Ibuprofen Inhibits Activation of Nuclear β-Catenin in Human Colon Adenomas and Induces the Phosphorylation of GSK-3β Free

Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the cyclooxygenase (COX)-1 and COX-2 enzymes, have an impressive history as effective chemopreventive agents for colorectal cancer (CRC; refs. 1, 2). These effects are largely dependent on their ability to inhibit the production of proliferative and inflammatory prostaglandins (PG), most notably prostaglandin E2 (PGE₂; ref. 3). More recently, specific COX-2 inhibitors were developed as a means to reduce the gastrointestinal toxicity associated with inhibiting COX-1. However, treatment with this drug class is associated with increased incidence of adverse cardiovascular events, even after short-term exposures (4, 5). Consequently, these negative side effects have limited their clinical application in chemoprevention (6–8). Therefore, there is renewed interest in developing nonselective NSAIDs for clinical use in chemoprevention, especially those chemically modified to circumvent gastrointestinal side effects.

Several double-blinded, placebo-controlled, and case-controlled studies have reported that both selective COX-2 inhibitors, such as celecoxib and rofecoxib, and nonselective inhibitors, such as sulindac and aspirin, are effective in reducing adenoma recurrence, as well as the risk for CRC (9–16). Apart from their abilities to inhibit COX activity and prostaglandin production, both selective and nonselective COX-2 inhibitors can target β-catenin, a key mediator of colon tumorigenesis. NSAIDs, including celecoxib, sulindac, and aspirin, can increase the phosphorylation of β-catenin, thus decreasing its nuclear accumulation and transcription of Wnt/β-catenin target genes, such as cyclin D1, c-myc, and PPARδ (17–22). Sulindac has also been shown to inhibit β-catenin expression in familial adenomatous polyposis (FAP)-related adenomas (21, 23).

In addition to their effects on Wnt/β-catenin signaling, a number of NSAIDs, including aspirin, diclofenac, sulindac, sulindac sulfone, indomethacin, celecoxib, and NS-398, have been shown to induce the degradation of the NF-κB inhibitor IκBα, resulting in the direct nuclear translocation of the p65 subunit of NF-κB in the absence of additional NF-κB activators (24–29). In fact, additional studies in human CRC cell lines have shown that aspirin and nonaspirin NSAIDs induce the translocation of NF-κB-p65 into the nucleolus, resulting in inhibition of NF-κB–related gene expression (25, 29, 30). It has thus been proposed that NSAID-mediated suppression of NF-κB target gene expression may contribute to the COX-2–independent growth inhibition and apoptosis induced by these drugs (26, 31, 32). Thus, the identification of additional chemopreventive agents that target both β-catenin- and NF-κB–mediated signaling in human tumors may lead to more effective therapeutic approaches.

In the following study, we evaluated the effects of the nonspecific COX-2 inhibitor ibuprofen on the Wnt/β-catenin pathway in human sporadic colon adenomas. Notably, it was found that ibuprofen treatment inhibited the accumulation of nuclear β-catenin expression, an outcome that correlated with reduced levels of cyclin D1. In colon cancer cells in vitro, S-ibuprofen, the active form in cell culture, along with a panel of other NSAIDs, increased phosphorylation-dependent inactivation of β-catenin. In addition, S-ibuprofen induced degradation of IκBα together with phosphorylation and nuclear localization of NF-κB-p65. Despite its nuclear localization, however, the activation of NF-κB target genes was suppressed, possibly related to the observed increase in phosphorylation of GSK-3β. These data suggest that ibuprofen may provide an effective alternative, or useful adjunct, to selective COX-2 inhibitors for use in chemoprevention.

Formalin-fixed, paraffin-embedded (FFPE) sections of adenomas from 37 patients taking daily NSAIDs (ibuprofen or aspirin) for up to 25 years and from 39 patients not taking daily NSAIDs were obtained from the Mayo Clinic. In total, there were 76 samples composed of 32 tubular adenomas and 5 tubulovillous adenomas from patients taking NSAIDs as well as 27 tubular adenomas and 12 tubulovillous adenomas from patients not taking NSAIDs. There was also a separate group of 17 adenomas from FAP patients given sulindac for 1 year. Samples were obtained in accordance with University of Connecticut Health Center Institutional Review Board (IRB) guidelines and use of these samples was approved by the IRB at the Mayo Clinic.

Staining was performed as described in our previous study (33). Briefly, FFPE tissues were de-paraffinized and incubated with 1% hydrogen peroxide for 20 minutes at room temperature. Tissues were subjected to antigen retrieval and blocked with 10% normal goat serum (Vector Laboratories) in PBS-Brij solution. Tissues were then incubated overnight at 4°C with anti-β-catenin (1:2,000; Sigma-Aldrich), anti-cyclin D1 (1:100; Immuno-Biological Laboratories, a kind gift from Dr. A. Arnold), antiproliferating cell nuclear antigen (anti-PCNA; 1:150; Novacastra Laboratories), or anti-p21 (1:100; Neomarkers) antibodies. Tissues were washed and incubated with biotinylated anti-mouse or anti-rabbit secondary antibodies for 30 minutes at room temperature, followed by incubation with avidin–biotin complex reagent (Vector Laboratories) for 30 minutes at room temperature. Signal was detected with DAB solution (3,3′-diaminobenzidine; Vector Laboratories), and tissues were counterstained with hematoxylin. For scoring of β-catenin and cyclin D1 nuclear immunostaining, the following scale was used: grade 1, 1%–10% positive nuclei; grade 2, 10%–20% positive nuclei, grade 3, 20%–50% positive nuclei; grade 4, >50% positive nuclei. Samples were considered negative if there were no positively staining nuclei.

SW480 and DLD-1 cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin. Cells were treated with 1 mmol/L S-ibuprofen, 5 mmol/L aspirin, 100 μmol/L sulindac sulfide, 600 μmol/L sulindac sulfone, 600 μmol/L indomethacin (all Sigma-Aldrich), or 1 μmol/L PGE₂ (Cayman Chemical) for 24 hours. Control cells were treated with vehicle (DMSO or 100% ethanol) alone.

The amount of PGE₂ present in cells was quantified using the PGE₂ Monoclonal EIA Kit (Cayman Chemical) according to the manufacturer's instructions. Briefly, cells were grown in 24-well plates to 80% confluence. Following drug treatments (as above, for 24 hours), cell culture supernatants were collected and immediately incubated for 18 hours at 4°C with a PGE₂-acetylcholinesterase conjugate (PGE₂ tracer) and a PGE₂ monoclonal antibody in plates precoated with goat anti-mouse IgG. Following washing to remove unbound reagents, the plates were developed with Ellman's reagent, which contains the substrate for acetylcholinesterase, and read at 405 nm. The absorbance readings were proportional to the amount of bound PGE₂ tracer and inversely proportional to the amount of PGE₂ in the samples. A standard curve was also measured using known concentrations of PGE₂ to calculate the absolute amounts of PGE₂ in the samples.

Following drug treatment (as above, for 24 hours), cells were lysed in a buffer containing 20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitors (50 mmol/L NaF, 10 mmol/L sodium β-glycerophosphate, 5 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 1 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonylfluoride). Following centrifugation at 14,000 rpm for 15 minutes at 4°C, the supernatant was removed and quantified for total protein. Thirty micrograms of protein was incubated at 95°C for 5 minutes and 4× sample loading buffer containing 8% β-mercaptoethanol and loaded onto a 10%–15% polyacrylamide gel. Proteins were transferred to an Immobilon-P membrane (Millipore Corp.) and blocked in 5% nonfat dry milk in TBS-T (1× TBS, 0.1% Tween-20) for 1 hour. Blots were incubated overnight at 4°C with phospho-β-catenin (1:2,000), phospho-NF-κB-p65 (1:1,000), PARP (1:1,000), total NF-κB-p65 (1:1,000), phospho-GSK-3β (1:1,000), total GSK-3β (1:1,000), total IκBα (1:1,000), phospho-AKT (1:1,000), or total AKT (1:1,000) antibodies (Cell Signaling), as well as β-catenin (1:2,000; Sigma-Aldrich), cyclin D1 (1:1,000; IBL), Bcl-2 (1:1,000; BD Transduction Laboratories), survivin (1:1,000; Lab Vision Corp.), β-tubulin (1:2,000; Sigma-Aldrich), or β-actin (1:6,000; Santa Cruz Biotechnology) antibodies. Blots were then washed multiple times and incubated with goat anti-mouse IgG HRP (horseradish peroxidase; 1:10,000; Upstate Biotechnology) or donkey anti-rabbit IgG HRP (1:4,000; Santa Cruz Biotechnology) for 45 minutes at room temperature. HRP was visualized with enhanced luminal reagent (Millipore Corp.).

Cells were grown on 22 × 22-mm glass coverslips in 6-well plates to approximately 50% confluence and then treated with drugs (as above, for 24 hours). Cells were washed with cold 1× PBS and then fixed in 4% paraformaldehyde for 10 minutes, followed by permeabilization in 0.5% Triton X-100 in PBS for 5 minutes. Cells were then blocked in 3% bovine serum albumin (BSA; 10 mg/mL) in PBS for 1 hour at room temperature, followed by incubation with anti-β-catenin antibody (1:100; Sigma-Aldrich) in 1% BSA in PBS for 1 hour at room temperature. Cells were washed in PBS and then incubated with secondary antibody (goat anti-mouse IgG Alexa 568; 1:200 in 1% BSA in PBS; Molecular Probes) for 1 hour at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:10,000). Cells on coverslips were mounted onto glass slides using Prolong Gold Antifade Reagent (Molecular Probes) and visualized using an Olympus IX50 fluorescence microscope (Olympus Corp.).

For associations comparing adenoma phenotype and NSAID use, statistical analyses were performed using a 2-tailed Fisher's exact test. To determine statistical significance of PGE₂ levels, analyses were performed using ANOVA between groups. The values of P < 0.05 were considered to be statistically significant.

The sample set of human adenomas was composed of 76 sporadic adenomas, 37 from patients taking daily ibuprofen or aspirin for 1 year or more and 39 from patients with no history of NSAID use. In addition, there were 17 adenomas from FAP patients given sulindac for 1 year. To evaluate the effects of 1 year or more of ibuprofen or aspirin use on adenoma phenotype, we performed statistical analyses comparing dysplasia or size with NSAID use. As shown in Table 1, there was no significant correlation found between degree of dysplasia and NSAID use (P = 0.12, 2-tailed Fisher's exact test) or between size and NSAID use (P = 0.21, 2-tailed Fisher's exact test). However, given the limited number of dysplastic adenomas observed in the present study (5 of a total of 76), a conclusive determination of the effects of NSAID use on histologic tumor grade is not possible. This indicates that in this sample set, NSAID use did not significantly impact the pathologic features of the adenomas.

NSAIDs, including aspirin, sulindac, and indomethacin, have been shown to decrease nuclear levels of β-catenin in colon cancer cells (17–19). To determine whether long-term NSAID use in this adenoma sample set could affect cellular localization of β-catenin, immunohistochemical analysis of β-catenin was performed in the 76 sporadic adenomas and 17 FAP adenomas. As shown in the representative examples in Figure 1A, in adenomas from non-NSAID patients, there was strong nuclear staining of β-catenin (a, b) whereas NSAID use significantly decreased nuclear β-catenin expression (c, d). Nuclear β-catenin was quantified and these data are shown in Table 2. Overall, 14 of 36 (39%) adenomas in the non-NSAID group had nuclear β-catenin staining compared with only 3 of 34 (9%) adenomas from patients with a history of NSAID intake (P = 0.0047). In addition, the 3 adenomas from the NSAID group with positive nuclear β-catenin staining were all from patients taking daily aspirin, indicating that daily ibuprofen use completely inhibited nuclear β-catenin. Neither ibuprofen nor aspirin use had an effect on cell proliferation, as assessed by PCNA immunostaining, or p21 expression. In the adenomas from FAP patients treated with sulindac, 9 of 17 (53%) had nuclear β-catenin staining, indicating that sulindac was only variably effective in inhibiting translocation of β-catenin in these patients. However, it was not possible to determine whether the percentage of positive nuclear β-catenin cells in the adenomas was suppressed by sulindac because there was no nontreated group for comparison.

When β-catenin translocates to the nucleus and binds to TCF/LEF (T-cell factor/lymphoid enhancer factor) transcription factors, it drives the transcription of a panel of Wnt target genes including cyclin D1, c-myc, COX-2, and PPARδ (34–36). Thus, we wanted to determine whether nuclear β-catenin localization in the adenoma samples might have downstream effects on the expression of cyclin D1. As shown in the representative photomicrographs (Fig. 1B), immunohistochemical analysis revealed strong nuclear expression of cyclin D1 in non-NSAID adenomas that had nuclear β-catenin staining (a, b); however, in adenomas from patients in the NSAID exposure group, the absence of nuclear β-catenin was associated with diminished nuclear cyclin D1 immunostaining (c, d). As shown in Table 2, however, there was no significant correlation between nuclear cyclin D1 expression and NSAID use (P > 0.05); however 12 of 16 (75%) adenomas with nuclear β-catenin expression also had nuclear expression of cyclin D1 (P = 0.022). This finding is illustrated in greater detail in Figure 1C, which shows a representative example of β-catenin (a–c) and cyclin D1 (d–f) colocalization in serial sections of a representative non-NSAID adenoma. This indicates that nuclear β-catenin does affect Wnt target gene expression.

To further investigate the effect of ibuprofen on β-catenin cellular localization, we treated human colon cancer cells with S-ibuprofen and a panel of other NSAIDs for comparison. Treatment of SW480 or DLD-1 cells with 1 mmol/L S-ibuprofen, 5 mmol/L aspirin, 100 μmol/L sulindac sulfide, 600 μmol/L sulindac sulfone, or 600 μmol/L indomethacin for 24 hours significantly inhibited PGE₂ production compared with vehicle alone, indicating that COX-1/2 enzymes were being effectively targeted by drug treatment (Fig. 2A and B).

To determine the effect of this panel of NSAIDs on β-catenin expression, immunofluorescence microscopy was carried out on SW480 cells, which have mutant APC and consequent high levels of nuclear β-catenin. As shown in Figure 3A, strong membrane, cytoplasmic, and nuclear staining of β-catenin was present in untreated cells. However, treatment with NSAIDs greatly decreased the overall intensity of β-catenin staining, most notably with S-ibuprofen, sulindac sulfide, and indomethacin treatment. These data indicate that ibuprofen can inhibit the accumulation of nuclear β-catenin, as well as suppress the levels of total cellular β-catenin, despite the loss of APC protein. Figure 3B shows that in cytoplasmic extracts from SW480 cells, S-ibuprofen, aspirin, sulindac sulfide, and indomethacin could increase the accumulation of the phosphorylated form of β-catenin, suggesting that these drugs decrease nuclear levels of β-catenin by increasing its cytoplasmic degradation.

To explore the possibility that ibuprofen may affect other signaling pathways in addition to the Wnt/β-catenin cascade, NF-κB signaling was examined in human SW480 colon cancer cells. NSAIDs, including aspirin, sulindac, and diclofenac, have been shown to activate NF-κB in a variety of CRC cells (24, 26). As shown in Figure 4A, 24-hour treatment of SW480 cells with increasing concentrations of S-ibuprofen resulted in a dose-dependent decrease in the levels of the NF-κB inhibitor protein IκBα. Furthermore, a dose-dependent increase in the phosphorylated form (serine-536) of the p65 subunit of the NF-κB transcription factor complex was observed (Fig. 4A). Although phosphorylation of NF-κB-p65 at this serine residue has been shown to be important in the kinetics of its nuclear import (37), further direct evidence of NF-κB-p65 nuclear localization was required. As shown in Figure 4B, 24-hour treatment with S-ibuprofen resulted in nuclear translocation and accumulation of NF-κB-p65 in SW480 cells, as assessed by immunofluorescence staining.

After showing that ibuprofen treatment (in the absence of additional NFκB activation) resulted in nuclear localization of NF-κB-p65, we next examined the possible effects of prolonged ibuprofen treatment on NF-κB target gene expression. As shown in Figure 5A, despite the observed nuclear localization, expression of the NF-κB target genes, Bcl-2, survivin, and cyclin D1, were decreased in a time-dependent manner following treatment of SW480 cells with S-ibuprofen, an effect that occurred through 72 hours. In addition, S-ibuprofen caused a time-dependent increase in apoptosis in SW480 cells treated, assessed by PARP cleavage (Fig. 5A). Recent studies have indicated that the serine/threonine kinase GSK-3β positively regulates NF-κB–mediated gene expression (38, 39). Thus, we investigated the possibility that ibuprofen treatment may inhibit the activity of GSK-3β, which is present constitutively in an active form, and phosphorylation of its serine-9 residue by Akt results in the inhibition of its signaling activity (40). As shown in Figure 5A, there was a time-dependent increase in the inhibitory phosphorylation of GSK-3β at serine-9. In addition, as shown in Figure 5B, we observed a rapid (within minutes) increase in the phosphorylation of GSK-3β at serine-9 in SW480 cells following treatment with S-ibuprofen. Interestingly, there was a concomitant increase in the active, phosphorylated form of Akt (serine-476), consistent with its established role in GSK-3β inhibition (40–42).

The adverse cardiovascular effects associated with specific COX-2 inhibitors have limited their application in cancer chemoprevention, especially in high-risk individuals (43–46). Therefore, a renewed interest in the development of less toxic, nonselective NSAIDs has emerged. Our study found significant inhibition of nuclear β-catenin expression in sporadic colon adenomas from patients with a history of daily intake of ibuprofen or aspirin. One important distinguishing feature of our study is related to the patient population, comprised primarily of sporadic adenoma cases. Currently, the only clinical human data showing that NSAIDs can suppress β-catenin expression are in FAP patients (21, 23). Of note, no adenomas from subjects in our study who reported daily ibuprofen use exhibited the presence of nuclear β-catenin, indicating that this drug could completely suppress its nuclear translocation. Surprisingly, adenomas from FAP patients maintained on sulindac for up to 1 year showed only a modest reduction in the levels of nuclear β-catenin. However, pretreatment adenomas were not available from either population group and thus it is difficult to establish a definitive comparison in response between these groups. It remains a possibility that sulindac treatment suppressed nuclear β-catenin translocation if compared with pretreatment adenomas.

Studies in human colon cancer cells have found that inhibition of nuclear β-catenin by NSAIDs, such as sulindac and indomethacin, results in a dramatic downregulation of its transcriptional targets, including cyclin D1 and c-myc (22, 47). Consistent with these in vitro findings, our study also found an association between nuclear β-catenin expression and positive cyclin D1 staining in the sporadic colon adenomas. In fact, 75% of adenomas with nuclear β-catenin staining also exhibited nuclear cyclin D1 expression. Furthermore, the suppression of nuclear β-catenin by either ibuprofen or aspirin was associated with a reduction in nuclear cyclin D1 expression. These data indicate that nuclear expression of β-catenin in adenomas can activate proliferative Wnt target genes, which can also be suppressed by NSAID intake.

To further investigate the effects of ibuprofen on β-catenin localization, additional studies were performed using human colon cancer cell lines. As anticipated, it was found that S-ibuprofen, as well as a panel of additional NSAIDs including aspirin, sulindac sulfide, sulindac sulfone, and indomethacin, significantly inhibited PGE₂ production in 2 human colon cancer cell lines, SW480 and DLD-1, both of which have mutant APC (48). However, we selected the SW480 cells for further analysis on the basis of their strong nuclear localization of β-catenin (48). In agreement with our in vivo results, S-ibuprofen inhibited nuclear translocation of β-catenin in SW480 cells, as well as stimulated its phosphorylation at serine-33,37 and threonine-41, indicating its increased cytoplasmic degradation. NSAIDs have been shown to induce β-catenin phosphorylation by acting as phosphatase inhibitors (17). Specifically, aspirin has been shown to negatively regulate protein phosphatase 2A (PP2A) enzymatic activity through phosphorylation of the inhibitory tyrosine-307 in PP2A (17). Thus, ibuprofen may also act as a phosphatase inhibitor to stabilize the phosphorylation of β-catenin.

In addition to its effects on β-catenin, S-ibuprofen treatment also resulted in the degradation of IκBα in SW480 cells. This was accompanied by phosphorylation of the p65 subunit of NF-κB (Fig. 4A). This signaling pathway has been established to play a key role in controlling the expression of a myriad of genes involved in inflammation, differentiation, proliferation, and apoptotic cell death (49). In resting cells, the heterodimeric NF-κB transcription factor complex is sequestered in the cytoplasm by the inhibitor protein IκBα. Cellular stimulation by various agents can result in the phosphorylation and proteosomal degradation of IκBα, causing the phosphorylation, release, and nuclear translocation of the NF-κB complex (49). As shown in Figure 4B, further evaluation of NF-κB-p65 staining, using immunofluorescence analysis, revealed its punctate, nuclear localization after treatment with S-ibuprofen. Previous studies have shown that nucleolar sequestration of the p65 subunit of NF-κB is essential for aspirin-mediated apoptosis (26, 50, 51). Furthermore, nucleolar sequestration of NF-κB-p65 may contribute to the suppression of NF-κB target gene activation by preventing proper binding of the NF-κB complex to the promoters of these genes (51). Future studies will be aimed at determining whether S-ibuprofen treatment results in nucleolar localization of NF-κB-p65.

Consistent with these findings, we also observed that treatment of SW480 cells in vitro with S-ibuprofen (for up to 72 hours) resulted in decreased expression of a limited set of NF-κB target genes, including Bcl-2, survivin, and cyclin D1 (Fig. 5A). Furthermore, the suppression of NF-κB target gene activation in SW480 cells was accompanied by a time-dependent increase in apoptosis. These data, however, do not directly address the possibility that S-ibuprofen directly mediates the nucleolar localization of NF-κB-p65. Thus, we sought an alternative mechanism to explain the phosphorylation and nuclear localization of NF-κB-p65 that occurs despite a marked decrease in the expression of this subset of NF-κB target genes.

Further examination of protein lysates from SW480 cells treated for 72 hours with S-ibuprofen (Fig. 5A) revealed a time-dependent increase in inhibitory phosphorylation of GSK-3β at serine-9, an effect that occurred within minutes of treatment (Fig. 5B). GSK-3β is a multifunctional serine/threonine kinase that was described initially as a regulator of glycogen synthase activity (52, 53). Elevated levels of GSK-3β have been found both in human colon cancer cell lines and in primary colon tumors (54). Importantly, there is accumulating evidence for a direct relationship between GSK-3β activity and the NF-κB signaling pathway (55–57). For example, several in vitro studies in MEFs and in pancreatic cancer cell lines have shown that GSK-3β regulates NF-κB signaling downstream of IκBα degradation and nuclear translocation of NF-κB (39, 55). In addition, GSK-3β is required for the expression of certain NF-κB target genes (58) and NF-κB–mediated cell survival (39). An earlier study also provided evidence that the treatment of SW480 cells with aspirin resulted in increased phosphorylation of β-catenin, as well as phosphorylation of GSK-3β at serine-9 (59). As noted earlier, it has also been shown that aspirin inhibits the phosphatases PP2A (17, 60). In the present study, we examined whether treatment of SW480 cells with S-ibuprofen may lead to an increase in inhibitory PP2A phosphorylation levels. However, we were unable to establish time- or dose-dependent differences in PP2A phosphorylation after S-ibuprofen treatment (data not shown). Although we did not establish a direct association between GSK-3β and PP2A, there are many other phosphatase enzymes that may possibly affect S-ibuprofen–induced GSK-3β inhibitory phosphorylation (61). Future studies will be directed at examining the possibility that other phosphatases may affect GSK-3β signaling with respect to S-ibuprofen treatment.

As noted earlier, the long-term use of traditional NSAIDs has been associated with gastrointestinal side effects. This toxicity is generally attributed to the key role that COX-1 plays in maintaining the integrity of the intestinal mucosa. The use of modified NSAIDs, including those containing nitric oxide that purportedly maintain mucosal integrity in a manner similar to that of endogenous prostaglandins, may circumvent gastrointestinal toxicity (5). Other approaches include the use of dual COX and 5-lipoxygenase inhibitors, as well as NSAIDs that are capable of releasing hydrogen sulfide (62). Thus, continued development of these novel strategies to circumvent gastrointestinal toxicity while still affording chemopreventive benefit is clearly needed to expand the clinical safety and efficacy of this important drug class.

To our knowledge, our study is the first to show that ibuprofen is effective at inhibiting β-catenin nuclear translocation in both human adenomas and in colon cancer cells in vitro. It is also the first to show that an NSAID can suppress β-catenin in sporadic adenomas following long-term treatment in patients. This indicates that the Wnt pathway in colon adenomas is sensitive to NSAID exposure, regardless of whether the lesions are from FAP patients or are sporadic in origin. In addition, our current data suggest that S-ibuprofen treatment results in the phosphorylation and nuclear translocation of NF-κB. However, this effect is associated with a concomitant loss in transcriptional activity. This reduced transcriptional activity of NF-κB may be due to the novel finding of increased S-ibuprofen–dependent phosphorylation of GSK-3β, leading to inhibition of NF-κB signaling. Future studies will establish the precise role that ibuprofen treatment plays in augmenting the GSK-3β–NF-κB signaling axis. In addition, because the Wnt/β-catenin and NF-κB signaling pathways share overlapping target genes, it will be important to establish the relative contribution of these pathway(s) to the transcriptional changes observed after ibuprofen treatment. In summary, we believe that these findings warrant the development of case-controlled studies for CRC chemoprevention using ibuprofen.

No potential conflicts of interest were disclosed.

This work was supported by National Institute of Health grants 5RO1CA125691 and 5R01CA114635 (to D.W. Rosenberg)

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