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. Author manuscript; available in PMC: 2008 Apr 30.
Published in final edited form as: J Hepatol. 2006 Dec 21;46(5):849–857. doi: 10.1016/j.jhep.2006.11.017

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

Jaideep Behari 1, Gang Zeng 2, Wade Otruba 1, Michael Thompson 2, Peggy Muller 2, Amanda Micsenyi 2, Sandeep S Sekhon 3, Lorenzo Leoni 4, Satdarshan P S Monga 1,2
PMCID: PMC1924913  NIHMSID: NIHMS22414  PMID: 17275129

Abstract

Background

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

Methods

Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/β-catenin pathway components, and downstream target gene expression.

Results

Celecoxib at high doses affected β-catenin protein by inducing its degradation via GSK3β and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated β-catenin protein secondary to decrease in 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 glutamine synthetase 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 (at physiological doses) on HCC cells were accompanied by the down-regulation of β-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer.

Keywords: Hepatocellular carcinoma, β-catenin, R-Etodolac, Celecoxib, cyclooxygenase 2

Introduction

Hepatocellular 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 urgent need 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 its aberrant activation is observed in HCCs and hepatoblastomas, [4–10]. The mechanisms of aberrant 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 attractive therapeutic target in HCC.

Previous studies have demonstrated upregulation of the enzyme COX-2 in HCC and a role of this enzyme in hepatocarcinogenesis [14, 15]. The selective COX-2 inhibitor, Celecoxib, can inhibit the growth and proliferation of HCC cell lines [16]. However, COX-2 inhibition is associated with cardiovascular and renal side effects [17, 18]. In addition COX-2 inhibitors affect 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 are not metabolically interconvertible [19]. The biochemical and pharmacological effects of Etodolac 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 minimizing COX-dependent side effects. Antineoplastic effects of R-Etodolac have been recently shown in 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 at physiological doses on hepatoma cell lines, which was associated with their diminished survival and proliferation.

Materials and Methods

Tissue culture and reagents

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

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

Cell viability assay

Equal 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 phosphate buffered saline (PBS) was added to each well. After appropriate incubation, cells were washed, lysed with isopropanol and the solubilized product spectrophotometrically quantified at 490nm.

Proliferation assay with [3H] Thymidine

Equal 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 added to 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 measured in a liquid scintillation counting system (Beckman Coulter, Fullerton, CA).

Light microscopy

Equal numbers of cells per well were seeded onto coverslips in 6-well plates. 2ml EMEM medium with appropriate concentration of DMSO or R-Etodolac was added and plates incubated 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 were prepared using Adobe Photoshop software.

Apoptosis assay

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

RNA isolation and real time PCR

Total 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 to Komoroski 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 analysis

Western blot analysis was performed as previously described [7, 9]. The following primary IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; Cell Signaling Technology, Danvers, MA; and Abcam Inc., Cambridge, MA) were used in this study (concentrations): mouse monoclonal anti-β-catenin (1:200); mouse monoclonal anti-active-beta-catenin (ser37/thr41-hypophosphorylated-form) (1:200); mouse monoclonal anti-E-cadherin (1:200); mouse monoclonal anti-GSK-3β (1:200); rabbit polyclonal anti-phospho-GSK-3β (Ser9) (1:200); rabbit polyclonal anti-glutamine synthetase (1:200); rabbit polyclonal anti-adenomatous polyposis 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 Western Lightning™ chemiluminescence kit (PerkinElmer Life Sciences, Boston, MA).

β-catenin/Tcf Transcription Reporter Assay

HepG2 cells were plated in six-well plates, grown to 80–90% confluence, and transiently transfected 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 is used as control for measuring nonspecific activation of the reporter. All transfections were performed with FuGene HD reagent (Roche, Indianapolis, IN) and 1.8μg of TOPFlash or FOPFlash plasmids. To normalize transfection efficiency, cells were co-transfected with 0.2μg of the internal control reporter Renilla reniformis luciferase driven under the TK promoter (pRL-TK; Promega, Madison, WI). Cells were treated with medium with or without R-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 the manufacturer’s protocols (Promega). Relative luciferase activity was reported as fold induction after normalization for transfection efficiency. Experiments were performed in triplicate.

Immunoprecipitation

500μg of protein was utilized for coprecipitation studies as described elsewhere [9]. 30μl of the samples were resolved on ready gels and immunoblotting performed as described above.

Immunofluorescence microscopy

Cells were grown on glass coverslips to 60% confluence, treated with R-Etodolac for varying time 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 of gelvatol and viewed on a Nikon Eclipse epifluorescence microscope and images obtained on a Sony CCD camera.

Statistical analysis

The autoradiographs were scanned, analyzed by NIH Image 1.58 software. The mean integrated optical density (IOD) values from at least 3 experiments were compared for statistical significance by the Student’s two-tailed t test and P<0.05 was considered statistically significant.

Results

Effect of Celecoxib on HCC cells

To test the effect of Celecoxib on HCC cell proliferation or viability, Hep3B cells were treated with 10 or 20 μg/ml of Celecoxib for 8 or 12d. Celecoxib resulted in dramatically sparse cultures as compared to the controls. We then examined thymidine incorporation as a measure of DNA synthesis 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 apoptotic nuclei after Celecoxib treatment as early as 2d after treatment (Figure 1B). The increase in apoptosis following Celecoxib treatment was significant (p<0.0001) and existed throughout the course of the treatment (Figure 1C).

Figure 1.

Figure 1

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Effect of Celecoxib on proliferation and survival of Hep3B cells. (A) Thymidine incorporation assays with Hep3B cells treated with DMSO as control or with 10 μg/ml or 20 μg/ml of Celecoxib control for 8 days. The results represent means + SD from three experiments. (B) Immunofluorescence micrographs showing results of TUNEL assays with Hep3B cells treated with 10 mg/ml Celecoxib for 8 days (panels 1–3) or DMSO as control (panels 4–6). Panels 1,4, 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 8 days of treatment with Celecoxib. The normalized ratio is the number of TUNEL-positive cells per high power field in treated samples divided by the number of positive cells per high power field in the controls.

Effect of Celecoxib on β-catenin levels in HCC cells

In 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 of celecoxib on β-catenin and its phosphorylation state. β-catenin can be phosphorylated at serine and threonine residues between positions 33 and 45 and phosphorylated β-catenin is targeted for 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 in two species of β-catenin protein, the wild-type form and a truncated 75 kDa form. Since the truncated form of the β-catenin protein lacks the regulatory serine and threonine residues between positions 33 and 45, the truncated protein is resistant to degradation and accumulates in the cell [27, 28].

β-Catenin suppression has been associated with increased apoptosis and decreased proliferation 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 in total GSK3β (Figure 2A). A dramatic increase in the ser45/thr41-phoshorylated-β-catenin, which is targeted for degradation, was also apparent.

Figure 2.

Figure 2

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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 (right panel) 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 protein in HepG2 cells are shown as β-catenin (wild type 96 kDa form) and tβ-catenin (75 kDa truncated form). The results of β-actin are shown as internal loading control. (B) Densitometric analysis of western immunoblots, with results of β-catenin normalized to β-actin. Results represent averages of at least three experiments ± S.D.

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 serine residue at position 9 [29]. We observed a small increase in total GSK3β level but significant decrease in its inactive GSK3β (ser9) form. Taken together, these results suggest that in the presence of Celecoxib, β-catenin protein is targeted for destruction via GSK3β-mediated phosphorylation.

Effect of R-Etodolac on survival and proliferation of HCC cells

To test whether R-Etodolac, which lacks COX-2 inhibitory activity, had any antitumor effects as Celecoxib, we treated Hep3B and HepG2 cells with different drug doses for 48h. Rounded and detached cells along with overall cell sparseness were visible at 48h after R-Etodolac treatment (Figure 3A). In both cell lines, dose-dependent and significant decrease in viability was observed by MTT assay (Figure 3B and 3C), increase in apoptosis by TUNEL assays (not shown), and anti-proliferative effect by thymidine incorporation assays. In Hep3B cells, a significant decrease in thymidine incorporation was observed at a concentration of 400μM and in HepG2 cells at 100μM (Figure 3D and 3E).

Figure 3.

Figure 3

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Inhibition of survival and proliferation of HCC cells by R-Etodolac. (A) Light micrographs of Hep3B cells treated with DMSO alone (control), or with varying concentrations of R-Etodolac for 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 concentrations of 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 with DMSO 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).

Effect of R-Etodolac on total and activated β-catenin levels and on downstream target genes

Western blot analysis on protein extracts prepared from cells grown in the presence of 400μM R-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. Significant decrease was also seen in levels of cyclin-D1 protein, a known β-catenin target during cell proliferation (Figure 4A).

Figure 4.

Figure 4

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Effect of R-Etodolac on total and activated β-catenin levels. (A) Western blot analysis with total β-catenin antibody and protein extracts from Hep3B and HepG2 cells treated with DMSO alone (lane C) or 400 μM R-Etodolac (lane D) for 24 hours. The 96 kDa wild type β-catenin protein and the 75 kDa truncated form (tβ-catenin) in HepG2 cells are indicated. Membranes were sequentially stripped and reprobed with cyclin D1 antibody followed by β-actin as an internal 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 24 hours. 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 activation was employed in HepG2 cells treated for 24h with or without R-Etodolac (400 μM). Luciferase activity 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 transfection efficiency and the results are expressed as relative Firefly/Renilla luciferase activity. The results are mean ± S.D. of three experiments. (D) Western blot analysis with glutamine synthetase (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 stripped and reprobed with β-actin antibody as an internal loading control.

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

We then utilized the TOPFlash reporter assay to measure β-catenin/Tcf-dependent transcriptional activation in R-Etodolac-treated HepG2 cells. Treatment with 400 μM R-Etodolac for 24h showed significantly down-regulated TOPFlash reporter activity, without affecting 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 target genes is observed in response to R-Etodolac at 24h of treatment, and precedes the biological responses such as diminished cell viability or proliferation.

Mechanism of β-catenin down-regulation by R-Etodolac

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

Figure 5.

Figure 5

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Mechanisms of down-regulation of β-catenin by R-Etodolac. (A) Expression levels of the CTNNB1 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 three experiments. (B) Western blot analysis with total GSK-3β antibody of protein extracts from Hep3B cells treated with DMSO alone (lane C) or 400 μM R-Etodolac (lane D) for 24 h. The mouse monoclonal antibody recognizes both GSK-3β and GSK-3β as indicated in the figure with arrows. The membrane was stripped and reprobed with β-actin as an internal loading control. (E) Western blot analysis with the phosphorylated Ser9-GSK-3β antibody of protein extracts 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.

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

Effect of R-Etodolac on β-catenin-E-cadherin interaction

Next, 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 treatment also showed an increase in membrane localization of β-catenin at 24h (Figure 6B).

Figure 6.

Figure 6

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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 of membranes with E-cadherin antibody of protein extracts from Hep3B cells treated with DMSO alone (lanes C) or 400 μM R-Etodolac (lanes D) for 24 h. (B) Immunofluorescence microscopy showing 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 the E-cadherin immunoblot shown in 5A, normalized to total β-catenin levels, showing increased normalized association between β-catenin and E-cadherin in R-Etodolac treated cells.

Discussion

Aberrations in several molecular signaling pathways and mutations in several genes have been described to underlie development of HCC [30]. Several groups have reported mutations in β-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 be associated with wild-type β-catenin nuclear accumulations in up to 90% HCCs [13]. Successful knockdown of β-catenin using antisense oligonucleotides or drugs has been associated with diminished survival and proliferation of tumor cells [29, 30]. Taken together, these results suggest that upregulated expression and activation of β-catenin is associated with a significant subset of HCCs, and thus represents an attractive target for therapy. Our study shows that both Celecoxib and R-Etodolac affect total and activated β-catenin levels with impact on proliferation and survival of hepatoma cells.

The proposed mechanisms of action of COX-2 inhibition in HCC include promotion of apoptosis 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 an increase in the ser45/thr41-phosphorylated-β-catenin, a form destined for degradation. This appears 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, which is known to phosphorylate β-catenin and hence its degradation [25, 36]. Our findings are thus consistent with these prior studies.

The therapeutic potential of COX-2 agents has been tempered by high dose requirement and associated serious adverse effects. Thus, it is attractive to consider alternate agents that lack the adverse consequences of COX-2 inhibition. R-Etodolac has been shown to have antitumor effects in several cancer cell lines and lacks COX-inhibitory activity [21–23]. Our results demonstrate 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. The 400μM concentration of R-Etodolac used in our study has been readily achieved in CLL patients in phase-II trials without adverse effects [21].

We employed two widely used human hepatoma cell lines for our studies. R-Etodolac decreased the levels of wild-type β-catenin in Hep3B cells and both the wild-type and truncated forms in HepG2 cells. In addition, decreased levels of active-β-catenin were also observed in Hep3B cells. Consistent with decreased β-catenin levels, decreased expression of a Tcf binding site 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 be associated with decreased survival and proliferation of normal and tumor cells [7, 10, 37, 38]. It is 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 of hepatoma 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 modification by phosphorylation at serine/threonine residues by GSK-3β, which forms a protein complex with 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 of drug treatment suggesting activation of GSK3β for β-catenin phosphorylation and degradation. This is supported by decreased levels of ser37/thr41-hypophsophorylated-β-catenin and identifies 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 was observed in both cell types and appears to be the mechanism of sustained β-catenin suppression of 96kDa in both cells and tβ-catenin in HepG2 cells. Whether, R-Etodolac directly inhibits β-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 identified β-catenin as a negative downstream target of PPAR-γ and that PPAR-γ agonists diminished γ-catenin expression [39]. In addition, PPAR-γ activation is known to induce GSK3β mediated β-catenin degradation as well [40]. Indeed, a recent study has shown R-Etodolac to transactivate PPAR-γ, which might explain its observed negative impact on CTNNB1 expression [41]. Finally, our immunoprecipitation and immunofluorescence results indicate that R-Etodolac enhances the interaction between the cell membrane bound E-cadherin and the remainder β-catenin after 24h of the treatment and thus may represent an additional mode of anti-β-catenin activity.

Thus R-Etodolac shows distinct modes of β-catenin down-regulation and anti-hepatoma effects. 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-catenin membrane localization in association with gradual affect on its gene expression. In hepatoma cells harboring stable β-catenin, the chief mode of action appears to be acute and profound suppression of β-catenin gene along with significant decrease in its activity. In conclusion, our results suggest that development of therapeutic agents that target β-catenin may be an attractive approach to treatment of HCC. Furthermore, drugs like R-Etodolac, which have HCC inhibitory effect independent of COX-inhibition, have potential advantage of avoiding canonical COX-2-inhibitory adverse effects.

Footnotes

Grant 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 by a National Institutes of Health (T32) Institutional Training Grant DK063922 (DCW).

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