R-ETODOLAC DECREASES BETA-CATENIN LEVELS ALONGWITH SURVIVAL AND PROLIFERATION OF HEPATOMA CELLS

Jaideep Behari1, Gang Zeng2, Wade Otruba1, Michael Thompson2, Peggy Muller2, AmandaMicsenyi2, Sandeep S. Sekhon3, Lorenzo Leoni4, and Satdarshan P. S. Monga1,21 Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213

2 Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213

3 Department of Medicine, Allegheny General Hospital, Pittsburgh, PA 15212

4 Salmedix Inc., San Diego, CA 92121.

AbstractBackground—Inhibition of hepatoma cells by cyclooxygenase (COX)-2 dependent andindependent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, aCOX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drugEtodolac, which lacks COX-inhibitory activity, on the Wnt/β-catenin pathway and human hepatomacells.

Methods—Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examinedfor viability, DNA synthesis, Wnt/β-catenin pathway components, and downstream target geneexpression.

Results—Celecoxib at high doses affected β-catenin protein by inducing its degradation viaGSK3β and APC along with diminished tumor cell proliferation and survival. R-Etodolac atphysiological doses caused decrease in total and activated β-catenin protein secondary to decreasein its gene expression and post-translationally through GSK3β activation. In addition, increased β-catenin-E-cadherin was also observed at the membrane. An associated inhibition of β-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutaminesynthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident.

Conclusion—The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (atphysiological doses) on HCC cells were accompanied by the down-regulation of β-catenindemonstrating a useful therapeutic strategy in hepatocellular cancer.

KeywordsHepatocellular carcinoma; β-catenin; R-Etodolac; Celecoxib; cyclooxygenase 2

Corresponding author: Satdarshan P. S. Monga, M.D. Assistant Professor of Pathology and Medicine, University of Pittsburgh Schoolof Medicine, S421-BST, 200 Lothrop Street, Pittsburgh, PA 15213, Telephone: 412-648-9550; Fax: 412-383-7969; Email: smonga+@pitt.eduGrant Support: Funded by American Cancer Society Grant RSG-03-141-01-CNE and National Institutes of Health Grant 1R01DK62277(S.P.S.M). Also supported by Cleveland Foundation and Rango’s Fund for Enhancement of Pathology Research. JB was supported bya National Institutes of Health (T32) Institutional Training Grant DK063922 (DCW).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

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Published in final edited form as:J Hepatol. 2007 May ; 46(5): 849–857.

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IntroductionHepatocellular carcinoma (HCC) is one of the most common malignancies in the world [1]and its incidence is increasing in the United States and worldwide [2,3]. Thus, there is an urgentneed to increase our understanding of its molecular pathogenesis for improved treatment.

The Wnt/β-catenin signaling pathway plays an important role in liver physiology and itsaberrant activation is observed in HCCs and hepatoblastomas, [4–10]. The mechanisms ofaberrant activation of β-catenin in HCC patients include mutations in the β-catenin gene(CTNNB1) (26–34%) or AXIN1/2 (5%), or upregulation of the frizzled-7 receptor (90%)[11–13]. β-Catenin activation and its role in regulating cell proliferation, makes it an attractivetherapeutic target in HCC.

Previous studies have demonstrated upregulation of the enzyme COX-2 in HCC and a role ofthis enzyme in hepatocarcinogenesis [14,15]. The selective COX-2 inhibitor, Celecoxib, caninhibit the growth and proliferation of HCC cell lines [16]. However, COX-2 inhibition isassociated with cardiovascular and renal side effects [17,18]. In addition COX-2 inhibitorsaffect tumors cells by COX-inhibition-independent mechanisms. Etodolac (1,8-diethyl-1,3,4,9-tetrahydropyrano-[3,4-b] indole-1-acetic acid) is a nonsteroidal anti-inflammatory drug that is marketed as a racemic mixture of the R- and S-enantiomers that arenot metabolically interconvertible [19]. The biochemical and pharmacological effects ofEtodolac are due to the S-enantiomer, while the R-enantiomer lacks COX-inhibitory activity[20]. Therapeutic use of the R-Etodolac, therefore, offers the potential advantage of minimizingCOX-dependent side effects. Antineoplastic effects of R-Etodolac have been recently shownin chronic lymphocytic leukemia (CLL), multiple myeloma, and prostate cancer [21–23]. Here,we demonstrate the anti-β-catenin properties of Celecoxib at high doses and of R-Etodolac atphysiological doses on hepatoma cell lines, which was associated with their diminishedsurvival and proliferation.

Materials and MethodsTissue culture and reagents

Human HCC cell lines HepG2 and Hep3B were obtained from the American Type CultureCollection (Manassas, VA). Cells were cultured in Eagle’s minimum essential medium(EMEM, Cambrex, Walkersville, MD) supplemented with 10% [v/v] fetal calf serum, in anatmosphere containing 5% carbon dioxide at 37°C. Celecoxib (Pfizer, New York, NY), wasdissolved in sterile dimethyl sulfoxide (DMSO) and added to culture medium at finalconcentrations of 10 or 20 μg/ml. Cultures were incubated for 5, 8, and 12 days before beingharvested for analysis.

R-Etodolac (Salmedix, San Diego, CA) was dissolved in sterile DMSO at 250mM immediatelybefore use. Final concentrations of R-Etodolac ranged from 100 to 500μM. Equal concentrationof DMSO alone was added to control cultures. Cell cultures were incubated for 24 or 48 hours.

Cell viability assayEqual number of cells per well were seeded onto 6-well plates and incubated until 60%confluent and treated with R-Etodolac or DMSO for 48h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO), 10% weight/volume in phosphatebuffered saline (PBS) was added to each well. After appropriate incubation, cells were washed,lysed with isopropanol and the solubilized product spectrophotometrically quantified at490nm.

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Proliferation assay with [3H] ThymidineEqual number of cells per well were plated onto 6-well plates and cultured until 60% confluent.Cells were treated with R-Etodolac or DMSO as described above. [3H]Thymidine was addedto the medium for 48h at 2.5μCi/ml. Cells were fixed with 1ml 5% trichloroacetic acid, rinsed,dried and 1ml of 0.33N sodium hydroxide added. [3H]Thymidine-incorporation was measuredin a liquid scintillation counting system (Beckman Coulter, Fullerton, CA).

Light microscopyEqual numbers of cells per well were seeded onto coverslips in 6-well plates. 2ml EMEMmedium with appropriate concentration of DMSO or R-Etodolac was added and platesincubated for 48h. Cells were viewed with a Zeiss Axioskop 40 microscope (20X and 40X).Digital images were acquired with a Nikon Coolpix 4500 digital camera. Collages wereprepared using Adobe Photoshop software.

Apoptosis assayCells were grown on coverslips in 6-well tissue culture plates until 50–60 percent confluentand treated with celecoxib as described above. Apoptotic cells were detected by the terminaldeoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining using thefluorescence method with the APO-BrdU TUNEL assay kit (Invitrogen, Carlsbad, CA)according to manufacturer’s instructions.

RNA isolation and real time PCRTotal cellular RNA was obtained by homogenizing cells in Trizol® reagent (Invitrogen,Carlsbad, CA). The sequences of primer pairs were as follows: Human β-catenin, 5′-TTGTTCAGCTTCTGGGTTCA-3′ (sense) and 5′-ATACCACCCACTTGGCAGAC-3′(antisense); β-actin, 5′-AGGCATCCTCACCCTGAAGTA-3′ (sense) and 5′-CACACGCAGCTCATTGTAGA-3′ (antisense). RT-PCR was performed according toKomoroski et al [24], on a ABI Prism 7000 Sequence Detection System (Applied Biosystems,Foster City, CA) and results normalized to β-actin.

Protein extraction and western blot analysisWestern blot analysis was performed as previously described [7,9]. The following primary IgGantibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Cell Signaling Technology, Danvers,MA; and Abcam Inc., Cambridge, MA) were used in this study (concentrations): mousemonoclonal anti-β-catenin (1:200); mouse monoclonal anti-active-beta-catenin (ser37/thr41-hypophosphorylated-form) (1:200); mouse monoclonal anti-E-cadherin (1:200); mousemonoclonal anti-GSK-3β (1:200); rabbit polyclonal anti-phospho-GSK-3β (Ser9) (1:200);rabbit polyclonal anti-glutamine synthetase (1:200); rabbit polyclonal anti-adenomatouspolyposis coli (APC) (1:100); rabbit anti-cyclin-D1 (1:200); mouse monoclonal anti-actin(1:5000). HRP-conjugated secondary antibodies (Chemicon International Inc., Temecula, CA)was used at concentrations of 1:10,00 to 1:50,000. Blots were visualized with WesternLightning™ chemiluminescence kit (PerkinElmer Life Sciences, Boston, MA).

β-catenin/Tcf Transcription Reporter AssayHepG2 cells were plated in six-well plates, grown to 80–90% confluence, and transientlytransfected with the plasmids TOPFlash and FOPFlash (Upstate Biotechnology, Lake Placid,NY). TOPFlash has three copies of the Tcf/Lef sites upstream of a thymidine kinase (TK)promoter and the firefly luciferase gene. FOPFlash has mutated copies of Tcf/Lef sites and isused as control for measuring nonspecific activation of the reporter. All transfections wereperformed with FuGene HD reagent (Roche, Indianapolis, IN) and 1.8μg of TOPFlash or

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FOPFlash plasmids. To normalize transfection efficiency, cells were co-transfected with0.2μg of the internal control reporter Renilla reniformis luciferase driven under the TKpromoter (pRL-TK; Promega, Madison, WI). Cells were treated with medium with or withoutR-Etodolac (400 μM) 24h after transfection and lysed with reporter lysis buffer (Promega).Luciferase assay was performed using the Dual Luciferase Assay System kit according to themanufacturer’s protocols (Promega). Relative luciferase activity was reported as fold inductionafter normalization for transfection efficiency. Experiments were performed in triplicate.

Immunoprecipitation500μg of protein was utilized for coprecipitation studies as described elsewhere [9]. 30μl ofthe samples were resolved on ready gels and immunoblotting performed as described above.

Immunofluorescence microscopyCells were grown on glass coverslips to 60% confluence, treated with R-Etodolac for varyingtime intervals, washed once with PBS, and fixed in 100% methanol for 3 minutes at −20 °C.Staining was performed as described elsewhere [9]. Nuclei were counterstained by 4′,6-Diamidino-2-phenylindole (DAPI). The coverslips were then placed on slides with a drop ofgelvatol and viewed on a Nikon Eclipse epifluorescence microscope and images obtained ona Sony CCD camera.

Statistical analysisThe autoradiographs were scanned, analyzed by NIH Image 1.58 software. The meanintegrated optical density (IOD) values from at least 3 experiments were compared forstatistical significance by the Student’s two-tailed t test and P<0.05 was considered statisticallysignificant.

ResultsEffect of Celecoxib on HCC cells

To test the effect of Celecoxib on HCC cell proliferation or viability, Hep3B cells were treatedwith 10 or 20 μg/ml of Celecoxib for 8 or 12d. Celecoxib resulted in dramatically sparse culturesas compared to the controls. We then examined thymidine incorporation as a measure of DNAsynthesis and proliferation, which identified a significant decrease (p<0.0001) in the drug-treated cells for 8 (Figure 1A) and 12 days (not shown). TUNEL assay showed several apoptoticnuclei after Celecoxib treatment as early as 2d after treatment (Figure 1B). The increase inapoptosis following Celecoxib treatment was significant (p<0.0001) and existed throughoutthe course of the treatment (Figure 1C).

Effect of Celecoxib on β-catenin levels in HCC cellsIn cholangiocarcinoma cells, celecoxib has been shown to modulate GSK3β and AKT levels[25], which in turn regulate β-catenin activity. Therefore, we investigated the effect ofcelecoxib on β-catenin and its phosphorylation state. β-catenin can be phosphorylated at serineand threonine residues between positions 33 and 45 and phosphorylated β-catenin is targetedfor degradation via the ubiquitin-proeteasome pathway [26].

Hep3B cells have homozygous normal β-catenin gene encoding the wild-type 96 kDa protein.HepG2 cells harbor a heterozygous deletion in exon 3 of the β-catenin gene, which results intwo species of β-catenin protein, the wild-type form and a truncated 75 kDa form. Since thetruncated form of the β-catenin protein lacks the regulatory serine and threonine residuesbetween positions 33 and 45, the truncated protein is resistant to degradation and accumulatesin the cell [27,28].

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β-Catenin suppression has been associated with increased apoptosis and decreasedproliferation during liver development and regeneration [8]. A modest but significant (p<0.01)decrease in β-catenin protein was observed at 8d of Celecoxib treatment (Figure 2A and 2B,left panels). There was a concomitant increase in APC gene product and a mild increase intotal GSK3β (Figure 2A). A dramatic increase in the ser45/thr41-phoshorylated-β-catenin,which is targeted for degradation, was also apparent.

Similar effects on hepatoma cells and β-catenin were observed in HepG2 (Figure 2A and 2B,right panels), with significant decrease in both the wild-type and truncated forms of the protein.GSK3β, a negative regulator of β-catenin, is itself inactivated by phosphorylation at the serineresidue at position 9 [29]. We observed a small increase in total GSK3β level but significantdecrease in its inactive GSK3β (ser9) form. Taken together, these results suggest that in thepresence of Celecoxib, β-catenin protein is targeted for destruction via GSK3β-mediatedphosphorylation.

Effect of R-Etodolac on survival and proliferation of HCC cellsTo test whether R-Etodolac, which lacks COX-2 inhibitory activity, had any antitumor effectsas Celecoxib, we treated Hep3B and HepG2 cells with different drug doses for 48h. Roundedand detached cells along with overall cell sparseness were visible at 48h after R-Etodolactreatment (Figure 3A). In both cell lines, dose-dependent and significant decrease in viabilitywas observed by MTT assay (Figure 3B and 3C), increase in apoptosis by TUNEL assays (notshown), and anti-proliferative effect by thymidine incorporation assays. In Hep3B cells, asignificant decrease in thymidine incorporation was observed at a concentration of 400μM andin HepG2 cells at 100μM (Figure 3D and 3E).

Effect of R-Etodolac on total and activated β-catenin levels and on downstream target genesWestern blot analysis on protein extracts prepared from cells grown in the presence of 400μMR-Etodolac showed dramatic difference in wild type β-catenin protein level in Hep3B cells,and both wild-type and truncated forms of the protein in HepG2 cells at 24h. Significantdecrease was also seen in levels of cyclin-D1 protein, a known β-catenin target during cellproliferation (Figure 4A).

To test whether the active form of β-catenin, which mediates target gene activation, was alsodecreased we performed western blot analysis with a monoclonal antibody specific for theactivated form of β-catenin (lacking phosphorylation at ser37/thr41 residues). Decrease inactivated or ser37/thr41-hypophsophorylated-β-catenin was observed at 24h (Figure 4B).

We then utilized the TOPFlash reporter assay to measure β-catenin/Tcf-dependenttranscriptional activation in R-Etodolac-treated HepG2 cells. Treatment with 400 μM R-Etodolac for 24h showed significantly down-regulated TOPFlash reporter activity, withoutaffecting FOPflash activity (Figure 4C).

Next, we measured the protein levels of another β-catenin target gene glutamine synthetase(GS) by western blotting. We found GS levels to be also significantly decreased in R-Etodolac-treated Hep3B cells (Figure 4D).

Thus a dramatic decrease in total and activated β-catenin levels and expression of its targetgenes is observed in response to R-Etodolac at 24h of treatment, and precedes the biologicalresponses such as diminished cell viability or proliferation.

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Mechanism of β-catenin down-regulation by R-EtodolacTo test the mechanism of decrease in β-catenin protein levels by R-Etodolac, we examined theexpression of CTNNB1 by real-time-PCR at 24h and 48h. In Hep3b cells, there was asignificant decrease in expression at 24h with maximal down-regulation of 4-fold observed by48h. In HepG2 cells there was a more robust 10–13 fold decrease in β-catenin gene expressionobserved at both 24h and 48h after drug treatment (Figure 5A).

Since GSK-3β is crucial in β-catenin phosphorylation and its eventual degradation, totalGSK3β levels were examined. In Hep3B cells, no changes in total GSK3β were evident (Figure5B), however, a decrease in its ser9-phoshorylated (inactive) form was observed after 24hsuggesting a dramatic increase in GSK3β activation in treated cells (Figure 5C).

Effect of R-Etodolac on β-catenin-E-cadherin interactionNext, we asked whether treatment with R-Etodolac altered interaction of β-catenin with E-cadherin at the membrane. Coprecipitation studies showed around 25% increase in β-catenin-E-cadherin association at 24h, despite an overall decrease in total β-catenin protein at this time(Figure 6A and 6C). Thus, R-Etodolac appears to increase association of the remaining β-catenin with E-cadherin. Immunofluorescence microscopy following R-Etodolac treatmentalso showed an increase in membrane localization of β-catenin at 24h (Figure 6B).

DiscussionAberrations in several molecular signaling pathways and mutations in several genes have beendescribed to underlie development of HCC [30]. Several groups have reported mutations inβ-catenin in 26–34% of human HCCs resulting in aberrant nuclear localization of β-catenin[12,27]. Recently, upregulated expression of the Frizzled 7 receptor, has been shown to beassociated with wild-type β-catenin nuclear accumulations in up to 90% HCCs [13]. Successfulknockdown of β-catenin using antisense oligonucleotides or drugs has been associated withdiminished survival and proliferation of tumor cells [29,30]. Taken together, these resultssuggest that upregulated expression and activation of β-catenin is associated with a significantsubset of HCCs, and thus represents an attractive target for therapy. Our study shows that bothCelecoxib and R-Etodolac affect total and activated β-catenin levels with impact onproliferation and survival of hepatoma cells.

The proposed mechanisms of action of COX-2 inhibition in HCC include promotion ofapoptosis and inhibition of angiogenesis and tumor cell migration [16,33,34]. Recently,inhibition of β-catenin by Celecoxib was shown in colon cancer cells [35]. In the current study,Celecoxib at high, non-therapeutic doses showed 2-fold decrease in total β-catenin with anincrease in the ser45/thr41-phosphorylated-β-catenin, a form destined for degradation. Thisappears to be secondary to increased APC protein, which is essential for β-catenin degradation.In addition, this is probably mediated by GSK3β phosphorylation via the AKT pathway, whichis known to phosphorylate β-catenin and hence its degradation [25,36]. Our findings are thusconsistent with these prior studies.

The therapeutic potential of COX-2 agents has been tempered by high dose requirement andassociated serious adverse effects. Thus, it is attractive to consider alternate agents that lackthe adverse consequences of COX-2 inhibition. R-Etodolac has been shown to have antitumoreffects in several cancer cell lines and lacks COX-inhibitory activity [21–23]. Our resultsdemonstrate that R-Etodolac inhibits survival and proliferation of HCC cells lines in a dose-dependent manner and may be potentially useful in HCC treatment or chemoprophylaxis. The400μM concentration of R-Etodolac used in our study has been readily achieved in CLLpatients in phase-II trials without adverse effects [21].

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We employed two widely used human hepatoma cell lines for our studies. R-Etodolacdecreased the levels of wild-type β-catenin in Hep3B cells and both the wild-type and truncatedforms in HepG2 cells. In addition, decreased levels of active-β-catenin were also observed inHep3B cells. Consistent with decreased β-catenin levels, decreased expression of a Tcf bindingsite reporter activity and protein levels of target genes GS and cyclin D1 were seen at 24h.These changes preceded diminished tumor cell proliferation and survival observed at 48h.Again, these findings are consistent with the studies showing β-catenin inhibition to beassociated with decreased survival and proliferation of normal and tumor cells [7,10,37,38]. Itis quite possible that there are additional pathways of action of R-Etodolac against HCC cells,which might be contributing towards the overall decreased viability of proliferation ofhepatoma cells. However, our results suggest a clear effect on β-catenin with ensuing anti-hepatoma phenotype.

A well-described mechanism of β-catenin regulation involves its posttranslational modificationby phosphorylation at serine/threonine residues by GSK-3β, which forms a protein complexwith the proteins APC and axin. While total GSK3β levels were unaltered in response to R-Etodolac, a significant decrease in its inactive form GSK-3β (pSer9) was observed at 24h ofdrug treatment suggesting activation of GSK3β for β-catenin phosphorylation and degradation.This is supported by decreased levels of ser37/thr41-hypophsophorylated-β-catenin andidentifies a mechanism decreased of 96kDa species of β-catenin in Hep3B and HepG2 cells.

Another novel finding of our study is the demonstration of transcriptional inhibition of β-catenin by R-Etodolac as a second mechanism of β-catenin down-regulation. This wasobserved in both cell types and appears to be the mechanism of sustained β-catenin suppressionof 96kDa in both cells and tβ-catenin in HepG2 cells. Whether, R-Etodolac directly inhibitsβ-catenin transcription or acts via inhibition of another transcription factor is not yet known.However, a possible mechanism could be via PPAR-γ transactivation. Gerhold et al identifiedβ-catenin as a negative downstream target of PPAR-γ and that PPAR-γ agonists diminishedγ-catenin expression [39]. In addition, PPAR-γ activation is known to induce GSK3β mediatedβ-catenin degradation as well [40]. Indeed, a recent study has shown R-Etodolac totransactivate PPAR-γ, which might explain its observed negative impact on CTNNB1expression [41]. Finally, our immunoprecipitation and immunofluorescence results indicatethat R-Etodolac enhances the interaction between the cell membrane bound E-cadherin andthe remainder β-catenin after 24h of the treatment and thus may represent an additional modeof anti-β-catenin activity.

Thus R-Etodolac shows distinct modes of β-catenin down-regulation and anti-hepatomaeffects. In hepatoma cells with wild-type βv-catenin, the acute effect appears to be post-translational via GSK3β activation to induce β-catenin degradation and remnant β-cateninmembrane localization in association with gradual affect on its gene expression. In hepatomacells harboring stable β-catenin, the chief mode of action appears to be acute and profoundsuppression of β-catenin gene along with significant decrease in its activity. In conclusion, ourresults suggest that development of therapeutic agents that target β-catenin may be an attractiveapproach to treatment of HCC. Furthermore, drugs like R-Etodolac, which have HCCinhibitory effect independent of COX-inhibition, have potential advantage of avoidingcanonical COX-2-inhibitory adverse effects.

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Figure 1.Effect of Celecoxib on proliferation and survival of Hep3B cells. (A) Thymidine incorporationassays with Hep3B cells treated with DMSO as control or with 10 μg/ml or 20 μg/ml ofCelecoxib control for 8 days. The results represent means + SD from three experiments. (B)Immunofluorescence micrographs showing results of TUNEL assays with Hep3B cells treatedwith 10 mg/ml Celecoxib for 8 days (panels 1–3) or DMSO as control (panels 4–6). Panels1,4, nuclear counterstain with propidium iodide; Panels 2,5, TUNEL staining; Panels 3,6,merged images. Magnification 600x. (C) Results of TUNEL apoptosis assays at 2, 5, and 8days of treatment with Celecoxib. The normalized ratio is the number of TUNEL-positive cellsper high power field in treated samples divided by the number of positive cells per high powerfield in the controls.

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Figure 2.Changes in the levels of β-catenin and Wnt pathway components, induced by Celecoxib. (A)Western blot analysis with protein lysates from Hep3B cells (left panel) and HepG2 cells (rightpanel) treated with DMSO alone (lane C), or with Celecoxib (D1, 10 μg/ml; D2, 20 μg/ml)with antibodies against total β-catenin, Serine-45/Threonine-41 phosphorylated form of β-catenin, total GSK-3β, Ser9-GSK3β, or APC as indicated. The two species of β-catenin proteinin HepG2 cells are shown as β-catenin (wild type 96 kDa form) and tβ-catenin (75 kDatruncated form). The results of β-actin are shown as internal loading control. (B) Densitometricanalysis of western immunoblots, with results of β-catenin normalized to β-actin. Resultsrepresent averages of at least three experiments ± S.D.

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Figure 3.Inhibition of survival and proliferation of HCC cells by R-Etodolac. (A) Light micrographs ofHep3B cells treated with DMSO alone (control), or with varying concentrations of R-Etodolacfor 48 h, showing dose-dependent alteration in cellular morphology. Magnification 600x. (B,C) Results of MTT assays with Hep3B and HepG2 cells treated with varying concentrationsof R-Etodolac for 48 hours. The results represent means of three experiments ± S.D. (* P <0.05). (D, E) Thymidine incorporation assays with Hep3B and HepG2 cells, treated withDMSO control, or with varying concentrations of R-Etodolac for 48 hours, showing dose-dependent inhibition of proliferation. The results are means ± S.D. of three experiments (*P <0.05).

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Figure 4.Effect of R-Etodolac on total and activated β-catenin levels. (A) Western blot analysis withtotal β-catenin antibody and protein extracts from Hep3B and HepG2 cells treated with DMSOalone (lane C) or 400 μM R-Etodolac (lane D) for 24 hours. The 96 kDa wild type β-cateninprotein and the 75 kDa truncated form (tβ-catenin) in HepG2 cells are indicated. Membraneswere sequentially stripped and reprobed with cyclin D1 antibody followed by β-actin as aninternal loading control. (B) Western blot analysis with total and active-β-catenin (ser37/thr41-hypophsophorylated) antibody of protein extracts from Hep3B cells treated R-Etodolac for 24hours. Membranes were stripped and reprobed with β-actin as an internal loading control. (C)TOPFlash reporter assay as a measure of β-Catenin/Tcf-dependent transcriptional activationwas employed in HepG2 cells treated for 24h with or without R-Etodolac (400 μM). Luciferaseactivity in FOPFlash was measured as control for nonspecific activation of the reporter system.A vector containing the Renilla luciferase was used as internal control for transfectionefficiency and the results are expressed as relative Firefly/Renilla luciferase activity. Theresults are mean ± S.D. of three experiments. (D) Western blot analysis with glutaminesynthetase (GS) antibody and protein extracts from Hep3B cells treated with DMSO alone(lane C) or 400 μM R-Etodolac (lane D) for 24 hours. Membranes were sequentially strippedand reprobed with β-actin antibody as an internal loading control.

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Figure 5.Mechanisms of down-regulation of β-catenin by R-Etodolac. (A) Expression levels of theCTNNB1 gene by RT-PCR. Results from Hep3B and HepG2 cells treated with DMSO alone(Ctr) or 400 μM R-Etodolac (Drug) for 24 h or 48 h. Results represent means ± S. D. of threeexperiments. (B) Western blot analysis with total GSK-3β antibody of protein extracts fromHep3B cells treated with DMSO alone (lane C) or 400 μM R-Etodolac (lane D) for 24 h. Themouse monoclonal antibody recognizes both GSK-3β and GSK-3β as indicated in the figurewith arrows. The membrane was stripped and reprobed with β-actin as an internal loadingcontrol. (E) Western blot analysis with the phosphorylated Ser9-GSK-3β antibody of proteinextracts from Hep3B cells treated with DMSO alone (lane C) or 400 μM R-Etodolac (lane D)for 24 h. Membranes were stripped and reprobed with β-actin as an internal loading control.

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Figure 6.Alterations in the β-catenin-E-cadherin interaction in the presence of R-Etodolac. (A)Representative result of immunoprecipitation with β-catenin antibody followed by probing ofmembranes with E-cadherin antibody of protein extracts from Hep3B cells treated with DMSOalone (lanes C) or 400 μM R-Etodolac (lanes D) for 24 h. (B) Immunofluorescence microscopyshowing increased membrane localization of β-catenin in R-Etodolac treated Hep3B cells.Cells were treated with DMSO alone (A–C) or 400 μM R-Etodolac (D–F) for 24 h. A and D,DAPI alone; B and E, β-catenin; C and F, merged images. (C) Densitometric analysis of theE-cadherin immunoblot shown in 5A, normalized to total β-catenin levels, showing increasednormalized association between β-catenin and E-cadherin in R-Etodolac treated cells.

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