Keng-Mean Lin

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. 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Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). with by the with cells in at a of 2 cells with including aspirin sulindac and for the the cells with and the at and the at for of the by the and of the with and with to for 2 at with with as at of The and of The results as the factor the of the of the control cells Aspirin and sodium salicylate, but not indomethacin, inhibit 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, M.-J. Yamamoto Y. S. Mercurio F. Barbosa M. M.H. Gaynor R.B. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Recently, we these and demonstrated that the of aspirin and sodium are mediated by the binding of these agents to IKKβ to decrease its kinase activity M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). aspirin and sulindac both have anti-inflammatory and we sulindac also inhibit activation of the NF-κB pathway. a result previous that sulindac sulfide can apoptosis in the colon cancer cell which A. Y. L. L. 1996; PubMed Scopus Google Scholar). we the of aspirin, salicylate, indomethacin, and the expression of an that two NF-κB The of sulindac to inhibit the proliferation of colon cancer cell to L. J. Clin. Invest. 1995; PubMed Scopus Google Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google Scholar). these sulindac not cell and its cell proliferation are the L. J. Clin. Invest. 1995; PubMed Scopus Google Scholar, Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google Scholar, J. S. W. T. G. F. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). of the sulfide of sulindac inhibit cell proliferation L. J. Clin. Invest. 1995; PubMed Scopus Google Scholar, Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google Scholar). In the we a of for both sulindac and sulindac sulfone and a of for sulindac These of sulindac and its metabolites in cellular proliferation not The of aspirin and the of these agents in the of required for their anti-inflammatory properties M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). NF-κB cells that with known of the NF-κB pathway. These either L. S. G.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the kinases NIK D. Nature. 1997; PubMed Scopus Google or MEKK1 L. Maniatis T. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Hagler J. Maniatis T. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google or the human virus Tax M.-J. Yamamoto Y. S. Mercurio F. Barbosa M. M.H. Gaynor R.B. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of these gene expression to A and These not stimulate the expression of an NF-κB not of the cells with either aspirin or NF-κB gene expression of the cells with either sulindac or sulindac sulfone also induction of NF-κB gene expression 1 Sulindac sulfide in a decrease in gene expression Similar levels of inhibition with these agents NF-κB gene expression by NIK expression in cells with either aspirin, sodium salicylate, or the sulfide and sulfone metabolites of sulindac 1 In two other non-steroidal anti-inflammatory and not reduce of gene expression 1 MEKK1 and Tax activation of the NF-κB pathway also inhibited by treatment of cells with either aspirin, sodium salicylate, or sulindac sulfone 1 Sulindac sulfide in a of inhibition of the NF-κB pathway with aspirin, salicylate, or sulindac sulfone A and Tax or MEKK1 induction of gene expression 1 These results suggest that sulindac and its sulfone and sulfide aspirin and sodium salicylate, inhibit activation of the NF-κB pathway in response to a variety of of this pathway. it to the mechanism by which sulindac inhibited the NF-κB pathway. either cells with TNFα, or cells with with nuclear these cells to either or treatment of cells binding to the NF-κB 1 and but not to a NF-κB not Stimulation of NF-κB binding in response to inhibited by of the cells with either aspirin, salicylate, or sulindac 2 but not or 2 and Similar inhibition of NF-κB binding by treatment of cells with either aspirin, salicylate, or sulindac the NF-κB pathway by NIK 2 of these agents binding in the These results that aspirin and salicylate, inhibited NF-κB nuclear to sulindac the degradation of cells with either or of cells with a NIK expression treatment levels at with the levels in cells 2 1 and of the cells with either aspirin, salicylate, or sulindac degradation 2 In treatment of cells with either or in degradation and Similar inhibition of degradation in cells with either aspirin, salicylate, or sulindac 2 These results that decreases in either the kinase activity of or the ubiquitination or degradation of be for sulindac-mediated inhibition of the NF-κB pathway. the cells in A and also for activity. with an IKKα that results in the of the M.-J. Yamamoto Y. S. Mercurio F. Barbosa M. M.H. Gaynor R.B. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). These for kinase activity a amino acids treatment of cells activity 2 1 and and this inhibited by treatment of cells with either aspirin, salicylate, or sulindac 2 In and not inhibit activity 2 and Similar results with these agents in cells 2 A at serine residues and not by not in IKKα protein levels in these 2 These results that aspirin and salicylate, inhibit activity to degradation and NF-κB nuclear to the of sulindac that to inhibit activity. Sulindac to with cells by treatment of these cells with kinase activity with an IKKα that the and a This that the of sulindac is in the where sulindac cellular growth properties we sulindac to in activity in two colon cancer and The growth of and cells is inhibited by treatment with either sulindac or sulindac sulfide A. Y. L. L. 1996; PubMed Scopus Google Scholar, L. J. Clin. Invest. 1995; PubMed Scopus Google Scholar). cells not and sulindac sulfide inhibition of their proliferation is due to a mechanism of inhibiting A. Y. L. L. 1996; PubMed Scopus Google Scholar). Both sulindac and aspirin, but not indomethacin, in activity in and cells. in IKKα protein levels in these cells with aspirin or sulindac These results that both sulindac and aspirin can induction of activity in colon cell lines. The ability of these anti-inflammatory agents to apoptosis in cells cells not contain we the mechanism of apoptosis to be and not by treatment with or indomethacin. In agents that inhibit the NF-κB pathway including aspirin, and be to apoptosis in cells. in sulindac apoptosis in of cells of of aspirin and also apoptosis of cells of apoptosis in the cells either or not of with these These results be with potential inhibition of the NF-κB pathway to apoptosis in cells that is of in prostaglandin sulindac inhibited both IKKα and IKKβ kinase we encoding either or of these kinases cells. The cells with either aspirin, salicylate, indomethacin, or of of these kinases with for their ability to phosphorylate the of these agents the kinase activity of either the or IKKα In treatment of cells with aspirin, salicylate, or but not or in inhibition of both the and the IKKβ These results that aspirin and salicylate, inhibited IKKβ kinase activity. of these agents the of either the IKKα or IKKβ proteins A and In we the of sulindac and its metabolites sulindac sulfide and sulindac sulfone of both activity and IKKβ activity. NIK activity 1 and and this inhibited by treatment of cells with aspirin and sulindac and but not Both sulindac sulfone and sulindac sulfide inhibited kinase activity. The of these agents IKKβ kinase activity of an IKKβ protein. NIK IKKβ kinase activity 1 and and its inhibited by 1 not of sulindac and Sulindac sulfide inhibited IKKβ activity at but not and Sulindac sulfone inhibition of IKKβ that of at 1 but not and treatment not inhibit IKKβ activity of sulindac or its metabolites IKKβ protein levels These results that sulindac and its metabolites inhibit IKKβ kinase activity. the of aspirin, salicylate, and the kinase activity of encoding the kinases or E. N. M.H. Cell. Full Text PDF PubMed Scopus Google Scholar, P. E. T. J. Nature. 1994; PubMed Scopus Google Scholar, S. M.H. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). we the ability of sulindac to inhibit a variety of other cellular of these kinases and the cells with either or to stimulate the activity of these treatment the kinase activity of both and treatment the activity of aspirin, salicylate, or treatment of cells the activity of these kinases or the levels of these proteins These results that the ability of sulindac and aspirin to inhibit IKKβ in that the activity of a variety of other kinases not by these sulindac or other anti-inflammatory agents inhibited IKKβ kinase IKKα and IKKβ proteins in in kinase aspirin, salicylate, indomethacin, inhibited IKKα kinase activity A aspirin, salicylate, and but not or inhibited IKKβ kinase activity to that of these kinases in of these A and These results that IKKβ is a for inhibition of the NF-κB pathway by aspirin, and we demonstrated that the anti-inflammatory agents and aspirin inhibit the NF-κB pathway by binding of aspirin and to IKKβ resulting in their for its binding to M.-J. Yamamoto Y. Gaynor R.B. Nature. 1998; 396: 77-80Crossref PubMed Scopus (1407) Google Scholar). In the anti-inflammatory agent not gene In the study, we the anti-inflammatory agent which is structurally related to indomethacin, and its metabolites sulindac sulfide and sulfone can inhibit gene we that sulindac and its sulfide are of the NF-κB pathway. The mechanism of of these agents is due to inhibition of IKKβ but not IKKα kinase activity. These results suggest that several non-steroidal anti-inflammatory agents are of the NF-κB pathway and by inhibiting IKKβ kinase activity. Aspirin and in addition to anti-inflammatory are of cellular proliferation A. Y. L. L. 1996; PubMed Scopus Google Scholar, L. J. Clin. Invest. 1995; PubMed Scopus Google Scholar, Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google Scholar, J. S. W. T. G. F. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). 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Nature. 1994; PubMed Scopus Google other pathways may also be by these the NF-κB pathway is in both the of the response and in cellular growth control (reviewed in 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 this pathway is also a potential for inhibition by aspirin and suggest that these agents inhibit IKKβ activity to degradation and in gene the of these agents to inhibit the NF-κB pathway not result in cellular The of aspirin in with with aspirin is to that in this P. The of Scholar). Sulindac and its sulfide have in the but levels in the where the sulfide is at least J. Google Scholar, Google Scholar). The of sulindac and sulindac sulfide in this likely the levels in the the we the of sulindac and aspirin inducing apoptosis in cells are due to inhibiting the NF-κB pathway or other The fact that cells are in the of A. Y. L. L. 1996; PubMed Scopus Google apoptosis in response to treatment with either sulindac or aspirin, that pathways other the mediated by likely Both sulindac and sulindac sulfide can reduce the proliferation the of and cell and apoptosis of colon cancer cell Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google Scholar). Sulindac has also been demonstrated to in cells with colon cancer J. S. W. T. G. F. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The of sulindac to inhibit the growth of colon cancer cell are to that inhibit the NF-κB pathway. sulindac-mediated a variety of other cellular have been demonstrated Y. A. 1996; Google Scholar, L. J. 1998; PubMed Scopus Google our results are with a role for sulindac inhibition of the NF-κB pathway as a potential mechanism that may be in inducing The the pathways that are inhibited by sulindac treatment to be further to the of its growth inhibitory and for of the and and for

Pathogenic infections and tissue injuries trigger the assembly of inflammasomes, cytosolic protein complexes that activate caspase-1, leading to cleavage of pro-IL-1β and pro-IL-18 and to pyroptosis, a proinflammatory cell death program. Although microbial recognition by Toll-like receptors (TLRs) is known to induce the synthesis of the major caspase-1 substrate pro-IL-1β, the role of TLRs has been considered limited to up-regulation of the inflammasome components. During infection with a virulent microbe, TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) are likely activated simultaneously. To examine the requirements and outcomes of combined activation, we stimulated TLRs and a specific NLR, nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3), simultaneously and discovered that such activation triggers rapid caspase-1 cleavage, leading to secretion of presynthesized inflammatory molecules and pyroptosis. This acute caspase-1 activation is independent of new protein synthesis and depends on the TLR-signaling molecule IL-1 receptor-associated kinase (IRAK-1) and its kinase activity. Importantly, Listeria monocytogenes induces NLRP3-dependent rapid caspase-1 activation and pyroptosis, both of which are compromised in IRAK-1-deficient macrophages. Our results reveal that simultaneous sensing of microbial ligands and virulence factors by TLRs and NLRP3, respectively, leads to a rapid TLR- and IRAK-1-dependent assembly of the NLRP3 inflammasome complex, and that such activation is important for release of alarmins, pyroptosis, and early IFN-γ production by memory CD8 T cells, all of which could be critical for early host defense.

We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca(2+) and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.

The Alliance for Cellular Signaling (AfCS) has chosen cultured adult mouse cardiac myocytes as a system for studying cellular signaling. We developed a reproducible protocol for the isolation and culture of large numbers of viable, rod-shaped myocytes. With this protocol, we routinely isolated 1.5to 1.7 million myocytes per heart, of which 65% to 74% were rod-shaped. After 24 hours in culture, myocytes were 80% rod-shaped, and 88% of the viable, rod-shaped myocytes originally plated were retained. Cultured myocytes demonstrated predictable ligand-induced changes in accumulation of cAMP, phosphorylation of signaling proteins, and excitation-contraction coupling (both calcium transients and contraction). By these criteria, the short-term cultured adult mouse cardiac myocytes are suitable for signaling studies. For future studies requiring the expression of exogenous proteins, protein mutants, or vector-based RNA interference (RNAi), we established a long-term culture system (72 hours) that relies on medium supplemented with 2, 3-butanedione monoxime as well as insulin, transferrin, and selenium. Myocytes cultured for 72 hours in this manner remained viable and were approximately 70% rod-shaped. Finally, using adenovirus-mediated gene transduction, we demonstrated expression of an exogenous β-galactosidase reporter gene for 72 hours in cultured myocytes.