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• W2038554369 abstract "Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IκB kinase β (IKKβ) that is required to activate the nuclear factor-κB (NF-κB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-κB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKβ kinase activity. Concentrations of sulindac that inhibit IKKβ activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-κB pathway. Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IκB kinase β (IKKβ) that is required to activate the nuclear factor-κB (NF-κB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-κB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKβ kinase activity. Concentrations of sulindac that inhibit IKKβ activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-κB pathway. nuclear factor-κB IκB kinase inhibitor of κB NF-κB inducing kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 glutathione S-transferase tumor necrosis factor α phosphate-buffered saline stress-activated protein kinase extracellular response kinase 2 human immunodeficiency virus amino acids mitogen-activated protein. The NF-κB1 pathway regulates the cellular response to a variety of stimuli including cytokines, bacterial and viral infection, and activation of cellular stress pathways (reviewed in Refs. 1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2910) Google Scholar and 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). The NF-κB pathway is also critical for the control of cellular growth properties (3Lu X. Xie W. Reed D. Bradshaw W.S. Simmons D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7961-7965Crossref PubMed Scopus (195) Google Scholar, 4Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2552) Google Scholar, 5Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1620) Google Scholar, 6Chu Z.-L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (819) Google Scholar, 7Wu M. Lee H. Bellas R.E. Schauer S.L. Arsura M. Katz D. FitzGerald M.J. Rothstein T.L. Sherr D.H. Sonenshein G.E. EMBO J. 1996; 15: 4682-4690Crossref PubMed Scopus (553) Google Scholar). For example, disruption of the gene encoding the p65 member of the NF-κB family leads to severe hepatic apoptosis indicating that at least in some cell types that NF-κB is a critical anti-apoptotic factor (5Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1620) Google Scholar). Inhibition of apoptosis is likely mediated by NF-κB induction of cellular genes such as cellular inhibitor of apoptosis-1 and -2 which inhibit the apoptotic process (4Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2552) Google Scholar, 6Chu Z.-L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (819) Google Scholar). The fact that high constitutive levels of NF-κB are found in the nucleus of some tumors further implicates activation of this pathway as a potential mechanism to stimulate cellular growth (8Sovak M.A. Bellas R.E. Kim D.W. Zanieski G.J. Rogers A.E. Traish A.M. Sonenshein G.E. J. Clin. Invest. 1997; 100: 2952-2960Crossref PubMed Scopus (641) Google Scholar, 9Nakshatri H. Bhat-Nakshatri P. Martin D.A. Goulet Jr., R.J. Sledge Jr., G.W. Mol. Cell. Biol. 1997; 17: 3629-3639Crossref PubMed Google Scholar). NF-κB is comprised of a family of related proteins that can either heterodimerize or homodimerize to facilitate their binding to a consensus DNA element to result in activation of gene expression (reviewed in Refs. 1Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2910) Google Scholar and 2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). The DNA binding and dimerization properties of NF-κB are mediated by a conserved domain in these proteins known as the Rel homology domain. NF-κB is normally sequestered in the cytoplasm of cells where it is bound by a family of inhibitory proteins known as IκB (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar, 10Beg A.A. Finco T.S. Nantermet P.V. Baldwin A.S.J. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar). These proteins that include IκBα, IκBβ, and IκBε contain multiple ankyrin repeats that are critical for their inhibitory function. The ability of the IκB to mask the nuclear localization signal of NF-κB prevents the nuclear translocation of these proteins (11Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (603) Google Scholar). A variety of stimuli including treatment of cells with TNFα, interleukin-1, phorbol esters, lipopolysaccharide, and the viral protein Tax results in activation of the NF-κB pathway (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5515) Google Scholar). These stimuli modulate signal transduction pathways that lead to the ability of upstream kinases including NIK and MEKK1 to activate the IκB kinases IKKα and IKKβ kinases (12Didonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1890) Google Scholar, 13Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1831) Google Scholar, 14Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar, 15Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1060) Google Scholar, 16Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar). Stimulation of the activity of these kinases results in their ability to phosphorylate two conserved serine residues in the amino terminus of the IκB proteins (17Alkalay I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10599-10603Crossref PubMed Scopus (387) Google Scholar, 18Brockman J.A. Scherer D.C. MsKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 19Brown K. Gerstberger S. Carlson L. Fransozo G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 20Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar, 21Didonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 22Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (927) Google Scholar, 23Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israel A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar). This resultant phosphorylation of IκB leads to its ubiquitination and its degradation by the proteasome resulting in the nuclear translocation of NF-κB (19Brown K. Gerstberger S. Carlson L. Fransozo G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1307) Google Scholar, 20Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1159) Google Scholar). Aspirin and sodium salicylate, but not several other anti-inflammatory agents including indomethacin, can inhibit activation of the NF-κB pathway (24Grilli M. Pizzi M. Memo M. Spano P. Science. 1996; 274: 1383-1385Crossref PubMed Scopus (739) Google Scholar, 25Kopp E. Ghosh S. Science. 1994; 265: 956-959Crossref PubMed Scopus (1592) Google Scholar, 26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). Inhibition of the NF-κB pathway by aspirin and salicylate is the result of their specific binding to IKKβ which inhibits its kinase activity (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). The effects of aspirin and salicylate prevent IκB degradation and the nuclear translocation of NF-κB (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). In addition to its role as an anti-inflammatory agent, aspirin can also help to prevent the development of colon cancer (27Thun M.J. Namboodiri M.M. Heath Jr., C.W. N. Engl. J. Med. 1991; 325: 1593-1596Crossref PubMed Scopus (1635) Google Scholar, 28Suh O. Mettlin C. Petrelli N.J. Cancer (Phila.). 1993; 72: 1171-1177Crossref PubMed Scopus (281) Google Scholar, 29Giovannucci E. Rimm E.B. Stampfer M.J. Colditz G.A. Ascherio A. Willett W.C. Ann. Intern. Med. 1994; 121: 241-246Crossref PubMed Scopus (730) Google Scholar, 30Giovannucci E. Egan K.M. Hunter D.J. Stampfer M.J. Colditz G.A. Willett W.C. Speizer F.E. N. Engl. J. Med. 1995; 333: 609-614Crossref PubMed Scopus (975) Google Scholar, 31Rosenberg L. Palmer J.R. Zauber A.G. Warshauer M.E. Stolley P.D. Shapiro S. J. Natl. Cancer Inst. 1991; 83: 355-358Crossref PubMed Scopus (525) Google Scholar). Sulindac is a non-steroidal anti-inflammatory agent that is structurally related to indomethacin and inhibits cyclooxygenase activity to prevent prostaglandin synthesis (32Duggan D.E. Hooke K.F. Risley E.A. Shen T.Y. Van Arman C.G. J. Pharmacol. Exp. Ther. 1977; 201: 8-13PubMed Google Scholar, 33Duggan D.E. Hooke K.F. Hwang S.S. Drug Metab. Dispos. 1980; 8: 241-246PubMed Google Scholar, 34Shen T.Y. Winter C.A. Adv. Drug Res. 1977; 12: 90-245PubMed Google Scholar, 35Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar). In the colon, sulindac is converted by bacteria to the metabolites sulindac sulfide and sulindac sulfone. Sulindac sulfide is the most active metabolite of sulindac and is concentrated in the colonic epithelium at concentrations that are at least 20-fold higher than those seen in the serum which are about 10–15 μm (33Duggan D.E. Hooke K.F. Hwang S.S. Drug Metab. Dispos. 1980; 8: 241-246PubMed Google Scholar). Sulindac sulfide, but not sulindac sulfone, blocks prostaglandin synthesis by non-selective inhibition of cyclooxygenase 1 and cyclooxygenase 2 (33Duggan D.E. Hooke K.F. Hwang S.S. Drug Metab. Dispos. 1980; 8: 241-246PubMed Google Scholar). However, alternative mechanisms of sulindac action other than inhibition of prostaglandin function have been suggested. For example, sulindac sulfone can inhibit mammary carcinogenesis (36Thompson H.J. Jiang C. Lu J. Mehta R.G. Piazza G.A. Paranka N.S. Pamukcu R. Ahnen D.J. Cancer Res. 1997; 57: 267-271PubMed Google Scholar), and sulindac sulfide inhibits the proliferation of colon cancer cell lines that do not express cyclooxygenases (37Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Riggs B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (593) Google Scholar). Sulindac, like aspirin, has anti-inflammatory properties and has also been demonstrated to induce the regression of adenomatous colon polyps to help prevent the development of colon cancer (27Thun M.J. Namboodiri M.M. Heath Jr., C.W. N. Engl. J. Med. 1991; 325: 1593-1596Crossref PubMed Scopus (1635) Google Scholar, 28Suh O. Mettlin C. Petrelli N.J. Cancer (Phila.). 1993; 72: 1171-1177Crossref PubMed Scopus (281) Google Scholar, 29Giovannucci E. Rimm E.B. Stampfer M.J. Colditz G.A. Ascherio A. Willett W.C. Ann. Intern. Med. 1994; 121: 241-246Crossref PubMed Scopus (730) Google Scholar, 30Giovannucci E. Egan K.M. Hunter D.J. Stampfer M.J. Colditz G.A. Willett W.C. Speizer F.E. N. Engl. J. Med. 1995; 333: 609-614Crossref PubMed Scopus (975) Google Scholar, 31Rosenberg L. Palmer J.R. Zauber A.G. Warshauer M.E. Stolley P.D. Shapiro S. J. Natl. Cancer Inst. 1991; 83: 355-358Crossref PubMed Scopus (525) Google Scholar, 38Labayle D. Fischer D. Vielh P. Drouhin F. Pariente A. Bories C. Duhamel O. Trousset M. Attali P. Gastroenterology. 1991; 101: 635-639Abstract Full Text PDF PubMed Google Scholar, 39Rigau J. Pique J.M. Rukbio E. Planas R. Tarrech J.M. Bordas J.M. Ann. Intern. Med. 1991; 115: 952-954Crossref PubMed Scopus (211) Google Scholar, 40Giardiello F.M. Hamilton S.R. Krush A.J. Piantadosi S. Hylind L.M. Celano P. Booker S.V. Robinson C.R. Offerhaus G.J. N. Engl. J. Med. 1993; 328: 1313-1316Crossref PubMed Scopus (1540) Google Scholar, 41Rao C.V. Rivenson A. Simi B. Zang E. Kelloff G. Steele V. Reddy B.S. Cancer Res. 1995; 55: 1464-1472PubMed Google Scholar, 42Winde G. Schmid K.W. Schlegel W. Fischer R. Osswald H. Bunte H. Dis. Colon Rectum. 1995; 38: 813-830Crossref PubMed Scopus (115) Google Scholar, 43Pasricha P.J. Bedi A. O'Connor K. Rashid A. Akhtar A.J. Zahurak M.L. Piantadosi S. Hamilton S.R. Giardiello F.M. Gastroenterology. 1995; 109: 994-998Abstract Full Text PDF PubMed Scopus (238) Google Scholar). The effects of sulindac on preventing colon cancer are likely mediated by stimulating cellular apoptotic pathways (37Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Riggs B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (593) Google Scholar, 44Shiff S.J. Qiao L. Tsai L.L. Rigas B. J. Clin. Invest. 1995; 96: 491-503Crossref PubMed Scopus (417) Google Scholar, 45Goldberg Y. Nassif H.I. Pittas A. Tsai L.L. Dynlacht B.D. Rigas B. Shiff S.J. Oncogene. 1996; 12: 893-901PubMed Google Scholar, 46Mahmoud N.N. Boolbol S.K. Dannenberg A.J. Mestre J.R. Billinski R.T. Martucci C. Newmark H.L. Chadburn A. Bertagnolli M.M. Carcinogenesis. 1998; 19: 87-91Crossref PubMed Scopus (131) Google Scholar, 47Qiao L. Shiff S.J. Rigas B. Int. J. Cancer. 1998; 76: 99-104Crossref PubMed Scopus (30) Google Scholar, 48Ruschoff J. Wallinger S. Dietmaier W. Bocker T. Brockhoff G. Hofstadter F. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11301-11306Crossref PubMed Scopus (154) Google Scholar). Since sulindac and aspirin have similar pharmacologic properties, we investigated whether sulindac-like aspirin could also inhibit kinases that regulate the NF-κB pathway to mediate in part its anti-inflammatory and pro-apoptotic properties. In this study, we demonstrate that sulindac and its metabolites, sulindac sulfide and sulindac sulfone, inhibit the activation of the NF-κB pathway by inhibiting IKKβ kinase activity. This result suggests that inhibition of components of the NF-κB pathway may at least in part be involved in the anti-inflammatory properties and the growth properties inhibitory of these agents. COS, HCT-15, and HT-29 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified 5% CO2 and 95% air incubator at 37 °C. Aspirin, sodium salicylate, and sulindac were obtained from Sigma and dissolved in 1 mTris-HCl, pH 8.0, to make 1 m stock solution. In transfection assays, aspirin and salicylate were used at a concentration of 5 mm sulindac; sulindac and sulindac sulfone were used at a concentration of 1 mm; sulindac sulfide was used at a concentration of 200 μm, and indomethacin and ibuprofen were used at a concentration of 25 μm. Indomethacin and ibuprofen were dissolved in ethanol to make a 100 mm stock solution (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). All the anti-inflammatory agents were added into cell culture medium for 16–18 h for the luciferase reporter gene expression assay or for 2 h prior to TNFα treatment and assays of IKK activity (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). The expression plasmids hemagglutinin-tagged IKKα and FLAG-tagged IKKβ, and their constitutively active mutants (SS/EE), were described previously (49Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The HIV-1 long terminal repeat luciferase reporter contains the human immunodeficiency virus long terminal repeat with two binding sites for NF-κB. Approximately 80% confluent COS, cells in 60-mm plates were transfected with the 3 μg of DNA using Fugene 6 (Roche Molecular Biochemicals). Cells were harvested 24–36 h after the transfection. Histidine and FLAG-tagged baculovirus-produced IKKα and IKKβ proteins were purified by nickel-agarose chromatography and immunoprecipitated with the M2 monoclonal antibody and then assayed in kinase assays as described (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). Transfected COS cells were suspended in lysis buffer containing 40 mm Tris, pH 8.0, 500 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 5 mm EGTA, 10 mmβ-glycerophosphate, 10 mm NaF, 1 mm sodium vanadate, and protease inhibitor tablet (Roche Molecular Biochemicals). Immunoprecipitation was performed with 300 ng of M2 FLAG antibody or 50 μl of 12CA5 supernatant to precipitate the wild-type and mutant hemagglutinin-IKKα and wild-type and mutants FLAG-IKKβ using 200 μg of transfected cell lysate. This was followed by the addition of 20 μl of protein A-agarose. After washing with the lysis buffer, the immunocomplex was then incubated with a kinase assay buffer containing 50 mm Tris, pH 8.0, 100 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 10 μm ATP, 10 mm β-glycerophosphate, 10 mm NaF, 1 mm Na3VO4, 5 μCi of [γ-32P]ATP, and 5 μg of glutathione S-transferase-IκBα (aa 1–54) at 30 °C for 30 min. The kinase reaction mixture was resolved on a SDS-polyacrylamide gel electrophoresis and detected by autoradiography (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). COS cells were treated with various concentrations of sulindac in vivo for 2 h before TNFα-treatment and assays of endogenous IKK activity. To assay endogenous IKK activity, cell lysates (200 μg of protein) were immunoprecipitated with a rabbit polyclonal antibody directed against IKKα (Santa Cruz Biotechnology) that immunoprecipitates the IKKα/IKKβ heterodimer followed by assays of kinase activity using the GST-IκB substrate (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). Apoptosis assay was measured with Cell Death Detection ELISAPLUS kit (Roche Molecular Biochemicals) using procedures suggested by the manufacturer with modifications. Briefly, HCT-15 cells were cultured in 6-well plates at a density of 2 × 106 cells per well overnight. Cells were treated with reagents including aspirin (5 mm), salicylate (5 mm), sulindac (1 mm), ibuprofen (25 μm), and indomethacin (25 μm) for the indicated periods. After incubation, the cells were washed twice with PBS and harvested from the plates. Cells were lysed 30 min at room temperature, and the lysates were centrifuged at 200 × g for 5 min. Then 20 μl of the supernatant was transferred into streptavidin-coated 96-well plates provided by the manufacturer, and 80 μl of the immunoreagents containing anti-histone conjugated with biotin and anti-DNA conjugated with peroxidase was added to each well. After incubation for 2 h at room temperature, plates were washed three times with wash buffer. Peroxidase was determined photometrically with 2,2′-azinodi[3-ethylbenzthiazoline sulfonate] as substrate using microtiter enzyme-linked immunosorbent assay plate reader at wavelength of 410 nm. The values from duplicate samples were averaged and subtracted from those of background (samples without lysates). The results were expressed as the enrichment factor using the following formula: absorbance of the sample (dying/dead cells)/absorbance of the control cells (without treatment). Aspirin and sodium salicylate, but not indomethacin, inhibit NF-κB-directed gene expression (24Grilli M. Pizzi M. Memo M. Spano P. Science. 1996; 274: 1383-1385Crossref PubMed Scopus (739) Google Scholar, 25Kopp E. Ghosh S. Science. 1994; 265: 956-959Crossref PubMed Scopus (1592) Google Scholar, 49Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Recently, we extended these studies and demonstrated that the effects of aspirin and sodium salicylate are mediated by the direct binding of these agents to IKKβ to decrease its kinase activity (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). Since aspirin and sulindac both have anti-inflammatory and anti-proliferative effects, we investigated whether sulindac could also inhibit activation of the NF-κB pathway. Such a result could help explain previous data demonstrating that sulindac sulfide can induce apoptosis in the colon cancer cell line HCT-15 which lacks cyclooxygenases (37Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Riggs B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (593) Google Scholar). First, we compared the effects of sulindac, aspirin, salicylate, indomethacin, and ibuprofen on the expression of an HIV-1 long terminal repeat reporter construct that contains two NF-κB sites. The concentration of sulindac necessary to inhibit the proliferation of colon cancer cell lines ranges from 400 to 1200 μm (44Shiff S.J. Qiao L. Tsai L.L. Rigas B. J. Clin. Invest. 1995; 96: 491-503Crossref PubMed Scopus (417) Google Scholar,45Goldberg Y. Nassif H.I. Pittas A. Tsai L.L. Dynlacht B.D. Rigas B. Shiff S.J. Oncogene. 1996; 12: 893-901PubMed Google Scholar, 47Qiao L. Shiff S.J. Rigas B. Int. J. Cancer. 1998; 76: 99-104Crossref PubMed Scopus (30) Google Scholar). At these concentrations, sulindac does not cause cell death, and its effects on cell proliferation are reversible after removing the drug (44Shiff S.J. Qiao L. Tsai L.L. Rigas B. J. Clin. Invest. 1995; 96: 491-503Crossref PubMed Scopus (417) Google Scholar, 45Goldberg Y. Nassif H.I. Pittas A. Tsai L.L. Dynlacht B.D. Rigas B. Shiff S.J. Oncogene. 1996; 12: 893-901PubMed Google Scholar, 47Qiao L. Shiff S.J. Rigas B. Int. J. Cancer. 1998; 76: 99-104Crossref PubMed Scopus (30) Google Scholar, 48Ruschoff J. Wallinger S. Dietmaier W. Bocker T. Brockhoff G. Hofstadter F. Fishel R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11301-11306Crossref PubMed Scopus (154) Google Scholar). Approximately 4–6-fold lower concentrations of the sulfide metabolite of sulindac (100–200 μm) inhibit cell proliferation (44Shiff S.J. Qiao L. Tsai L.L. Rigas B. J. Clin. Invest. 1995; 96: 491-503Crossref PubMed Scopus (417) Google Scholar, 45Goldberg Y. Nassif H.I. Pittas A. Tsai L.L. Dynlacht B.D. Rigas B. Shiff S.J. Oncogene. 1996; 12: 893-901PubMed Google Scholar, 47Qiao L. Shiff S.J. Rigas B. Int. J. Cancer. 1998; 76: 99-104Crossref PubMed Scopus (30) Google Scholar). In the analysis described below, we used a concentration of 1,000 μm for both sulindac and sulindac sulfone and a concentration of 200 μm for sulindac sulfide. These concentrations of sulindac and its metabolites resulted in reversible effects on cellular proliferation (data not shown). The concentrations of aspirin (1–5 mm), indomethacin (25 μm), and ibuprofen (25 μm) were based on the pharmacologic concentrations of these agents in the serum of patients required for their anti-inflammatory properties (26Yin M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). An NF-κB reporter was transfected into COS cells that were treated with known activators of the NF-κB pathway. These included either TNFα (50Osborn L. Kunkel S. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1347) Google Scholar), the MAP3 kinases NIK (51Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-548Crossref PubMed Scopus (1157) Google Scholar) or MEKK1 (52Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, 53Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar), or the human T-cell leukemia virus type I transactivator Tax (49Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Each of these activators stimulated NF-κB-directed gene expression from 10- to 40-fold (Fig. 1, A and B). These activators did not stimulate the expression of an HIV-1 reporter containing mutated NF-κB sites (data not shown). Treatment of the cells with either aspirin or salicylate reduced TNFα-directed NF-κB gene expression approximately 4-fold (Fig.1 A). Treatment of the cells with either sulindac or sulindac sulfone also reduced TNFα induction of NF-κB gene expression about 4-fold (Fig. 1 A). Sulindac sulfide resulted in approximately a 10-fold decrease in NF-κB-directed gene expression (Fig.1 A). Similar levels of inhibition were seen with these agents when NF-κB gene expression was activated by NIK expression in cells treated with either aspirin, sodium salicylate, sulindac, or the sulfide and sulfone metabolites of sulindac (Fig. 1 A). In contrast, two other non-steroidal anti-inflammatory agents, indomethacin and ibuprofen, did not reduce TNFα stimulation of NF-κB-directed gene expression (Fig. 1 A). MEKK1 and Tax activation of the NF-κB pathway was also inhibited 4–6-fold by treatment of cells with either aspirin, sodium salicylate, sulindac, or sulindac sulfone (Fig. 1 B). Sulindac sulfide consistently resulted in a 2–3-fold greater level of inhibition of the NF-κB pathway than seen with aspirin, salicylate, or sulindac sulfone (Fig. 1, A and B). Neither indomethacin nor ibuprofen reduced Tax or MEKK1 induction of NF-κB-directed gene expression (Fig. 1 B). These results suggest that sulindac and its sulfone and sulfide metabolites, like aspirin and sodium salicylate, inhibit activation of the NF-κB pathway in response to a variety of well characterized inducers of this pathway. Next it was important to address the mechanism by which sulindac inhibited the NF-κB pathway. Nuclear extract was prepared from either untreated COS cells, COS cells treated with TNFα, or COS cells transfected with NIK. Gel retardation analysis was performed with nuclear extract prepared from these cells using oligonucleotides corresponding to either NF-κB- or SP-1-binding sites. TNFα treatment of cells induced binding to the wild-type NF-κB oligonucleotide (Fig.2 A, lanes 1 and 2) but not to a mutant NF-κB oligonucleotide (data not shown). Stimulation of NF-κB binding in response to TNFα was inhibited by incubation of the cells with either aspirin, salicylate, or sulindac (Fig. 2 A, lanes 3–5) but not ibuprofen or indomethacin (Fig. 2 A, lanes 6 and 7). Similar inhibition of NF-κB binding by treatment of cells with either aspirin, salicylate, or sulindac was seen when the NF-κB pathway was activated by NIK transfection (Fig. 2 A, lanes 9–14). None of these agents altered SP1 binding in the gel retardation assay (Fig.2 A, lower panel). These results indicated that sulindac, like aspirin and salicylate, inhibited NF-κB nuclear translocation. Assays were performed to investigate whether sulindac altered the degradation of IκBα when COS cells were treated with either TNFα or following transfection of COS cells with a NIK expression vector. TNFα treatment markedly reduced IκBα levels at 30 min post-treatment when compared with the IκBα levels seen in untreated COS cells (Fig. 2 B, lanes 1 and 2). Treatment of the COS cells with either aspirin, salicylate, or sulindac prevented TNFα-induced IκBα degradation (Fig. 2 B, lanes 3–5). In contrast, treatment of cells with either indometh" @default.
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• W2038554369 title "Sulindac Inhibits Activation of the NF-κB Pathway" @default.
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