Nonsteroidal Anti-inflammatory Sulindac Research Articles

W1744 The Extracellular Calcium-Sensing Receptor Regulates the Proliferation of Normal Colonic Epithelial Cells and Reverses the Malignant Phenotype of Colon Cancer Cells

Background: EGFR family members are all thought to signal through the MAPK and PI3K/ Akt pathways. Upregulation of EGFR, HER2, HER3 and MAPK signaling is well documented in colorectal tumors. Similarly, PI3K activity is elevated in the vast majority of colorectal cancers and over-expression of Akt isoforms has also been detected in CRC. We previously reported that the sulfide and sulfone metabolites of the non-steroidal anti-inflammatory drug (NSAID) sulindac downregulate the expression of EGFR, inhibit EGFR signaling through the MAPK pathway, and induce apoptotic cell death in CRC cells. Goals: 1) To examine the effects of sulindac metabolites (SMs) on protein levels and phosphorylation status of the other EGFR family members, HER2 and HER3, and the downstream signaling effector, Akt and 2) To determine if downregulation of EGFR family members is required for the apoptotic effects of SMs. Results: SMs downregulated both total and phosphorylated forms of HER2 and HER3 in a manner similar to their effects on EGFR. This downregulation was seen by 12h after treatment and persisted through at least 48h. Unexpectedly bothmetabolites significantly increased Akt phosphorylation as early as 1h after treatment in all cell lines tested. SW620 CRC cells which lack EGFR and do not signal through HER2 remained sensitive to both the apoptotic effects and the pAkt stimulatory effects of SMs. siRNA treatment resulted in a 76% knockdown of HER3 expression in HT29 (EGFR+) cells and 84% in SW620 (EGFR-) cells. HER3 knockdown failed to induce significant levels of apoptosis in either cell line but it did not prevent the apoptotic effect or the pAkt induction by SMs. Pre-treatment of HT29 cells with LY294002 prevented Akt phosphorylation by SMs and potentiated the growth inhibitory effects of sulindac sulfide in this cell line. Conclusion: These results demonstrate that I) EGFR, HER2, and HER3 appear to be similarly downregulated by SMs; II) Downregulation of EGFR family members does not appear to be sufficient for the apoptotic effect of sulindacmetabolites; III) Sulindacmetabolites induce an unexpected stimulation of Akt phosphorylation through an EGFR family member-independent effect and IV) Prevention of NSAID-induced AKT phosphorylation might potentiate the growth inhibitory activity of these drugs.

Sulindac Metabolites Induce Proteosomal and Lysosomal Degradation of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. In response to ligand, EGFR is internalized and degraded by the ubiquitin-proteasome/lysosome pathway. We previously reported that metabolites of the nonsteroidal anti-inflammatory drug sulindac downregulate the expression of EGFR and inhibit basal and EGF-induced EGFR signaling through extracellular signal-regulated kinase 1/2. We now have evaluated the mechanisms of sulindac metabolite-induced downregulation of EGFR. EGF-induced downregulation of EGFR occurs within 10 minutes and lasts for 24 hours. By contrast, downregulation of EGFR by sulindac sulfide and sulindac sulfone was first evident at 4 and 24 hours, respectively, with maximal downregulation at 72 hours. Pretreatment with either the lysosomal inhibitor chloroquine or the proteosomal inhibitor MG132 blocked sulindac metabolite-induced downregulation of EGFR. Sulindac metabolites also increased the ubiquitination of EGFR. Whereas sulindac metabolites inhibited phosphorylation of EGFR pY1068, they increased phosphorylation of EGFR pY1045, the docking site where c-Cbl binds, thereby enabling receptor ubiquitination and degradation. Immunofluorescence analysis of EGF and EGFR distribution confirmed the biochemical observations that sulindac metabolites alter EGFR localization and EGFR internalization in a manner similar to that seen with EGF treatment. Expression of ErbB family members HER2 and HER3 was also downregulated by sulindac metabolites. We conclude that downregulation of EGFR expression by sulindac metabolites is mediated via lysosomal and proteosomal degradation that may be due to drug-induced phosphorylation at pY1045 with resultant ubiquitination of EGFR. Thus, sulindac metabolite-induced downregulation of EGFR seems to be mediated through mechanism(s) similar, at least in part, to those involved in EGF-induced downregulation of EGFR.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit vascular smooth muscle cell proliferation via differential effects on the cell cycle.

Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of both atherosclerosis and restenosis. Recent studies suggest that high-dose salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in-vitro and in-vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAIDs) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains to be shown. In this study, we demonstrate that the NSAIDs aspirin, sodium salicylate, diclofenac, ibuprofen, indometacin and sulindac induce a dose-dependent inhibition of proliferation in rat A10 VSMCs in the absence of significant cytotoxicity. Flow cytometric analyses showed that exposure of A10 cells to diclofenac, indometacin, ibuprofen and sulindac, in the presence of the mitotic inhibitor, nocodazole, led to a significant G0/G1 arrest. In contrast, the salicylates failed to induce a significant G1 arrest since flow cytometry profiles were not significantly different from control cells. Cyclin A levels were elevated, and hyperphosphorylated p107 was present at significant levels, in salicylate-treated A10 cells, consistent with a post-G1/S block, whereas cyclin A levels were low, and hypophosphorylated p107 was the dominant form, in cells treated with other NSAIDs consistent with a G1 arrest. The ubiquitously expressed cyclin-dependent kinase (CDK) inhibitors, p21 and p27, were increased in all NSAID-treated cells. Our results suggest that diclofenac, indometacin, ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase, whereas the growth inhibitory effect of salicylates probably affects the late S and/or G2/M phases. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit in the treatment of certain vasculoproliferative disorders.

Carcinogenic effect of the nsaid sulindac in the mouse proximal colon

Background Procarcinogenic effects of the non-steroidal anti-inflammatory drug (NSAID) sulindac have been described in the mouse proximal colon. The possible role of hypoxia inducible factors (HIF) in this process is poorly understood. Aim To study histological effects of sulindac in HIF1α knockout mice. Methods Mice were bred with a specific homozygous HIF1α deletion in the colonic epithelium (n = 16) with their genotype controls that express HIF1α in the colon (n = 19). They were given a diet containing 320 ppm sulindac for 20 weeks and the dietary controls (n = 17) received mouse chow without sulindac. Biopsies from proximal to distal mouse colons were examined by light microscopy. The average number of biopsies analysed per mouse was 7.9 for the test and 8.5 for the control genotype. SPSS (Version 11) and StatXact 8 software were used for statistical analysis. Results Sulindac exposure was associated with acute and chronic inflammation. Neovascularisation, apoptosis, necrosis and fibrosis were not present. Neoplastic transformation was only seen in the mice receiving the sulindac diet (p < 0.0001). There was less colon inflammation in the HIF1α knockout mice compared with the genotype controls (p = 0.004). Conclusion HIF1α may have a pro-inflammatory role in sulindac-induced carcinogenesis in the mouse proximal colon. Procarcinogenic effects of the non-steroidal anti-inflammatory drug (NSAID) sulindac have been described in the mouse proximal colon. The possible role of hypoxia inducible factors (HIF) in this process is poorly understood. To study histological effects of sulindac in HIF1α knockout mice. Mice were bred with a specific homozygous HIF1α deletion in the colonic epithelium (n = 16) with their genotype controls that express HIF1α in the colon (n = 19). They were given a diet containing 320 ppm sulindac for 20 weeks and the dietary controls (n = 17) received mouse chow without sulindac. Biopsies from proximal to distal mouse colons were examined by light microscopy. The average number of biopsies analysed per mouse was 7.9 for the test and 8.5 for the control genotype. SPSS (Version 11) and StatXact 8 software were used for statistical analysis. Sulindac exposure was associated with acute and chronic inflammation. Neovascularisation, apoptosis, necrosis and fibrosis were not present. Neoplastic transformation was only seen in the mice receiving the sulindac diet (p < 0.0001). There was less colon inflammation in the HIF1α knockout mice compared with the genotype controls (p = 0.004). HIF1α may have a pro-inflammatory role in sulindac-induced carcinogenesis in the mouse proximal colon.

Carcinogenic effect of the NSAID sulindac in the mouse proximal colon

Background Procarcinogenic effects of the non-steroidal anti-inflammatory drug (NSAID) sulindac have been described in the mouse proximal colon. The possible role of hypoxia inducible factors (HIF) in this process is poorly understood. Aim To study histological effects of sulindac in HIF1α knockout mice. Methods Mice were bred with a specific homozygous HIF1α deletion in the colonic epithelium (n = 16) with their genotype controls that express HIF1α in the colon (n = 19). They were given a diet containing 320 ppm sulindac for 20 weeks and the dietary controls (n = 17) received mouse chow without sulindac. Biopsies from proximal to distal mouse colons were examined by light microscopy. The average number of biopsies analysed per mouse was 7.9 for the test and 8.5 for the control genotype. SPSS (Version 11) and StatXact 8 software were used for statistical analysis. Results Sulindac exposure was associated with acute and chronic inflammation. Neovascularisation, apoptosis, necrosis and fibrosis were not present. Neoplastic transformation was only seen in the mice receiving the sulindac diet (p Conclusion HIF1α may have a pro-inflammatory role in sulindac-induced carcinogenesis in the mouse proximal colon.

Polyamines as mediators of APC-dependent intestinal carcinogenesis and cancer chemoprevention

Combination chemoprevention for cancer was proposed a quarter of a century ago, but has not been implemented in standard medical practice owing to limited efficacy and toxicity. Recent trials have targeted inflammation and polyamine biosynthesis, both of which are increased in carcinogenesis. Preclinical studies have demonstrated that DFMO (difluoromethylornithine), an irreversible inhibitor of ODC (ornithine decarboxylase) which is the first enzyme in polyamine biosynthesis, combined with NSAIDs (non-steroidal anti-inflammatory drugs) suppresses colorectal carcinogenesis in murine models. The preclinical rationale for combination chemoprevention with DFMO and the NSAID sulindac, was strengthened by the observation that a SNP (single nucleotide polymorphism) in the ODC promoter was prognostic for adenoma recurrence in patients with prior sporadic colon polyps and predicted reduced risk of adenoma in those patients taking aspirin. Recent results from a phase III clinical trial showed a dramatic reduction in metachronous adenoma number, size and grade. Combination chemoprevention with DFMO and sulindac was not associated with any serious toxicity. A non-significant trend in subclinical ototoxicity was detected by quantitative audiology in a subset of patients identified by a genetic marker. These preclinical, translational and clinical data provide compelling evidence for the efficacy of combination chemoprevention. DFMO and sulindac is a rational strategy for the prevention of metachronous adenomas, especially in patients with significant risk for colorectal cancer. Toxicities from this combination may be limited to subsets of patients identified by either past medical history or clinical tests.

Combination of sulindac and antimicrobial eradication of Helicobacter pylori prevents progression of gastric cancer in hypergastrinemic INS-GAS mice.

Helicobacter pylori infection causes severe dysplasia manifested as gastrointestinal intraepithelial neoplasia (GIN) after 28 weeks post-H. pylori infection (WPI) in cancer-prone, hypergastrinemic male INS-GAS mice. We examined the efficacy of the nonsteroidal anti-inflammatory drug sulindac (400 ppm in drinking water) alone, the CCK2/gastrin receptor antagonist YM022 (45 mg/kg/wk) alone, and sulindac or YM022 combined with H. pylori eradication therapy to prevent H. pylori-associated gastric cancer in male INS-GAS mice. Treatments started at 22 WPI, and mice were euthanized at 28 WPI. In uninfected mice, all treatments significantly delayed development of spontaneous GIN (P < 0.05). In H. pylori-infected mice, sulindac alone or YM022 alone had no protective effect on H. pylori-associated GIN. Importantly, sulindac exacerbated the severity of H. pylori-associated gastritis despite decreased gastric prostaglandin E(2) levels. However, sulindac combined with H. pylori antimicrobial eradication reduced the incidence of GIN (P < 0.05), whereas YM022 combined with antimicrobial eradication did not reduce GIN. In infected mice, sulindac or YM022 treatment did not alter gastric expression of the proinflammatory cytokines Ifn-gamma and Tnf-alpha and mucosal cell proliferation. Sulindac or YM022 combined with antimicrobial eradication down-regulated mRNA levels of Ifn-gamma and Tnf-alpha and mucosal cell proliferation (P < 0.05). We conclude that sulindac enhances H. pylori gastritis and may promote inflammation-mediated gastric carcinogenesis. The combination of sulindac and antimicrobial H. pylori eradication was beneficial for reducing proinflammatory cytokine mRNA in the stomach and preventing progression from severe dysplasia to gastric cancer in H. pylori-infected INS-GAS mice.

Sulindac inhibits canonical Wnt signaling by blocking the PDZ domain of the protein Dishevelled.

A new application: The nonsteroidal anti-inflammatory drug sulindac interacts directly and specifically with the PDZ domain of the protein Dishevelled (Dvl), which is a key intracellular component of the Wnt signaling pathways. Sulindac binds to the conventional peptide-binding pocket of the domain (see picture), and may exert a cancer chemoprotective effect by blocking it, thereby inhibiting canonical Wnt signaling.

Novel cell culture model for prevention of carcinogenic risk in familial adenomatous polyposis syndrome

Clinical familial adenomatous polyposis (FAP) syndrome represents a high risk pre-invasive precursor for colon cancer, and is characterized by germ line mutation in the adenomatous polyposis coli (APC) tumor suppressor gene. Cellular models with relevant genetic and biological characteristics should provide important mechanistic leads for predisposition and preventive intervention. Cloned colon epithelial cell line from the Apc850 Min/+ mouse represented a model for FAP. Cell cycle progression, cellular apoptosis and anchorage-independent growth represented the biomarkers for carcinogenic risk. The Apc mutant 850Min COL-Cl1 cells exhibited decreased G0/G1:S+G2/M ratio, increased S+G2/M:subG0 ratio, and increased anchorage-independent colony formation, indicating loss of homeostatic growth control and gain of anchorage-independent growth. Growth of these cells in serum-depleted medium was promoted by mitogenic insulin and epidermal growth factor, and inhibited by anti-mitogenic transforming growth factor-beta1 and dexamethasone. Treatment with low dose combinations of synthetic enzyme inhibitor difluoro methylornithine (DFMO), synthetic non-steroidal anti-inflammatory drug sulindac (SUL), and naturally occurring epigallocatechin gallate (EGCG), and eicosapen-taenoic acid (EPA) produced cytostatic growth arrest and inhibited anchorage-independent colony formation. These data identify a novel cell culture model and validate a mechanism-based approach to prioritize combinations of effective chemopreventive compounds for prevention/therapy of colon cancer.

Investigation of the Ligand-binding Mechanism of Methionine Sulfoxide Reductase A of E.coli

The rise of free oxygen in the atmosphere over 2.5 billion years ago made it possible for large land-based plants and animals to thrive. But oxygen, and the energy it provides, comes at great cost. Aerobic metabolism generates highly reactive intermediates and by-products in the form of hydroxyl radicals, superoxide anions and hydrogen peroxide. Within the cell, these reactive oxygen species attack biological macromolecules, producing covalent modifications that can affect both function and structure. One amino acid residue in proteins that is particularly sensitive to oxidation is the sulfur-containing side chain of methionine. Fortunately, methionine oxidation can be reversed by the actions of peptide methionine sulfoxide reductase (MsrA), which reduces methionine sulfoxide back to methionine and restores function to damaged proteins. We have used multiple spectroscopic techniques to investigate the mechanism by which MsrA recognizes and binds to a wide range of oxidized substrates in need of repair. Substrates studied include proteins, peptides and the non-steroidal anti-inflammatory drug Sulindac. Competition experiments with the fluorescent reporter ANS suggest the existence of weak, but specific, hydrophobic interactions between MsrA and unstructured and/or hydrophobic ligands.

Combination Chemoprevention of Intestinal Carcinogenesis in a Murine Model of Familial Adenomatous Polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited syndrome in humans. The Apc Min/+ mouse, which expresses a mutant homolog of the adenomatous polyposis coli gene, is a model of FAP in humans. Treatment with the nonsteroidal anti-inflammatory drugs (NSAIDS) sulindac or celecoxib can suppress polyp development in FAP patients, but responses are generally transient and incomplete. Combination chemoprevention with the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) and either celecoxib or sulindac was evaluated in the Apc Min/+ mouse. Combinations of DFMO and either NSAID reduced intestinal tumor number by more than 80% (P < 0.0001) compared to untreated controls. In addition to the dramatic reduction in tumor number, the combination of DFMO and sulindac reduced the development of high-grade intestinal adenomas compared to sulindac alone (P = 0.003). The fraction of high-grade intestinal adenomas remaining after treatment was similar for the combination of DFMO and celecoxib and celecoxib alone. Only combinations of DFMO plus sulindac reduced total intestinal polyamine contents compared to untreated mice. These data support the rationale for treatment of FAP patients postcolectomy with DFMO combined with either celecoxib or sulindac but indicate that sulindac may be more effective than celecoxib in reducing intestinal polyamine contents and the incidence of high-grade intestinal adenomas when combined with DFMO.

Sulindac Derivatives That Activate the Peroxisome Proliferator-activated Receptor γ but Lack Cyclooxygenase Inhibition

A series of novel derivatives of the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide were synthesized as potential agonists of the peroxisome proliferator-activated receptor gamma (PPARγ). Nonpolar and aromatic substitutions on the benzylidene ring as well as retention of the carboxylic acid side chain were required for optimal activity. Compound 24 was as potent a compound as any other in the series with an EC50 of 0.1 μM for the induction of peroxisome proliferator response element (PPRE)-luciferase activity. Direct binding of compound 24 to PPARγ was demonstrated by the displacement of [3H]troglitazone, a PPARγ agonist, in a scintillation proximity assay. Compound 24 also stimulated the binding of PPARγ to a PPRE-containing oligonucleotide and induced expression of liver fatty-acid binding protein (L-FABP) and adipocyte fatty acid-binding protein (aP2), two established PPARγ target genes. Taken together, these compounds represent potential leads in the development of novel PPARγ agonists.

Interactions of sulindac and its metabolites with phospholipid membranes: An explanation for the peroxidation protective effect of the bioactive metabolite

Non-steroidal anti-inflammatory drugs (NSAIDs) treat inflammatory processes by inhibition of cycloxygenase (COX). However, their action against lipid peroxidation can be an alternative pathway to COX inhibition. Since inflammation and lipid peroxidation are cell-surface phenomena, the effects of NSAIDs on membrane models were investigated. Peroxidation was induced by peroxyl radical (ROO•) derived from AAPH and assessed in aqueous or lipid media using fluorescence probes with distinct lipophilic properties: fluorescein; HDAF and DPH-PA. The antioxidant effect of Sulindac and its metabolites was tested and related with their membrane interactions. Drug–membrane interactions included the study of: drug location by fluorescence quenching; drug interaction with membrane surface by zeta-potential measurements; and membrane fluidity changes by steady-state anisotropy. Results revealed that the active NSAID (sulindac sulphide) penetrates into the lipid bilayer and protects the membrane against oxy-radicals. The inactive forms (sulindac and sulindac sulphone) present weaker interactions with the membrane and are better radical scavengers in aqueous media.

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.

A Case of Cronkhite-Canada Syndrome With Colorectal Adenomas: Effect of the Nonsteroidal Anti-Inflammatory Drug Sulindac

Hizawa, Kazuoki M.D.1; Nakamori, Mari M.D.2; Yao, Takashi M.D.2; Matsumoto, Takayuki M.D.3; Iida, Mitsuo M.D.3 Author Information

Insensitivity to Aβ42-lowering Nonsteroidal Anti-inflammatory Drugs and γ-Secretase Inhibitors Is Common among Aggressive Presenilin-1 Mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.

Up-regulation of early growth response gene 1 (EGR-1) via ERK1/2 signals attenuates sulindac sulfide-mediated cytotoxicity in the human intestinal epithelial cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and inflammation and have also received considerable attention because of their preventive effects against human cancer. However, the drug application is sometimes limited by the severe gastrointestinal ulcers and mucosal complications. In the present study, NSAID sulindac sulfide was investigated for the cytotoxic injury in the intestinal epithelial cells in association with an immediate inducible factor, early growth response gene 1 (EGR-1). Previously we reported that sulindac sulfide can suppress tumor cell invasion by inducing EGR-1. Extending the previous study, EGR-1 induction by sulindac sulfide was observed both in the non-transformed and transformed human intestinal epithelial cell lines. In terms of signaling pathway, ERK1/2 MAP kinases and its substrate Elk-1 transcription factor were involved in the sulindac sulfide-induced EGR-1 gene expression. Moreover, sulindac sulfide stimulated the nuclear translocation of the transcription factor EGR-1, which was also mediated by ERK1/2 signaling pathway. The roles of EGR-1 signals in the apoptotic cell death were assessed in the intestinal epithelial cells. Suppression of EGR-1 expression retarded cellular growth and colony forming activity in the intestinal epithelial cells. Moreover, induced EGR-1 ameliorated sulindac sulfide-mediated apoptotic cell death and enhanced the cellular survival. Taken all together, sulindac sulfide activated ERK1/2 MAP kinases which then mediated EGR-1 induction and nuclear translocation, all of which played important roles in the cellular survival from NSAID-mediated cytotoxicity in the human intestinal epithelial cells, implicating the protective roles of EGR-1 in the NSAID-mediated mucosal injuries.

Nonsteroidal anti-inflammatory drugs and oxidative stress in cancer cells.

Nonsteroidal antiinflammatory drugs (NSAIDs) induce apoptosis in a variety of cancer cells, including those of colon, prostate, breast and leukemia. In addition, the classical NSAIDs sulindac and aspirin are promising chemopreventive agents against colon cancer. NSAIDs inhibit cyclooxygenases (COX) preventing the formation of prostaglandins, prostacyclin and thromboxane. NSAIDs also exert other biological effects, including generation of reactive oxygen species (ROS) and inhibition of NF-kappaB-mediated signals. Despite many suggested mechanisms for their anticancer effects, it remains uncertain how they induce cell cycle arrest and apoptosis in cancer cells. Furthermore, there is little information on the selectivity of NSAIDs-mediated anticancer effects, although this is one of the most important issues in cancer therapy. Increased understanding of the biological basis for the anticancer activity of NSAIDs and their selectivity is essential for future therapeutic advances. In this paper, we propose that increased ROS generation is one of the key mechanisms for NSAIDs-mediated anticancer effects on various cancer cells.

Impact of dietary amino acids and polyamines on intestinal carcinogenesis and chemoprevention in mouse models

Colon cancer in humans is influenced by both genetic and dietary risk factors. The majority of colon cancers have somatic mutations in the APC (adenomatous polyposis coli) tumour-suppressor gene. Dietary arginine enhances the risk of APC-dependent colon carcinogenesis in mouse models by a mechanism involving NOS2 (nitric oxide synthase 2), as elimination of NOS2 alleles suppresses this phenotype. DFMO (difluoromethylornithine), a specific inhibitor of polyamine synthesis, also inhibits dietary arginine-induced colon carcinogenesis in C57BL/6J-Apc(Min)/J mice. The primary consequence of dietary arginine is to increase the adenoma grade in these mice. Either loss of NOS2 alleles or inhibition of polyamine synthesis suppresses the arginine-induced increase in adenoma grade. In addition to promoting intestinal carcinogenesis, polyamines can also reduce the efficacy of certain intestinal cancer chemopreventive agents. The NSAID (non-steroidal anti-inflammatory drug) sulindac is a potent inhibitor of intestinal carcinogenesis in the C57BL/6J-Apc(Min)/J mouse model and is used to treat humans with FAP (familial adenomatous polyposis). Dietary putrescine reduces the ability of sulindac to suppress intestinal tumorigenesis in the mouse model. These data suggest that reducing polyamine metabolism and dietary polyamine levels may enhance strategies for colon cancer chemoprevention.