123576 hasConcept C41008148 @default.
• W2093123576 hasConcept C55493867 @default.

• next