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• W1995881800 abstract "Epidemiological studies have revealed that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer. Various mechanisms, including induction of apoptosis and inhibition of the growth and invasion of cancer cells, have been implicated in this anti-tumorigenic activity. In this study we focused on S100P, which is known to be overexpressed in clinically isolated tumors and which functions through both intracellular and extracellular mechanisms. We showed the up-regulation of S100P expression in human gastric carcinoma cells treated with various NSAIDs, including celecoxib. The celecoxib-mediated up-regulation of S100P was suppressed by the transfection of cells with small interfering RNA for activating transcription factor 4 (ATF4), a transcription factor involved in the endoplasmic reticulum stress response. Furthermore, deletion of ATF4 binding consensus sequence located in the promoter of the S100P gene resulted in inhibition of celecoxibmediated transcriptional activation of the gene. These results suggest that celecoxib up-regulates the expression of S100P through an ATF4-mediated endoplasmic reticulum stress response. Celecoxib inhibited the growth and induced apoptosis, and these actions could be either suppressed or stimulated by transfection of cells with S100P overexpression plasmid or small interfering RNA, respectively. Celecoxib also inhibited the invasive activity of the cells. Cromolyn, which inhibits the binding of S100P to its receptor, enhanced the celecoxib-mediated inhibition of cell invasion and growth but did not affect apoptosis. These results suggest that S100P affects apoptosis, cell growth, and invasion through either an intracellular or an extracellular mechanism and that the up-regulation of S100P expression by NSAIDs reduces their anti-tumorigenic activity. Epidemiological studies have revealed that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer. Various mechanisms, including induction of apoptosis and inhibition of the growth and invasion of cancer cells, have been implicated in this anti-tumorigenic activity. In this study we focused on S100P, which is known to be overexpressed in clinically isolated tumors and which functions through both intracellular and extracellular mechanisms. We showed the up-regulation of S100P expression in human gastric carcinoma cells treated with various NSAIDs, including celecoxib. The celecoxib-mediated up-regulation of S100P was suppressed by the transfection of cells with small interfering RNA for activating transcription factor 4 (ATF4), a transcription factor involved in the endoplasmic reticulum stress response. Furthermore, deletion of ATF4 binding consensus sequence located in the promoter of the S100P gene resulted in inhibition of celecoxibmediated transcriptional activation of the gene. These results suggest that celecoxib up-regulates the expression of S100P through an ATF4-mediated endoplasmic reticulum stress response. Celecoxib inhibited the growth and induced apoptosis, and these actions could be either suppressed or stimulated by transfection of cells with S100P overexpression plasmid or small interfering RNA, respectively. Celecoxib also inhibited the invasive activity of the cells. Cromolyn, which inhibits the binding of S100P to its receptor, enhanced the celecoxib-mediated inhibition of cell invasion and growth but did not affect apoptosis. These results suggest that S100P affects apoptosis, cell growth, and invasion through either an intracellular or an extracellular mechanism and that the up-regulation of S100P expression by NSAIDs reduces their anti-tumorigenic activity. Non-steroidal anti-inflammatory drugs (NSAIDs) 2The abbreviations used are: NSAID, non-steroidal anti-inflammatory drug; AARE, amino acid responsible element; ATF, activating transcription factor; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid; CHOP, C/EBP homologous transcription factor; COX, cyclooxygenase; ER, endoplasmic reticulum; ERK, extracellular-regulated kinase; GRP, glucose-regulated protein; MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; PG, prostaglandin; RAGE, receptor for activated glycation end products; siRNA, small interfering RNA; TPCK, N-p-tosyl-l-phenylalanine chloromethyl ketone; RT, reverse transcription. 2The abbreviations used are: NSAID, non-steroidal anti-inflammatory drug; AARE, amino acid responsible element; ATF, activating transcription factor; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid; CHOP, C/EBP homologous transcription factor; COX, cyclooxygenase; ER, endoplasmic reticulum; ERK, extracellular-regulated kinase; GRP, glucose-regulated protein; MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; PG, prostaglandin; RAGE, receptor for activated glycation end products; siRNA, small interfering RNA; TPCK, N-p-tosyl-l-phenylalanine chloromethyl ketone; RT, reverse transcription. comprise a useful family of therapeutics. In addition to their anti-inflammatory effects, recent epidemiological studies have revealed that prolonged NSAID use reduces the risk of cancer, whereas preclinical and clinical studies have indicated that some NSAIDs, in particular celecoxib, are effective in the treatment and prevention of cancer (1Wang W.H. Huang J.Q. Zheng G.F. Lam S.K. Karlberg J. Wong B.C. J. Natl. Cancer Inst. 2003; 95: 1784-1791Crossref PubMed Scopus (241) Google Scholar). The anti-tumorigenic activity of NSAIDs is believed to involve various mechanisms, including induction of apoptosis, cell growth suppression, inhibition of angiogenesis, and inhibition of metastasis (cell invasion suppression) (2Gupta R.A. Dubois R.N. Nat. Rev. Cancer. 2001; 1: 11-21Crossref PubMed Scopus (952) Google Scholar, 3Kismet K. Akay M.T. Abbasoglu O. Ercan A. Cancer Detect. Prev. 2004; 28: 127-142Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). NSAIDs act as inhibitors of cyclooxygenase (COX), an enzyme essential for the synthesis of prostaglandins (PGs). PGs, such as PGE2, inhibit apoptosis of cancer cells and stimulate their growth and invasion as well as promote angiogenesis (4Hoshino T. Tsutsumi S. Tomisato W. Hwang H.J. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 12752-12758Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 5Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2211) Google Scholar, 6Rolland P.H. Martin P.M. Jacquemier J. Rolland A.M. Toga M. J. Natl. Cancer Inst. 1980; 64: 1061-1070PubMed Google Scholar). Thus, it is certain that the anti-tumorigenic effect of NSAIDs was mediated mainly through the inhibition of COX. However, several lines of evidence now suggest that a COX-independent mechanism may also be involved (7Piazza G.A. Alberts D.S. Hixson L.J. Paranka N.S. Li H. Finn T. Bogert C. Guillen J.M. Brendel K. Gross P.H. Sperl G. Ritchie J. Burt R.W. Ellsworth L. Ahnen D.J. Pamukcu R. Cancer Res. 1997; 57: 2909-2915PubMed Google Scholar, 8Reddy B.S. Kawamori T. Lubet R.A. Steele V.E. Kelloff G.J. Rao C.V. Cancer Res. 1999; 59: 3387-3391PubMed Google Scholar). To investigate this COX-independent mechanism, we used DNA microarray techniques to systematically search for genes in human gastric carcinoma (AGS) cells whose expression was altered by the NSAID indomethacin in a COX-independent manner (9Mima S. Takehara M. Takada H. Nishimira T. Hoshino T. Mizushima T. Carcinogenesis. 2008; 10: 1994-2000Crossref Scopus (24) Google Scholar, 10Mima S. Tsutsumi S. Ushijima H. Takeda M. Fukuda I. Yokomizo K. Suzuki K. Sano K. Nakanishi T. Tomisato W. Tsuchiya T. Mizushima T. Cancer Res. 2005; 65: 1868-1876Crossref PubMed Scopus (64) Google Scholar). This analysis revealed that NSAIDs induce an endoplasmic reticulum (ER) stress response (11Tsutsumi 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 (214) Google Scholar). ER stress response is induced through transcription factors, such as activating transcription factor 6 (ATF6) 6 and ATF4 (12Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1088) Google Scholar, 13Ron D. J. Clin. Investig. 2002; 110: 1383-1388Crossref PubMed Scopus (736) Google Scholar, 14Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (790) Google Scholar), and we have previously reported that both ATF4 and ATF6 are activated by various NSAIDs, including indomethacin and celecoxib (15Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar, 16Namba T. Hoshino T. Tanaka K. Tsutsumi S. Ishihara T. Mima S. Suzuki K. Ogawa S. Mizushima T. Mol. Pharmacol. 2007; 71: 860-870Crossref PubMed Scopus (53) Google Scholar). In this study we focused our attention on S100P, the expression of which appears to be induced by indomethacin based on results from DNA microarray analysis (10Mima S. Tsutsumi S. Ushijima H. Takeda M. Fukuda I. Yokomizo K. Suzuki K. Sano K. Nakanishi T. Tomisato W. Tsuchiya T. Mizushima T. Cancer Res. 2005; 65: 1868-1876Crossref PubMed Scopus (64) Google Scholar). S100P is a member of the S100 family of EF-hand Ca2+-binding proteins (17Becker T. Gerke V. Kube E. Weber K. Eur. J. Biochem. 1992; 207: 541-547Crossref PubMed Scopus (154) Google Scholar). Overexpression of S100P has been observed in tumors clinically isolated from various tissue types, with the extent of the overexpression being positively correlated to the degree of malignancy (18Shyu R.Y. Huang S.L. Jiang S.Y. J. Biomed. Sci. 2003; 10: 313-319Crossref PubMed Google Scholar, 19Birkenkamp-Demtroder K. Olesen S.H. Sorensen F.B. Laurberg S. Laiho P. Aaltonen L.A. Orntoft T.F. Gut. 2005; 54: 374-384Crossref PubMed Scopus (195) Google Scholar, 20Arumugam T. Simeone D.M. Van Golen K. Logsdon C.D. Clin. Cancer Res. 2005; 11: 5356-5364Crossref PubMed Scopus (189) Google Scholar, 21Logsdon C.D. Simeone D.M. Binkley C. Arumugam T. Greenson J.K. Giordano T.J. Misek D.E. Kuick R. Hanash S. Cancer Res. 2003; 63: 2649-2657PubMed Google Scholar, 22Wang G. Platt-Higgins A. Carroll J. de Silva Rudland S. Winstanley J. Barraclough R. Rudland P.S. Cancer Res. 2006; 66: 1199-1207Crossref PubMed Scopus (133) Google Scholar, 23Averboukh L. Liang P. Kantoff P.W. Pardee A.B. Prostate. 1996; 29: 350-355Crossref PubMed Google Scholar). Overproduction of S100P appears to stimulate tumor malignancy through both intracellular and extracellular mechanisms (24Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1325) Google Scholar). Secreted S100P binds to its receptor, the receptor for activated glycation end products (RAGE), thereby stimulating the invasion and growth of cancer cells or inhibiting their apoptosis through activation of extracellular-regulated kinase (ERK) and nuclear factor-κB (NF-κB) (20Arumugam T. Simeone D.M. Van Golen K. Logsdon C.D. Clin. Cancer Res. 2005; 11: 5356-5364Crossref PubMed Scopus (189) Google Scholar, 25Arumugam T. Simeone D.M. Schmidt A.M. Logsdon C.D. J. Biol. Chem. 2004; 279: 5059-5065Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Fuentes M.K. Nigavekar S.S. Arumugam T. Logsdon C.D. Schmidt A.M. Park J.C. Huang E.H. Dis. Colon Rectum. 2007; 50: 1230-1240Crossref PubMed Scopus (135) Google Scholar, 27Arumugam T. Ramachandran V. Logsdon C.D. J. Natl. Cancer Inst. 2006; 98: 1806-1818Crossref PubMed Scopus (129) Google Scholar). Furthermore, S100P was suggested to function also in cells through its binding to ezrin and Casy/SIP (28Koltzscher M. Neumann C. Konig S. Gerke V. Mol. Biol. Cell. 2003; 14: 2372-2384Crossref PubMed Scopus (88) Google Scholar, 29Filipek A. Jastrzebska B. Nowotny M. Kuznicki J. J. Biol. Chem. 2002; 277: 28848-28852Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 30Hunter K.W. Trends Mol. Med. 2004; 10: 201-204Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). It has recently been reported that S100P induces the expression of cathepsin D; however, the mechanism responsible for this effect is still to be identified (31Whiteman H.J. Weeks M.E. Dowen S.E. Barry S. Timms J.F. Lemoine N.R. Crnogorac-Jurcevic T. Cancer Res. 2007; 67: 8633-8642Crossref PubMed Scopus (76) Google Scholar). It was recently reported that the expression of S100P is altered by some anti-tumor drugs (18Shyu R.Y. Huang S.L. Jiang S.Y. J. Biomed. Sci. 2003; 10: 313-319Crossref PubMed Google Scholar, 32Jiang F. Shults K. Flye L. Hashimoto Y. Van Der Meer R. Xie J. Kravtsov V. Price J. Head D.R. Briggs R.C. Leuk. Res. 2005; 29: 1181-1190Crossref PubMed Scopus (10) Google Scholar), although the underlying regulatory mechanism remains unknown. In this study we report that various NSAIDs up-regulate the expression of S100P through an ATF4-mediated ER stress response. Furthermore, our results suggest that up-regulation of S100P expression by NSAIDs negatively affects their anti-tumorigenic activity through inhibition of apoptosis, stimulation of cancer cell growth and invasion. Materials and Animals—RPMI 1640 medium was obtained from Nissui Pharmaceutical Co. Fetal bovine serum was purchased from Invitrogen. 1,2-Bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid (BAPTA-AM) was obtained from Dojindo Co. Cromolyn sodium salt, tunicamycin, normal mouse IgG, 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene ethanolate (U0126), N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), and staurosporine were purchased from Sigma-Aldrich. Indomethacin, diclofenac, and thapsigargin were obtained from Wako Co. Celecoxib and meloxicam were from LKT Laboratories Inc. An antibody against actin, IκB, or RAGE was purchased from Santa Cruz Biotechnology Inc., antibody against ERK was from Cell Signaling, and antibody against S100P was from R&D Systems Inc. The RNeasy kit, siRNAs, HiPerFect, and RNAiFect were from Qiagen. A first-strand cDNA synthesis kit was purchased from Amersham Biosciences. Lipofectamine (TM2000), zymogram developing buffer, and pcDNA3.1 plasmid were obtained from Invitrogen. The iQ SYBR Green Supermix was from Bio-Rad. S100P enzyme-linked immunosorbent assay kits were purchased from CircuLex. Matrigel was from BD Biosciences, and the 24-well transwells were from Costar. The Dual Luciferase Assay System was from Promega. Male nonobese diabetes/severe combined immunodeficiency mice (5 weeks of age) were obtained from the Charles River. 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 Institute of Health and were approved by the Animal Care Committee of Kumamoto University. Cell Culture and Overexpression of S100P—AGS, MKN45, and Kato III are human carcinoma cell lines derived from stomach. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 95% air with 5% CO2 at 37 °C. NSAIDs were dissolved in DMSO, and control experiments were performed in the same concentrations of DMSO alone. Cells were exposed to NSAIDs and other chemicals by changing the medium. Unless otherwise noted, cells were cultured for 24 h before use in experiments. The overexpression plasmid for S100P was constructed by insertion of S100P-encoding DNA fragments from the plasmid (S100P wild type (His tag), a gift from Dr. Gerke, University of Muenster (28Koltzscher M. Neumann C. Konig S. Gerke V. Mol. Biol. Cell. 2003; 14: 2372-2384Crossref PubMed Scopus (88) Google Scholar)) into pcDNA3.1. Transfection of AGS cells with the plasmid was then carried out using Lipofectamine (TM2000) according to the manufacturer's protocols. Stable transfectants expressing S100P were selected by immunoblotting analysis. Positive clones were maintained in the presence of 800 μg/ml G418. Real-time RT-PCR Analysis—Total RNA was extracted from cells using an RNeasy kit according to the manufacturer's instructions. Samples were reverse-transcribed using a first-strand cDNA synthesis kit. Synthesized cDNA was applied in real-time RT-PCR (Chromo 4 instrument (Bio-Rad)) experiments using iQ SYBR GREEN Supermix and analyzed with Opticon Monitor Software. The cycle conditions were 2 min at 50 °C followed by 10 min at 90 °C and finally 45 cycles each at 95 °C for 30 s and 63 °C for 60 s. Specificity was confirmed by electrophoretic analysis of the reaction products and by inclusion of template- or reverse transcriptase-free controls. To normalize the amount of total RNA present in each reaction, the actin gene was used as an internal standard. Primers were designed using the Primer3 Web site. Primers are listed as name, forward primer, and reverse primer: S100P, 5′-GATGCCGTGGATAAATTGCT-3′, 5′-ACTTGTGACAGGCAGACGTG-3′; cathepsin D, 5′-GACACAGGCACTTCCCTCAT-3′, 5′-CCTCCCAGCTTCAGTGTGAT-3′; actin, 5′-GGACTTCGAGCAAGAGATGG-3′, 5′-AGCACTGTGTTGGCGTACAG-3′. Luciferase Assay—DNA fragments of the S100P promoter (from –1200 to –1) were amplified by PCR and ligated into the XhoI-HindIII site of the Photinus pyralis luciferase reporter plasmid, pGL3, to generate pS100Pluc. A plasmid with deletion of the amino acid-responsible element (AARE) sequence (33Averous J. Bruhat A. Jousse C. Carraro V. Thiel G. Fafournoux P. J. Biol. Chem. 2004; 279: 5288-5297Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 34Cherasse Y. Maurin A.C. Chaveroux C. Jousse C. Carraro V. Parry L. Deval C. Chambon C. Fafournoux P. Bruhat A. Nucleic Acids Res. 2007; 35: 5954-5965Crossref PubMed Scopus (65) Google Scholar) (from –84 to –76) was generated by PCR. All plasmids were sequenced to exclude unexpected mutations. The luciferase assay was performed as described previously (11Tsutsumi 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 (214) Google Scholar, 35Aburaya M. Tanaka K. Hoshino T. Tsutsumi S. Suzuki K. Makise M. Akagi R. Mizushima T. J. Biol. Chem. 2006; 281: 33422-33432Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Cells were transfected with 1 μg of each of the P. pyralis luciferase reporter plasmids (pS100Pluc or its derivative) and 0.125 μg of the internal standard plasmid bearing the Renilla reniformis luciferase reporter (pRL-SV40). P. pyralis luciferase activity in cell extracts was measured using the Dual Luciferase Assay System and then normalized for R. reniformis luciferase activity. Gelatin Zymography—The proteolytic activity of matrix metalloproteinase (MMP)-9 was assessed by SDS-PAGE using zymogram gels containing 0.1% (m/v) gelatin, as described previously (36Taraboletti G. Sonzogni L. Vergani V. Hosseini G. Ceruti R. Ghilardi C. Bastone A. Toschi E. Borsotti P. Scanziani E. Giavazzi R. Pepper M.S. Stetler-Stevenson W.G. Bani M.R. Exp. Cell Res. 2000; 258: 384-394Crossref PubMed Scopus (42) Google Scholar). The culture medium was concentrated, and the protein concentration was determined according to the Bradford method (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar). Following electrophoresis at 4 °C, the gels were washed with 2.5% Triton X-100 for 30 min at room temperature and incubated with zymogram development buffer for 2 days at 37 °C. Bands were visualized by staining with Coomassie Brilliant Blue. Immunoblotting Analysis—Whole-cell extracts were prepared as described previously (38Tsutsumi 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 (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar). Samples were applied to polyacrylamide SDS gels and subjected to electrophoresis, and the resultant proteins were then immunoblotted with their respective antibodies. Analysis of Apoptosis by Fluorescence-activated Cell Sorting—Apoptosis was monitored by fluorescence-activated cell sorting analysis as described previously (39Alves da Costa C. Paitel E. Mattson M.P. Amson R. Telerman A. Ancolio K. Checler F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4043-4048Crossref PubMed Scopus (114) Google Scholar). Briefly, cells were collected by centrifugation. The pellets were fixed with 70% ethanol for 4 h at –20 °C, centrifuged, and re-suspended in phosphate-citrate buffer (0.2 m Na2HPO4 and 4 mm citric acid), then incubated for 20 min at room temperature. After centrifugation, the pellets were re-suspended in DNA staining solution (50 μg/ml propidium iodide and 10 μg/ml RNase A) and incubated for 20 min at room temperature. Samples were scanned with a FACSCalibur (BD Biosciences) cell sorter under conditions designed to measure only specific propidium iodide-mediated fluorescence. Apoptotic cells appeared as a hypodiploid peak due to nuclear fragmentation and loss of DNA. Invasion Assay—The invasion assay was performed as previously described (40Munoz-Najar U.M. Neurath K.M. Vumbaca F. Claffey K.P. Oncogene. 2006; 25: 2379-2392Crossref PubMed Scopus (170) Google Scholar) with some modifications. Serum-free RPMI 1640 medium containing 5 mg/ml Matrigel was applied to the upper chamber of a 24-well transwell and incubated at 37 °C for 4 h. The cell suspension was applied to the Matrigel, and the lower chamber of the transwell was filled with culture medium containing 10% fetal bovine serum and 5 μg/ml fibronectin. The plate was incubated at 37 °C for 24 h. Cells were removed from the upper surface of the membrane, and the lower surface of the membrane was stained for 10 min with 0.5% crystal violet in 25% methanol, rinsed with distilled water, and air-dried overnight. The crystal violet was then extracted with 0.1 m sodium citrate in 50% ethanol, and the absorbance was measured at 585 nm. siRNA Targeting of Genes—We used siRNA of 5′-GCCUAGGUCUCUUAGAUGAdTdT-3′ and 5′-UCAUCUAAGAGACCUAGGCdTdT-3′ or 5′-GCAACCAAUUAUCAGUUUAdTdT-3′ and 5′-UAAACUGAUAAUUGGUUGCdTdT-3′ as annealed oligonucleotides for repressing ATF4 or ATF6 expression, respectively. The siRNA for S100P was purchased from Qiagen. AGS cells were transfected with siRNA using RNAiFect or HiPerFect transfection reagents according to the manufacturer's instructions. Non-silencing siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′ and 5′-ACGUGACACGUUCGGAGAAdTdT-3′) was used as a negative control. Xenograft Tumor Growth—This assay was done as described (15Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar, 16Namba T. Hoshino T. Tanaka K. Tsutsumi S. Ishihara T. Mima S. Suzuki K. Ogawa S. Mizushima T. Mol. Pharmacol. 2007; 71: 860-870Crossref PubMed Scopus (53) Google Scholar) with some modification. Briefly, each nonobese diabetes/severe combined immunodeficiency mouse was inoculated subcutaneously in the both flanks with 1 × 107 AGS cells. After 3 weeks, the mice began to receive a single daily intraperitoneal administration of cromolym, a protocol that continued for the duration of the study. Tumors were measured every 7 days, and their volumes were calculated. Statistical Analysis—All values are expressed as the mean ± S.D. Two-way analysis of variance followed by the Tukey test or the Student's t test for unpaired results was used to evaluate differences between more than three groups or between two groups. Differences were considered to be significant for values of p < 0.05. NSAIDs Up-regulate S100P Expression—In a previous study we used DNA microarray analysis to search for genes whose expression is altered by indomethacin and found that S100P mRNA expression was up-regulated (10Mima S. Tsutsumi S. Ushijima H. Takeda M. Fukuda I. Yokomizo K. Suzuki K. Sano K. Nakanishi T. Tomisato W. Tsuchiya T. Mizushima T. Cancer Res. 2005; 65: 1868-1876Crossref PubMed Scopus (64) Google Scholar). In the present study we confirmed this result using a real-time RT-PCR technique. As shown in Fig. 1A, indomethacin up-regulated S100P mRNA expression in a dose-dependent manner. A similar result was obtained with the NSAID celecoxib, a finding that is particularly significant given its importance as an anti-cancer drug (Fig. 1B). Immunoblotting experiments revealed that celecoxib also up-regulates the expression of S100P at the protein level in both AGS cells and in another gastric cancer-derived cell line, MKN45 cells (Fig. 2, A and E). A similar response was observed in AGS cells with a number of other NSAIDs (indomethacin, meloxicam, and diclofenac; Fig. 2, B–D).FIGURE 2Up-regulation of S100P expression by NSAIDs. AGS (A–D and G), MKN-45 (E), or Kato III (F) cells were incubated with the indicated concentrations of NSAIDs for 12 h (A and E–G) or 24 h (B–D). Whole cell extracts were analyzed by immunoblotting with an antibody against S100P or actin (A–F). The level of S100P in the culture medium was determined by enzyme-linked immunosorbent assay. Values are given as the mean ± S.D. (n = 3). **, p < 0.01 (G).View Large Image Figure ViewerDownload Hi-res image Download (PPT) COX exists as two subtypes, COX-1 and COX-2. Given that celecoxib and meloxicam are COX-2 selective, the results shown in Fig. 2, A–D, suggest that NSAIDs up-regulate S100P expression irrespective of COX selectivity. We next examined the celecoxib-mediated up-regulation of S100P expression in Kato III cells, in which COX-1 but not COX-2 mRNA is expressed (41Saukkonen K. Nieminen O. van Rees B. Vilkki S. Harkonen M. Juhola M. Mecklin J.P. Sipponen P. Ristimaki A. Clin. Cancer Res. 2001; 7: 1923-1931PubMed Google Scholar). This phenotype was confirmed by RT-PCR (data not shown). As shown in Fig. 2F, celecoxib up-regulated the expression of S100P even in the Kato III cells; in other words, a COX-2-selective NSAID up-regulated S100P expression in cells lacking COX-2 expression, suggesting that this occurs independently of COX inhibition. For further confirmation of this point, we examined the effect of PGE2, revealing that it had no effect on the expression of S100P in the presence or absence of celecoxib (data not shown). As described above, secreted S100P regulates various cell functions through its binding to RAGE (20Arumugam T. Simeone D.M. Van Golen K. Logsdon C.D. Clin. Cancer Res. 2005; 11: 5356-5364Crossref PubMed Scopus (189) Google Scholar, 25Arumugam T. Simeone D.M. Schmidt A.M. Logsdon C.D. J. Biol. Chem. 2004; 279: 5059-5065Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Fuentes M.K. Nigavekar S.S. Arumugam T. Logsdon C.D. Schmidt A.M. Park J.C. Huang E.H. Dis. Colon Rectum. 2007; 50: 1230-1240Crossref PubMed Scopus (135) Google Scholar, 27Arumugam T. Ramachandran V. Logsdon C.D. J. Natl. Cancer Inst. 2006; 98: 1806-1818Crossref PubMed Scopus (129) Google Scholar, 28Koltzscher M. Neumann C. Konig S. Gerke V. Mol. Biol. Cell. 2003; 14: 2372-2384Crossref PubMed Scopus (88) Google Scholar). We monitored the level of S100P in the culture medium by enzyme-linked immunosorbent assay and found that it increased in a dose-dependent manner in response to celecoxib treatment (Fig. 2G). Mechanism for Up-regulation of S100P Expression by Celecoxib—As outlined above, the mechanism underlying the regulation of S100P expression is unknown. We have recently revealed that various NSAIDs including celecoxib induce an ER stress response, and the concentration of each NSAID required to mediate this response (15Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar) is similar to that which induces S100P expression (Fig. 2). We investigated the role of ER stress response in NSAID-induced S100P expression by examining the effect of other ER stress-inducing chemicals (thapsigargin and tunicamycin) on the expression of S100P mRNA. As shown in Fig. 3, A and B, both of these chemicals increased S100P mRNA. We also confirmed that both agents up-regulated the expression of glucose-regulated protein (GRP) 78 mRNA, a representative marker of the ER stress response at the concentration specified in Fig. 3, A and B (data not shown). In contrast, exposure to staurosporine, which does not produce such a response (15Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar, 16Namba T. Hoshino T. Tanaka K. Tsutsumi S. Ishihara T. Mima S. Suzuki K. Ogawa S. Mizushima T. Mol. Pharmacol. 2007; 71: 860-870Crossref PubMed Scopus (53) Google Scholar), had no significant effect on S100P mRNA expression (Fig. 3C), suggesting that the expression of S100P is indeed linked to the ER stress response. To confirm this we examined the effect of BAPTA-AM, an intracellular Ca2+ chelator, on celecoxib-mediated up-regulation of S100P. We have previously shown that NSAIDs increase the intracellular Ca2+ concentration and that this increase is required for the NSAID-induced ER stress response (15Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar, 42Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem." @default.
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