Wnt/β-Catenin Signaling Regulates Cytokine-Induced Human Inducible Nitric Oxide Synthase Expression by Inhibiting Nuclear Factor-κB Activation in Cancer Cells

The Wnt/β-catenin signaling pathway influences many cellular processes including cell adhesion, growth, and differentiation (1). Several lines of evidence have implicated aberrant Wnt/β-catenin signaling in the development of human cancers, especially colon cancer and hepatocellular carcinoma (2, 3). In colon cancer, mutation of the tumor suppressor gene adenomatous polyposis coli (APC) leads to stabilization of transcriptional regulator β-catenin, which accumulates in the cytoplasm, enters the nucleus, and interacts with nuclear T-cell factor 4 (Tcf-4)/lymphoid enhancer factor (LEF) to activate transcription of specific target genes leading to cell proliferation and carcinogenesis (4–6).

Nitric oxide (NO) is a diverse biological mediator with important roles in vascular biology, inflammation, and tumor biology (7–9). Studies have shown that NO promotes carcinogenesis at low levels and that NO inhibits tumor growth at high concentrations (10–12). NO has been shown to cause DNA damage and is mutagenic (13). Inducible nitric oxide synthase (iNOS) has been documented in cancer specimens (14) and precancerous chronic inflammatory conditions (15, 16). Several studies have also investigated the relationship between iNOS expression, vascular endothelial growth factor, and the tumor suppressor gene product p53 (17, 18). Exposure of human cancer cells to a NO donor resulted in p53 protein accumulation, which then acted in a negative feedback loop to trans-repress human iNOS (hiNOS) gene transcription in vitro and in vivo (19, 20). Hence, the double-edged sword of NO in tumor biology clearly depends on cell type, NO concentration, oxidative stress, and tumor milieu (21, 22).

The expression of iNOS is predominantly regulated at the transcriptional level by inflammatory cytokines that signal through nuclear factor κB (NF-κB) and other nuclear factors (23–27). NF-κB is also an important transcriptional factor that is involved in colorectal carcinogenesis (28, 29). Recently, we have shown that the hiNOS gene is up-regulated by Wnt/β-catenin signaling (30). Overexpression of β-catenin and Tcf-4 increased basal and cytokine-induced iNOS promoter activity in cancer cells that was dependent on intact cis-acting Tcf-4 binding element (TBE) motifs in the hiNOS promoter (30). However, another study has shown that β-catenin physically interacts with NF-κB and inhibits its activation in human colon and breast cancer (31). Therefore, we examined the effect of β-catenin signaling on NF-κB activation and tumor necrosis factor α (TNFα)–induced iNOS expression in human colon, liver, and lung cancer cells. Our findings indicate that β-catenin differentially regulates TNFα-induced hiNOS and other NF-κB target gene expression by physical interaction with NF-κB in colon and liver cancer cells.

Cell lines and reagents. The human colorectal cancer cell lines SW480, DLD1, RKO, and 293 were obtained from American Type Culture Collection; HCT116, HAβ18, and HAβ85 (gene-targeted HCT116 derivatives) were kindly given by Dr. Todd Waldman (Georgetown University, Washington, DC; ref. 32). They were cultured at 37°C in 5% CO₂ in medium containing 10% fetal bovine serum (FBS; Clontech), 100 units/mL penicillin, 100 μg/mL streptomycin, and 15 mmol/L HEPES. SW480, DLD1, RKO, and 293 cells were cultured in DMEM (Invitrogen), and HCT116, HAβ18, and HAβ85 in McCoy's 5A medium (Invitrogen). Unless indicated, cells were stimulated with a cytokine mixture consisting of 1,000 units/mL human TNF-α (R&D Systems), 100 units/mL IL-1β (R&D Systems), and 250 units/mL human IFNγ (R&D Systems or Roche Pharmaceuticals).

Plasmid constructs. The hiNOS −5.8 kb NF-κB-luciferase reporter plasmid (2× NF-κB; ref. 25), hiNOS −5.8 kb NF-κB oligonucleotide cloned into the unique Sma1 site of luciferase reporter plasmid pT109, which contains 109 bp of the herpes virus thymidine kinase promoter, driving expression of firefly luciferase, was confirmed by sequencing. NF-κB −5.8 wild-type (WT), 5′-AGAGGGCTTTCCCAGAACCA-3′; NF-κB −5.8 mutant, 5′-AGAGGGCTCGCCCAGAACCA-3′ (25). The reporter plasmids pTOP-FLASH and pFOP-FLASH, WT APC expression vector (PCMV-neo-APC), and β-catenin expression vector (PCI-neo β-catenin) were kindly provided by Dr. Bert Vogelstein. Expression plasmids encoding human p50 and FLAG-taggep65 (pFL65) NF-κB proteins were kindly provided by Dr. Joseph DiDonato (Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH; ref. 33).

Transient transfection assay. DNA transfections of cells were carried out in six-well plates (Corning) by using Lipofectamine (Invitrogen) and MIRUS Trans-IT reagent (Mirus) as previously described (30). To control for transfection efficiency between groups, 0.5 μg of a plasmid containing a cytomegalovirus promoter–driven β-galactosidase was added to each well. pRSV-Luc was used as positive control, and the promoterless plasmid pXP2 served as negative control. RKO β-catenin S33Y stable transfectants were established by transfection with β-catenin S33Y plasmid (2). Stable transfectants in SW480 cells were established by transfection with full-length APC plasmid (2).

Immunoprecipitation and Western blot analysis. Immunoprecipitation, SDS-PAGE, and Western blot were done as described previously (30). Antibodies used for Western blot were rabbit anti-hiNOS polyclonal (BD Biosciences), β-catenin (H-102), p65 (A), p50 (c-19), TRAF1 (H-132, Santa Cruz), and mouse anti–β-catenin monoclonal (Sigma).

Electrophoretic mobility shift assays. Nuclear protein extraction and gel shift assay were done as previously described (30). Antibody supershifts used 0.5 to 2 μg of anti-p65 (A)X (Santa Cruz) and anti-p50 (NLS)X (Santa Cruz).

Immunofluorescent staining. Cells were cultured on coverslips and washed twice with cold PBS, fixed with 2% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 and 10% FBS in PBS for 30 min at room temperature, and then incubated with the specific primary antibodies for p50 (Santa Cruz), β-catenin (Sigma), APC (BD), iNOS(BD), and cleaved PARP (Asp214, Cell Signaling; ref. 30). Slides were viewed with Olympus Provis and Leica TSL-SL microscopes.

Immunohistochemistry. Tissue samples were deparaffinized and subjected to alcohol gradient and washed thrice, then treated with 0.3% H₂O₂ in methanol followed by 10% horse serum for 30 min. Sections were incubated overnight with primary antibodies β-catenin (Sigma; 1:200 dilution), Fas (CH11 Upstate; 1:150 dilution), or iNOS (Abcam; 1:150 dilution) and then with secondary antibodies Bio-antimouse and rabbit IgG (1:200 dilution) for 30 min at room temperature; signal was detected using ABC Elite Kit (Vector Laboratories).

NO production assessment. Culture supernatants were collected and assayed for nitrite, a stable end product of NO oxidation, using the Griess reaction as described (34).

Statistical methods. Data are presented as the mean ± SE. Cultures were done in duplicate or triplicate, and experiments were done at least thrice. Data were analyzed by the Student t test or ANOVA, and P < 0.05 was considered statistically significant.

β-Catenin inversely correlated with cytokine-induced iNOS expression. To examine the level of cytokine-induced iNOS gene expression in HCT116, DLD1, and SW480 cell lines, we transfected the cells with the WT hiNOS −7.2 kb luciferase promoter plasmid and then treated them with a cytokine mixture of TNFα + IL-1β + IFNγ for 6 hours. The cytokine mixture induced hiNOS promoter activity in HCT116 and DLD1 cells, but significantly decreased hiNOS promoter activity below basal levels in SW480 cells (Fig. 1A). This was an unexpected finding and led us to investigate in other human cell lines. A similar differential expression of cytokine-induced hiNOS transcription was seen in HepG2 cells compared with Huh7 and Hep3B liver cancer cells, where again cytokine mixture stimulation decreased hiNOS promoter activity below basal levels (Fig. 1B). We also observed that the magnitude of cytokine-induced NO production was quite different, being dramatically increased in primary human and rat hepatocytes, lower in HepG2 and DLD1 cells, and no appreciable NO synthesis detectable by nitrate assay in SW480 cells (Fig. 1C). Interestingly, endogenous β-catenin expression was high in the SW480 cells and lower in the HCT116 and DLD1 cells (Supplementary Fig. S1A). These observations led us to hypothesize that the presence of β-catenin might inversely correlate and regulate cytokine-induced iNOS expression, possibly through interaction with the NF-κB pathway.

β-Catenin reduced TNFα-induced hiNOS-specific NF-κB reporter and binding activities. To determine whether β-catenin interacts with NF-κB and regulates hiNOS expression, we synthesized hiNOS-specific −5.8 kb NF-κB oligonucleotides and constructed two tandem copies of the NF-κB element at −5.8 kb in the hiNOS promoter for use in gel shift assay, as well as hiNOS-specific NF-κB reporter plasmid driving the minimal herpes thymidine kinase-luciferase promoter. We initially determined the effects of β-catenin overexpression on NF-κB p65–mediated activation of the hiNOS NF-κB reporter. A p65 expression plasmid (0.2 μg) was cotransfected with increasing doses of β-catenin expression plasmid (mutant β-cateninS33Y) along with the hiNOS NF-κBx2 reporter. β-Catenin overexpression inhibited the p65 activation of hiNOS NF-κB reporter in a dose-dependent manner (Fig. 2A). β-Catenin overexpression also abrogated the cytokine mixture–induced hiNOS promoter activity in the HCT116, DLD1, and HepG2 cells previously noted and actually produced a 30% to 50% drop in luciferase activity compared with basal cells (Fig. 2B). These findings suggest that β-catenin can antagonize cytokine-mediated induction of the NF-κB–dependent hiNOS gene.

Next, we examined for β-catenin/Tcf-4 transcriptional activity in HCT116 cells (β-catenin WT/mut) compared with HAβ18 (del/mut) and HAβ85 (WT/del) cell lines (32) by pTOP/pFOP transfection. The HAβ85 (WT/del) cells lack β-catenin signaling due to deletion of the mutant β-catenin allele. In these studies, the WT β-catenin is subject to degradation, whereas the mutant β-catenin is stabilized and active for nuclear translocation. The pTOP expression plasmid is a positive reporter plasmid for β-catenin transcriptional activity (containing three copies of an optimal Tcf binding motif), whereas the pFOP is a negative control mutant expression reporter. As predicted, the presence of a mutant β-catenin allele increased β-catenin/Tcf-4 transcriptional activity (pTOP reporter activity) in HCT116 and HAβ18 cell lines, but not in HAβ85 cells (Fig. 3A). As a negative control, the pFOP reporter was used and did not show any β-catenin activation. TNFα stimulation of the HAβ18 cells reduced resting hiNOS-specific NF-κB reporter activity below basal level, whereas it induced NF-κB reporter activity in the HAβ85 cells that lacked β-catenin signaling (Fig. 3B). Dominance of the mutant β-catenin in the HAβ18 cells also suppressed TNFα-induced p50 DNA binding (Fig. 3C,, lane 4, lower band). Immunofluorescent staining also indicated that activated β-catenin decreased TNFα-induced NF-κB p50 translocation to the nucleus of HAβ18 cells, compared with abundant p50 staining in the HAβ85 cells (Fig. 3D), which is consistent with the β-catenin–mediated repression of NF-κB reporter activity and gel shift binding (Fig. 3B and C).

Inactivation of β-catenin by integrating full-length APC in SW480 restored NF-κB activity. To support our hypothesis that high level of β-catenin was inhibiting NF-κB–mediated gene activation, we generated stably transformed SW480 cells overexpressing the APC gene. Because the SW480 cells contain a truncated APC gene and exhibited very high constitutive β-catenin levels (Supplementary Fig. S1A), we reasoned that adding back full-length APC could bind and degrade β-catenin in the multiprotein complex. If so, then NF-κB–mediated gene transcription should be restored in SW480-APC cells compared with control SW480-vector cells. Immunoblot of stably transformed SW480-APC cells showed strong APC, decreased β-catenin, and increased hiNOS protein compared with the SW480 cells expressing empty vector (Fig. 4A,, top). In addition, overexpression of APC led to decreased β-catenin transcriptional activity with pTOP transfection (Fig. 4A,, bottom). This was also consistent with the immunofluorescent staining, with SW480-APC exhibiting decreased β-catenin and increased iNOS staining (Fig. 4B). Likewise, SW480-APC showed a higher level of TNFα-induced p65 binding on gel shift (Fig. 4C), as well as increased TNFα-induced hiNOS mRNA and protein expressions (Fig. 4D). In general, iNOS protein exists as a homodimer. However, a doublet type of iNOS protein band is often exhibited in published Western blots, and some authors have suggested that the lower band may reflect monomers or iNOS degradation products (35).

β-Catenin physically interacts with NF-κB subunits in colon cancer cells. To document that β-catenin was physically binding to NF-κB proteins, we performed coimmunoprecipitation experiments. The SW480 cells have strong β-catenin expression, DLD1 and HCT116 have moderate β-catenin expression, whereas the RKO cells show nearly undetectable β-catenin protein levels (Supplementary Fig. S1A). On coimmunoprecipitation, β-catenin complexed with both p50 and p65 (Supplementary Fig. S1B). This confirms a direct interaction of β-catenin with NF-κB subunits in these cells, as has been shown in other cells (31), and provides a potential regulatory mechanism for modulating NF-κB signaling. We also performed immunoprecipitation of cell lysates from primary human hepatocytes and HepG2 cells and found that β-catenin complexed with either p65 or p50 (data not shown).

Effects of β-catenin on NF-κB–mediated transcription in other target genes. To show that the inhibitory effects of β-catenin on NF-κB activity were not limited to hiNOS, we also examined Traf1 gene expression, which is known to be regulated by NF-κB activity (31). Strong TNFα-induced Traf1 protein was seen in the HAβ85 cells that lack significant β-catenin transcriptional activity, whereas the HAβ18 cells did not show inducible Traf1 expression (Fig. 5A). Similarly, the RKO cancer cell line, which lacks β-catenin protein (Supplementary Fig. S1A), also showed strong TNFα-mediated Traf1 expression (Fig. 5B). In agreement with the notion of β-catenin–mediated inhibition of NF-κB activation, we also generated RKO cells stably transformed to overexpress β-catenin and observed a significant down-regulation of TNFα-induced Traf1 protein expression (Fig. 5C). Finally, consistent with β-catenin degradation by APC binding, inducible Traf1 expression was shown in the SW480-APC cells, but not in the control SW480-vector cells that exhibit high β-catenin (Fig. 5D).

β-Catenin inhibited NO-induced apoptosis in HAβ18 cells. To test the relationship between β-catenin and NO-induced apoptosis, HAβ18 and HAβ85 cells were treated with the NO donor S-nitroso-N-acetylpenicillamine. The results showed that NO induced activation of caspase-3 (Fig. 6A) and PARP (Fig. 6B) in the HAβ85 cells, but not in the HAβ18 cells, and suggests that the presence of mutated β-catenin in the HAβ18 cells inhibits NO-induced apoptosis. A recent study also reported similar findings in stably transformed RKO-β-catenin cells (36).

Lithium chloride stabilizes β-catenin and inhibits TNFα-mediated NF-κB activation. As another approach to support our hypothesis, we used lithium chloride (LiCl), a well-known inhibitor of glycogen synthase kinase 3β (GSK-3β), to block β-catenin phosphorylation and degradation. LiCl was added to the carcinoid cell line NCI H727 and dramatically induced β-catenin expression (Supplementary Fig. S2A). LiCl also decreased TNFα-induced gel shift for NF-κB (Supplementary Fig. S2B, lane 4). Finally, LiCl also inhibited TNFα-stimulated p65 staining in cultures of a freshly isolated carcinoid metastasis to the liver from a pancreatic carcinoid primary tumor (Supplementary Fig. S2C).

β-Catenin inversely correlated with iNOS or Fas expression in human liver tumors. Both iNOS and Fas are target genes of the NF-κB pathway, which are also regulated by β-catenin signaling in cancer cells. To determine if there was also an inverse correlation between β-catenin and iNOS or Fas expression in vivo, we stained 25 samples of hepatocellular carcinoma or metastatic colorectal cancer to liver by immunohistochemistry. We observed that β-catenin inversely correlated with iNOS or Fas expression in these tumor tissue samples (Fig. 6C; Supplementary Table S1). Cases #1 and #2 are primary hepatocellular carcinoma tumor samples with opposite findings: In case #1, β-catenin levels are low and the tumor shows strong staining for both iNOS and Fas, whereas in case #2, high β-catenin levels inversely result in low iNOS and Fas expression (Fig. 6C). In 10 hepatocellular carcinoma tumors that were resected, all of the β-catenin–positive tumors were negative for iNOS or Fas staining, whereas the β-catenin–negative tumors stained for both Fas and iNOS (Supplementary Table S1). For the resected hepatic colorectal cancer metastases, 9 of 11 Fas-negative tumors were β-catenin positive whereas all of the Fas-positive tumors were negative for β-catenin staining. Similar findings were observed for iNOS staining.

Previously we identified that the hiNOS (NOS2) gene was regulated by Wnt/β-catenin signaling and identified two TBEs in the hiNOS promoter that accounted for a direct induction of basal and cytokine-induced hiNOS promoter activity dependent on intact TBE (30). However, in the course of those studies, we observed several different human liver and colon cancer cells that actually exhibited decreased iNOS gene expression after cytokine stimulation, which markedly contrasted with primary human and rodent hepatocytes, as well as the findings in several other human cancer cell lines. Because the level of activated β-catenin varies among different cancers (1, 2) and because NF-κB has been shown to have an important role in cytokine-mediated hiNOS transcription (24–26) as well as inflammation-associated cancer (37, 38), we hypothesized that an inverse effect might exist between β-catenin signaling and NF-κB–dependent hiNOS gene transcription. This study presents major and novel findings: (a) β-catenin signaling in tumor cell lines inversely correlated with NF-κB–dependent hiNOS gene expression; (b) overexpression of β-catenin inhibited cytokine-induced hiNOS transcription to below basal levels mediated by β-catenin interaction with NF-κB p50 and p65 proteins; (c) high levels of endogenous β-catenin in tumor cells repressed TNFα-induced NF-κB activation that was reversed by APC overexpression or enhanced by GSK-3β inhibition with LiCl; (d) the inverse relationship of β-catenin and NF-κB–dependent gene expression was not confined to hiNOS and was also seen with other NF-κB–dependent genes such as Traf1 and Fas and; (e) the inverse correlation of β-catenin levels and NF-κB activation was also seen in vivo in primary hepatocellular carcinoma, colorectal cancer metastases, and neuroendocrine cancer.

NF-κB plays a key role in inflammation and carcinogenesis (28, 29). NF-κB is also a central transcription factor mediating TNFα- and IL-1β–induced iNOS gene expression in rat and human hepatocytes (25, 26, 33, 39, 40). Studies indicate that higher levels of iNOS and NO contribute to apoptosis through cytotoxicity (41), whereas lower NO levels may be involved in carcinogenesis (42). NF-κB activation is up-regulated by inflammatory cytokines and growth factors although it is constitutively activated in many cancer cells (43).

Although our previous work showed that the hiNOS gene was directly induced by Wnt/β-catenin signaling (30), we did not examine the relationship between differential β-catenin levels and NF-κB–induced hiNOS expression after cytokine stimulation. Our findings in the current study showed that β-catenin expression indeed reduced cytokine-induced and p65-driven hiNOS-specific NF-κB reporter activity (Fig. 2). Moreover, β-catenin overexpression abolished cytokine-induced hiNOS promoter activity in the HCT116, DLD1, and HepG2 cells. Hence, high levels of β-catenin antagonized cytokine-mediated induction of the NF-κB–dependent hiNOS gene.

By using a pair of cell lines derived from the HCT116 human colon cancer cells, we found that stabilization of β-catenin by mutation in the HAβ18 cells (CTNNB1 −/Δ45) decreased TNFα-induced p50 nuclear translocation, NF-κB p50 DNA binding, and hiNOS-specific NF-κB reporter activity (Fig. 3B–D). In contrast, the absence of β-catenin signaling in the HAβ85 cells produced the opposite results (Fig. 3B–D) and augmented NF-κB–dependent hiNOS and Traf1 protein expression (Fig. 5A). Through these experiments, we can conclude that stabilized expression of β-catenin attenuates hiNOS expression by inhibiting NF-κB pathway signaling.

In addition to testing mutant β-catenin cell lines, we also used SW480 colon cancer cells stably expressing WT APC (SW480-APC) to restore the truncated APC in the endogenous SW480 cells. The SW480-APC cells exhibited decreased β-catenin protein by Western blot and immunoflourescent staining (Fig. 4A and B) and enhanced basal and TNFα-induced hiNOS expression and NF-κB DNA binding activity (Fig. 4A–D). These results also support our finding of an inverse correlation between β-catenin and TNFα-induced NF-κB–dependent gene expression. These results also highlight that mutations in either β-catenin or APC (leading to altered cytosolic β-catenin levels) can modulate NF-κB–dependent target gene expression.

Two other recent studies have shown a relationship between β-catenin and NF-κB signaling (31, 37). Deng and colleagues (31) first showed that β-catenin inhibited NF-κB activity in human colon and breast cancer cell lines; however, they did not examine for β-catenin or NF-κB–dependent gene expression in primary liver cancer specimens. Subsequently, Sun and colleagues (37) reported a cross talk between NF-κB and β-catenin in bacterial-colonized HCT116 intestinal epithelial cells. In this study, constitutively expressed β-catenin indirectly stabilized IκBα and thereby inhibited NF-κB. In our study, we did not find a major change in IκBα levels among cancer cell lines with different degrees of β-catenin expression (Supplementary Fig. S1). Instead, we provided evidence by coimmunoprecipitation for a direct interaction between β-catenin and both p50 and p65 proteins. Neither of these studies determined the effect of β-catenin on hiNOS gene expression. Further, we developed stably transformed SW480 cells overexpressing APC and showed that this resulted in lower β-catenin protein levels, lower β-catenin transcriptional activity, and higher NF-κB DNA binding activity, and subsequently increased hiNOS mRNA and protein expressions in colon cancer cells. This is very important because it shows that manipulation of Wnt signaling by either APC or β-catenin can influence NF-κB–mediated target gene expression. Taken together, the inverse correlation between β-catenin and hiNOS expression indicates that activated β-catenin predominantly controls TNFα-induced hiNOS expression and NO production via interaction between β-catenin and NF-κB in cancer cells.

In contrast, Bandino and colleagues (44) recently reported that EGTA/LiCl inactivation of GSK-3β increased β-catenin and resulted in NF-κB–dependent activation of iNOS in rat hepatocytes. Their experimental design examined cell adherens junction disruption by collagenase liver perfusion and did not address the role of β-catenin signaling in cytokine-stimulated iNOS expression or the direct effects of β-catenin on NF-κB–dependent gene activation in tumor cells.

To strengthen the significance of the findings in the cancer cells in vitro, we also investigated the relationship between β-catenin levels and NF-κB–dependent hiNOS and Fas expression in vivo in resected hepatocellular carcinoma and metastatic colorectal cancer liver tumors. We observed that β-catenin inversely correlated with iNOS or Fas expression in these tumors (Fig. 6C; Supplementary Table S1). The in vivo tumor findings suggest that the cross talk between Wnt/β-catenin and NF-κB signaling occurs in the setting of inflammation-associated liver cancer and has also been reported in breast cancer specimens (45). We acknowledge that this study does not provide correlation between the level of β-catenin/iNOS expression and the aggressiveness of the cancer and overall patient prognosis. Such a study would require long-term follow-up in a larger group of patients.

Our results add insight to the complexity of the Wnt/β-catenin and NF-κB/iNOS/NO signaling pathways involved in carcinogenesis (38, 46–48). Kinzler and Vogelstein (48) defined the important role of Wnt/β-catenin in colon cancer, and Pikarsky and colleagues (38) pointed out that NF-κB functions as a tumor promoter in inflammation-associated cancer. Hofseth and colleagues (11) highlighted the interaction between NO and p53 as a crucial pathway in inflammatory-mediated carcinogenesis, and also reported that cyclooxygenase-2 (COX-2) is a target of Wnt/β-catenin signaling (49). Another study showed that matrix metalloproteinases mediate NO-induced dissociation of β-catenin from membrane-bound E-cadherin and the formation of nuclear β-catenin/LEF-1 complex in mouse colonic epithelial cells (50). Our data show that Wnt/β-catenin signaling in cancer cells can diminish the overproduction of cytokine-induced iNOS, which contributes to apoptosis by NO-mediated cytotoxic mechanisms (41), and is consistent with a study wherein β-catenin overexpression blocked NO-induced apoptosis in colon cancer cells (49). Further efforts will study the working model of Wnt/β-catenin, NF-κB, iNOS/NO, and COX-2 contribution to carcinogenesis.

No potential conflicts of interest were disclosed.

Grant support: NIH grants R01-GM52021/RO1-DK62313 (D.A. Geller).

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