Involvement of Intracellular Ca2+ Levels in Nonsteroidal Anti-inflammatory Drug-induced Apoptosis
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m502956200
ISSN1083-351X
AutoresKen‐ichiro Tanaka, Wataru Tomisato, Tatsuya Hoshino, Tomoaki Ishihara, Takushi Namba, Mayuko Aburaya, Takashi Katsu, Keitarou Suzuki, Shinji Tsutsumi, Tohru Mizushima,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoWe recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells. We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells. Nonsteroidal anti-inflammatory drugs (NSAIDs) 1The abbreviations used are: NSAIDs, nonsteroidal anti-inflammatory drugs; COX, cyclooxygenase; PG, prostaglandin; PGE2, prostaglandin E2; ER, endoplasmic reticulum; CHOP, CCAAT/enhancer-binding protein homologous transcription factor; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid; Ho 342, Hoechst 33342; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DePsipher, 5,5′,6,6′-tetrachloro-1, 1′,3,3′-tetraethylbenzimidaozolylcarbocyanine iodide; Z-Leu-Leu-H, carbobenzoxy-l-leucyl-l-leucinal; AMC, aminomethylcoumarin; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. account for nearly 5% of all prescribed medications (1Smalley W.E. Ray W.A. Daugherty J.R. Griffin M.R. Am. J. Epidemiol. 1995; 141: 539-545Crossref PubMed Scopus (273) Google Scholar). The anti-inflammatory action of NSAIDs is mediated through their inhibitory effect on cyclooxygenase (COX) activity. COX is an enzyme essential for the synthesis of prostaglandins (PGs), which have a strong capacity to induce inflammation. On the other hand, NSAID use is associated with gastrointestinal complications (2Hawkey C.J. Gastroenterology. 2000; 119: 521-535Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), with about 15-30% of chronic users of NSAIDs suffering from gastrointestinal ulcers and bleeding (3Barrier C.H. Hirschowitz B.I. Arthritis Rheum. 1989; 32: 926-932PubMed Google Scholar, 4Gabriel S.E. Jaakkimainen L. Bombardier C. Ann. Intern. Med. 1991; 115: 787-796Crossref PubMed Scopus (1240) Google Scholar, 5Fries J.F. Miller S.R. Spitz P.W. Williams C.A. Hubert H.B. Bloch D.A. 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Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (103) Google Scholar). Therefore, elucidation of the mechanisms governing NSAID-induced cell death is important for understanding the mechanisms by which NSAIDs cause gastric lesions and may allow the development of new safer NSAIDs. We recently showed that all of the NSAIDs tested have membrane permeabilization activity, which is implicated in NSAID-induced apoptosis and necrosis (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). However, the mechanism by which the membrane permeabilization activity of NSAIDs causes cell death and in particular apoptosis in gastric mucosal cells remains unknown. Accumulation of unfolded protein in the endoplasmic reticulum (ER) induces the ER stress response, otherwise known as the unfolded protein response. Cells initially adapt to the accumulation of unfolded proteins by inducing the expression of ER-resident molecular chaperones such as glucose-regulated protein 78 (14Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar, 15Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar, 16Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 17Reddy R.K. Lu J. Lee A.S. J. Biol. Chem. 1999; 274: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, if this adaptation does not prove sufficient, the apoptotic response is initiated, which is primarily the induction of the CCAAT/enhancer-binding protein homologous transcription factor (CHOP) (18Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1690) Google Scholar). We recently reported that various NSAIDs induce not only glucose-regulated protein 78 but also CHOP expression. In addition to this, we showed, using CHOP-deficient mice and a dominant negative form of CHOP, that this CHOP induction is essential for NSAID-induced apoptosis (19Tsutsumi S. Gotoh T. Tomisato W. Mima S. Hoshino T. Hwang H.J. Takenaka H. Tsuchiya T. Mori M. Mizushima T. Cell Death Differ. 2004; 11: 1009-1016Crossref PubMed Scopus (216) Google Scholar). Therefore, NSAIDs seem to induce apoptosis by acting as ER stressors. However, the mechanism by which NSAIDs induce the ER stress response has remained unknown. Other groups have pointed out the involvement of mitochondrial dysfunction in NSAID-induced apoptosis; some NSAIDs (such as celecoxib) stimulate the release of cytochrome c from mitochondria and decrease the mitochondrial membrane potential (20Ding H. Han C. Zhu J. Chen C.S. D'Ambrosio S.M. Int. J. Cancer. 2005; 113: 803-810Crossref PubMed Scopus (106) Google Scholar, 21Zhang Z. Lai G.H. Sirica A.E. Hepatology. 2004; 39: 1028-1037Crossref PubMed Scopus (94) Google Scholar). Various mechanisms have been proposed for NSAID-induced mitochondrial dysfunction, such as inactivation of phosphatidylinositol 3-kinase/3-phosphoinositide-dependent kinase-1/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (20Ding H. Han C. Zhu J. Chen C.S. D'Ambrosio S.M. Int. J. Cancer. 2005; 113: 803-810Crossref PubMed Scopus (106) Google Scholar, 21Zhang Z. Lai G.H. Sirica A.E. Hepatology. 2004; 39: 1028-1037Crossref PubMed Scopus (94) Google Scholar, 22Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). 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Commun. 2003; 304: 445-454Crossref PubMed Scopus (393) Google Scholar). In addition, calpain, a Ca2+-dependent cysteine protease, plays an important role in the Ca2+-induced apoptosis pathway. Calpain activates or inhibits Bax and Bid or Bcl-2 and Bcl-XL, respectively, by their cleavage, resulting in stimulation of the release of cytochrome c from mitochondria and a decrease in the mitochondrial membrane potential. Then cytochrome c activates caspase-9, which in turn activates caspase-3 and induces apoptosis. It is also suggested that calpain directly activates some caspases (26Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 552-565Crossref PubMed Scopus (2447) Google Scholar, 29Hajnoczky G. Davies E. Madesh M. Biochem. Biophys. Res. Commun. 2003; 304: 445-454Crossref PubMed Scopus (393) Google Scholar, 30Mandic A. Viktorsson K. Strandberg L. Heiden T. Hansson J. Linder S. Shoshan M.C. Mol. Cell. Biol. 2002; 22: 3003-3013Crossref PubMed Scopus (222) Google Scholar, 31Wood D.E. Thomas A. Devi L.A. Berman Y. Beavis R.C. Reed J.C. Newcomb E.W. Oncogene. 1998; 17: 1069-1078Crossref PubMed Scopus (308) Google Scholar, 32Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1044) Google Scholar). On the other hand, recent reports show that Ca2+ ionophores induce the ER stress response and CHOP expression, suggesting that increases in the intracellular Ca2+ level can induce the ER stress response and CHOP expression (26Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell. Biol. 2003; 4: 552-565Crossref PubMed Scopus (2447) Google Scholar, 33Nozaki S. Sledge Jr. G.W. Nakshatri H. Oncogene. 2001; 20: 2178-2185Crossref PubMed Scopus (101) Google Scholar). Permeabilization of cytoplasmic membranes causes an increase in intracellular Ca2+ levels by stimulating Ca2+ influx across the cytoplasmic membrane. Some NSAIDs were shown to increase intracellular Ca2+ levels (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar, 34Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar). Therefore, it is reasonable to speculate that increases in intracellular Ca2+ levels are involved in NSAID-induced apoptosis (i.e. that the increased intracellular Ca2+ level connects the membrane permeabilization and CHOP induction activities of NSAIDs). Furthermore, it is also possible that activation of calpain connects the increases in intracellular Ca2+ levels to mitochondrial dysfunction in the presence of NSAIDs. In this study, we showed, in primary cultures of guinea pig gastric mucosal cells, that all NSAIDs tested increased intracellular Ca2+ levels, which accompanied the induction of apoptosis. An intracellular Ca2+ chelator partially inhibited CHOP induction, release of cytochrome c from mitochondria, the decrease in mitochondrial membrane potential, and apoptotic cell death in the presence of celecoxib (the most potent NSAID for apoptosis induction). Furthermore, an inhibitor of calpain partially suppressed the mitochondrial dysfunction and apoptotic cell death but did not affect CHOP induction in the presence of celecoxib. These results suggest that the celecoxib-dependent increase in intracellular Ca2+ levels and the resulting calpain activation and CHOP induction are involved in celecoxib-induced apoptosis in gastric mucosal cells. Chemicals, Media, and Animals—Fetal bovine serum was from Invitrogen. RPMI 1640 and Hanks' solution were obtained from Nissui Pharmaceutical Co. Pronase E and type I collagenase were purchased from Kaken Pharmaceutical Co. and Nitta Gelatin Co., respectively. Pluronic F127, fluo-3/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM), and BAPTA were from Dojindo Co. Nimesulide and flurbiprofen were from Cayman Chemical Co. Indomethacin was from Wako Co. Hoechst 33342 (Ho 342), ibuprofen, diclofenac, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), mefenamic acid, ketoprofen, and flufenamic acid were from Sigma. 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidaozolylcarbocyanine iodide (DePsipher) was from Trevigen. Celecoxib was from LKT Laboratories Inc. Etodolac was a gift kindly provided by Nippon Shinyaku Co. Peptides for the assay of caspase-3 or calpain and carbobenzoxy-l-leucyl-l-leucinal (Z-Leu-Leu-H) were from Peptide Institute, Inc. Egg phosphatidylcholine was from Kanto Chemicals Co. Antibodies against CHOP and actin were from Santa Cruz Biotechnology Inc. Antibody against cytochrome c was from PharMingen. Male guinea pigs weighing 200-300 g were purchased from Kyudo Co. The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and were approved by the Animal Care Committee of Kumamoto University. In Vitro Assay of Cytotoxicity—Gastric mucosal cells were isolated from guinea pig fundic glands as described previously (35Tomisato W. Hoshino T. Tsutsumi S. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2125-2133Crossref PubMed Scopus (13) Google Scholar). Isolated gastric mucosal cells were cultured for 12 h in RPMI 1640 containing 0.3% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in type I collagen-coated plastic culture plates under conditions of 5% CO2, 95% air and 37 °C. Nonadherent cells were removed, and the cells attached to the plate were used in the experiments. Guinea pig gastric mucosal cells prepared under these conditions have previously been characterized, with the majority (about 90%) of cells identified as pit cells (35Tomisato W. Hoshino T. Tsutsumi S. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2125-2133Crossref PubMed Scopus (13) Google Scholar). NSAIDs were dissolved in Me2SO or Na2CO3 (for indomethacin only) and control experiments (without NSAIDs) were performed in the presence of the same concentrations of Me2SO or Na2CO3. Cells were exposed to NSAIDs by changing the medium. Cell viability was determined by the MTT method. Apoptotic chromatin condensation was monitored as described previously (36Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). Cells were washed with PBS, stained with 10 μg/ml Ho 342, and observed under a fluorescence microscope. Immunoblotting Analysis—Whole cell extracts were prepared as described previously (36Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). The protein concentration of the samples was determined by the Bradford method. The samples were electrophoresed on polyacrylamide gels containing SDS, and the proteins were then transferred to membranes and detected using antibodies. Mitochondrial Membrane Potential Assay—Mitochondrial membrane potential was assayed using a fluorometric mitochondrial permeability assay kit (Trevigen) (36Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). Briefly, after treatment with NSAIDs, cells were treated with DePsipher (5 μg/ml) for 20 min at 37 °C and observed under a fluorescence microscope using 590 nm for red emission and 530 nm for green emission. Measurement of the Intracellular Ca2+ Level—Intracellular Ca2+ levels were monitored as described (37Kao J.P. Harootunian A.T. Tsien R.Y. J. Biol. Chem. 1989; 264: 8179-8184Abstract Full Text PDF PubMed Google Scholar). The cells were washed with assay buffer (115 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2, 20 mm HEPES, and 13.8 mm glucose). For Ca2+-free conditions, the modified assay buffer (115 mm NaCl, 5.4 mm KCl, 5 mm EGTA, 20 mm HEPES, and 13.8 mm glucose) was used instead of the normal assay buffer. The cells were then incubated with 4 μm fluo-3/AM in assay buffer supplemented with 0.1% bovine serum albumin, 0.04% pluronic F127, and 2 mm probenecid, for 40 min at 37 °C. After washing twice with assay buffer, cells were suspended in assay buffer supplemented with 2 mm probenecid. Cells were transferred to a water-jacketed cuvette (1.6 × 106 cells/cuvette), and the fluo-3 fluorescence was then measured with a Hitachi F-4500 spectrofluorophotometer by recording excitation signals at 490 nm and the emission signal at 530 nm at 1-s intervals. Maximum and minimum fluorescence values (Fmax and Fmin) were obtained by adding 10 μm ionomycin and 10 μm ionomycin under Ca2+-free conditions, respectively. The intracellular Ca2+ level was calculated according to the equation, [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where Kd is the apparent dissociation constant (400 nm) of the fluorescent dye-Ca2+ complex. Membrane Permeability Assay—Permeabilization of calcein-loaded liposomes was assayed as described previously (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). Liposomes were prepared using the reversed-phase evaporation method. Egg phosphatidylcholine (10 μmol, 7.7 mg) was dissolved in chloroform/methanol (1:2, v/v), dried, dissolved in 1.5 ml of diethyl ether, and added to 1 ml of 100 mm calcein-NaOH (pH 7.4). The mixture was sonicated to obtain a homogenous emulsion. The diethyl ether solvent was removed, and the resulting suspension of liposomes was centrifuged and washed twice with fresh buffer A (10 mm potassium buffer, containing 150 mm NaCl) to remove untrapped calcein. The final liposome precipitate was resuspended in 5 ml of buffer A. A 0.3-ml aliquot of this suspension was diluted with 19.7 ml of buffer A, and 400 μl of this diluted suspension was then incubated at 30 °C for 10 min in the presence of the NSAID under investigation. The release of calcein from liposomes was determined by measuring fluorescence intensity at 520 nm (excitation at 490 nm). Assay for K+ Efflux from Cells—K+ efflux from cells was monitored as described previously (38Katsu T. Kobayashi H. Hirota T. Fujita Y. Sato K. Nagai U. Biochim. Biophys. Acta. 1987; 899: 159-170Crossref PubMed Scopus (54) Google Scholar) with some modifications. Cells were washed twice with buffer A and then suspended in fresh buffer A (6 × 106 cells/ml). After incubation with NSAIDs for 30 min at 37 °C, K+ efflux from cells was measured with a K+ ion-selective electrode. Assay for Caspase Activity—The activity of caspase-3 was determined as described previously (36Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). Briefly, cells were collected by centrifugation and suspended in extraction buffer (50 mm PIPES (pH 7.0), 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, and 1 mm dithiothreitol). Suspensions were sonicated and centrifuged, after which the supernatants were incubated with fluorogenic peptide substrates (acetyl-DEVD-methylcoumarin amide) in reaction buffer (100 mm HEPES-KOH (pH 7.5), 10% sucrose, 0.1% CHAPS, and 1 mg/ml bovine serum albumin) for 15 min at 37 °C. The release of aminomethylcoumarin (AMC) was determined using a fluorescence spectrophotometer. One unit of protease activity was defined as the amount of enzyme required to release 1 pmol of AMC/min. Assay for Calpain Activity—The activity of calpain was determined as described previously (30Mandic A. Viktorsson K. Strandberg L. Heiden T. Hansson J. Linder S. Shoshan M.C. Mol. Cell. Biol. 2002; 22: 3003-3013Crossref PubMed Scopus (222) Google Scholar). Briefly, cells were collected by centrifugation, washed by Hanks' solution, and suspended with the same solution at 2.5 × 105 cells/ml. Suspensions were incubated with fluorogenic peptide substrates succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine 4-methylcoumaryl-7-amide for 3 min at 37 °C. The release of AMC was determined using a fluorescence spectrophotometer. Statistical Analyses—All values are expressed as the means ± S.E. One-way analysis of variance, followed by Scheffe's multiple comparison, was used for evaluation of differences between the groups. Student's t test for unpaired results was performed to evaluate differences between two groups. Differences were considered to be significant for values of p < 0.05. Close Relationship between NSAID-induced Apoptosis and Membrane Permeabilization—We recently reported that some NSAIDs (celecoxib, mefenamic acid, flufenamic acid, nimesulide, and flurbiprofen) cause not only apoptosis in primary cultures of guinea pig gastric mucosal cells but also membrane permeabilization in calcein-loaded liposomes (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). To examine the relationship between the apoptosis-inducing and membrane permeabilization activities of NSAIDs, in this study, we have examined these activities of other NSAIDs. As shown in Fig. 1A, treatment of primary cultures of guinea pig gastric mucosal cells with celecoxib for 16 h decreased cell viability in a dose-dependent manner, and this is consistent with our previous results (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). Each of the NSAIDs tested here (indomethacin, diclofenac, etodolac, ibuprofen, and ketoprofen) also decreased cell viability in a dose-dependent manner. Because cell death under these conditions was accompanied by apoptotic DNA fragmentation and chromatin condensation (data not shown), it is most likely mediated by apoptosis. Two subtypes of COX, COX-1 and COX-2, are responsible for the majority of COX activity in gastric mucosal and inflammatory tissues, respectively, and recently a number of COX-2-selective NSAIDs have been developed (39Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (698) Google Scholar). Among the NSAIDs tested in Fig. 1A, etodolac and celecoxib have selectivity for COX-2. No relationship was evident between NSAID-induced apoptosis and selectivity for COX-2 (Fig. 1A). We also confirmed that exogenously added PGE2 (either native PGE2 or 16,16-dimethyl-PGE2) did not affect NSAID-induced apoptosis even at a higher concentration of PGE2 in the culture medium than is present endogenously (10-9 m) (data not shown). These data show that NSAID-induced apoptosis is independent of COX-inhibition by NSAIDs and are consistent with our previous conclusion (12Tomisato W. Tsutsumi S. Hoshino T. Hwang H.J. Mio M. Tsuchiya T. Mizushima T. Biochem. Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (103) Google Scholar, 13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). Calcein fluoresces very weakly at high concentrations due to self-quenching, so the addition of membrane-permeabilizing drugs to a medium containing calcein-loaded liposomes should cause an increase in fluorescence by diluting out the calcein (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar). As shown in Fig. 2A, not only celecoxib but also other NSAIDs increased the calcein fluorescence, suggesting that they have membrane permeabilization activity. Combining the results from the previous (13Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (86) Google Scholar) and present studies, we obtained dose-response curves of 10 different NSAIDs for both induction of apoptosis and membrane permeabilization. To examine the relationship between NSAID-induced apoptosis and membrane permeabilization, we determined ED50 values of the 10 NSAIDs for apoptosis (concentrations of NSAIDs required for 50% inhibition of cell viability by apoptosis) and ED20 values for membrane permeabilizati
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