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Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells

Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany

¹Correspondence: Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe Universität Theodor Stern Kai 7, Frankfurt/Main 60590, Germany. E-mail: groesch{at}em.uni-frankfurt.de

SPECIFIC AIMS

The present study is aimed at investigating molecular pathways contributing to the anticarcinogenic effects of celecoxib in human colon carcinoma cells. Due to its pivotal role in colorectal carcinogenesis, we focused on the APC/ß-catenin-signaling pathway as a possible target of celecoxib.

Principal findings

1. Celecoxib affected the protein levels of ß-catenin and e-cadherin in human Caco-2 colon carcinoma cells
Beta-catenin and e-cadherin are components of the cell adherens complex in the cell membrane. Nuclear ß-catenin has been shown to interact with DNA-bound TCF or lef proteins to activate the transcription of several target genes, such as Cyclin D1 and COX-2. Depending on the presence of several cellular signals, newly synthesized ß-catenin can either be translocated into the cell membrane or the nuclei or directed to cytoplasmatic degradation. To address the question of whether or not interference with the APC/ß-catenin pathway may contribute to the anticarcinogenic effects of celecoxib, we treated human Caco-2 cells with 100 µM celecoxib for various periods and analyzed the protein levels of ß-catenin and e-cadherin in different cellular fractions. Western blot analysis of soluble extracts (Fig. 1 A) revealed a time-dependent accumulation of ß-catenin between 0–6 h, whereas further incubation for 8 h and 16 h resulted in strong degradation of the protein. Membrane ß-catenin levels decreased by 2 h after celecoxib treatment, whereas a reduction in membrane e-cadherin signal was not seen before 16 h of drug incubation (Fig. 1B ). To confirm the specificity of celecoxib-induced ß-catenin degradation, the cells were treated with increasing concentrations of celecoxib (0–100 µM) for 20 h. Moderately reduced ß-catenin protein levels were already observed at concentrations of 60 µM celecoxib, whereas clear degradation of ß-catenin was seen at concentrations of 80 µM and 100 µM celecoxib (Fig. 1C ). Furthermore, we observed increased nuclear ß-catenin levels between 0.5 and 4 h of treatment with 100 µM celecoxib (Fig. 1D ).

View larger version (36K): [in this window] [in a new window]   Figure 1. Western blot analysis of (A) soluble and (B) membrane ß-catenin protein levels after treatment of Caco-2 cells with 100 µM celecoxib for the indicated time periods. C) Western blot analysis of soluble ß-catenin protein levels after treatment of the cells with increasing concentrations of celecoxib for 20 h. D) Western blot analysis of nuclear ß-catenin protein levels after treatment of Caco-2 cells with 100 µM celecoxib for the indicated periods. ERK-2 or ponceau staining was used as a loading control.

This nuclear translocation of ß-catenin was not associated with increased TCF/lef-DNA binding activity as assessed by using electromobility shift assays (EMSA). The DNA binding activity clearly decreased already 2 h after celecoxib treatment (Fig. 2 A). To identify the components of the ß-catenin DNA binding complex, we performed supershift experiments using antibodies directed against TCF-4, TCF-1, and lef-1 (Fig. 2B ). Supershift signals were obtained using anti-lef-1 and anti-TCF-1 antibodies, whereas the anti-TCF-4 antibody and the p-c-jun antibody as an unspecific control did not supershift any binding signals. The specificity of the ß-catenin/TCF/lef signals was confirmed by performing unlabeled probe competition experiments (Fig. 2B ).

View larger version (64K): [in this window] [in a new window]   Figure 2. A) Determination of ß-catenin DNA binding activity after treatment of Caco-2 cells with 100 µM celecoxib using electrophoretic mobility shift assay. B) Unlabeled probe competition and supershift experiments with untreated Caco-2 nuclear extracts using antibodies directed against lef-1, TCF-1, TCF-4, and p-c-jun.

2. Celecoxib treatment affected the cellular distribution of ß-catenin
Next, we analyzed the mechanisms leading to the initial (2–6 h) increase of ß-catenin protein levels in soluble cell extracts after celecoxib treatment. To investigate whether new protein synthesis might be the cause of this increase, we preincubated Caco-2 cells with 10 µg/mL cycloheximide and 5 µg/mL actinomycin D for 30 min. Even in the presence of these agents, the celecoxib-induced initial increase in ß-catenin protein level was not inhibited, indicating that this effect is not due to new protein synthesis, but might be explained by translocation of ß-catenin from the outer cell membrane into the cytoplasm and the nuclei.

3. Celecoxib-induced degradation of ß-catenin is caused by both the proteasomal pathway and caspases
We analyzed the mechanisms leading to the degradation of ß-catenin 8, 10, and 16 h after treatment of Caco-2 cells with 100 µM celecoxib using Western blot analysis. Such concentrations of celecoxib were already described to induce apoptosis in different human cancer cell lines. To confirm this for Caco-2 cells, we assessed PARP (poly (ADP) -ribose polymerase) cleavage that occurred 8 h after treatment. Beta-catenin was described to be degraded either by the proteasomal pathway or by caspases owing to induction of apoptosis. To investigate the involvement of these pathways, we preincubated the cells for 30 min with the irreversible broad range caspase inhibitor Boc-D -FMK (100 µM) or the selective proteasome inhibitor lactacystine (10 µM). Lactacystine, and to a greater extent the caspase inhibitor, partly blocked the celecoxib-induced degradation of ß-catenin. In parallel, as expected, the caspase inhibitor prevented the cleavage of PARP, whereas lactacystine did not retard the progression of apoptosis. These experiments point out that the decrease of ß-catenin protein level after celecoxib treatment is on the one hand due to cleavage by caspases and on the other hand mediated by proteasomal degradation.

For the proteasomal degradation, ß-catenin is incorporated into a large complex including the APC protein, GSK-3ß-kinase and other proteins allowing ß-catenin to be phosphorylated by the GSK-3ß-kinase. Therefore, we determined the activity status of GSK-3ß after celecoxib treatment by analyzing phospho-GSK-3ß (inactive form) and total GSK-3ß levels using the Western blot method. Phospho-GSK-3ß levels declined in Caco-2 cells 2 h after celecoxib treatment, whereas the total-GSK-3ß protein levels remained unchanged. To investigate whether GSK-3ß activity has an impact on ß-catenin degradation after celecoxib treatment, we pretreated Caco-2 cells for 30 min with the selective GSK-3ß inhibitor lithiumchloride using a concentration of 20 mM and subsequently with 100 µM celecoxib for 4 and 6 h. The presence of LiCl prevented the early membrane ß-catenin degradation after celecoxib treatment. To gain more insight into the role of GSK-3ß and the proteasomal pathway in the celecoxib-induced degradation of ß-catenin, we used the human colon carcinoma cell line HCT-116 expressing mutated ß-catenin. Treatment of HCT-116 cells with 100 µM celecoxib for various periods led to moderate reduction in ß-catenin protein levels after 10 h in soluble extracts whereas clear degradation was not observed both in soluble and membrane extracts before 16 h of incubation. The early increase of ß-catenin levels in soluble extracts, observed in Caco-2 cells after celecoxib treatment did not occur in HCT 116 cells.

Recently, caspase-3 and to a very low extent caspase-6 and -7 were shown to directly cleave the ß-catenin protein. To determine the extent to which ß-catenin degradation after celecoxib treatment is up to caspase-3-dependent mechanisms, we used MCF-7 breast cancer cells, which are caspase-3 depleted. Treatment of these cells with 100 µM celecoxib for various periods led to moderately decreased protein levels of ß-catenin in soluble extracts between 6 and 24 h of treatment. An early increase of ß-catenin protein level in soluble extracts after 2–4 h was not as pronounced as with Caco-2 cells. In parallel, we determined PARP cleavage, which did not occur in MCF-7 cells due to the lack of caspase-3, -6, and -7 cleavage activities. Similar to the results obtained from Caco-2 cells, Western blot analysis of the respective membrane extracts of MCF-7 cells showed a clear decrease of ß-catenin levels 4 h after celecoxib treatment. The experiments suggest that caspases and the proteasomal pathway independently mediate the degradation of ß-catenin after celecoxib treatment since degradation was observed in both HCT-116 and MCF-7 cells (Fig. 3 ). To demonstrate that ß-catenin degradation is not cell type-restricted, we treated human LNCAP prostate cancer cells with 100 µM celecoxib for various periods and observed decreased ß-catenin protein levels after 24 h of drug incubation. Thus, ß-catenin degradation was seen in four different tumor cell types (Caco-2, HCT-116, MCF-7, and LNCAP), indicating that a cell type-specific celecoxib-induced degradation of ß-catenin can be ruled out.

View larger version (42K): [in this window] [in a new window]   Figure 3. Molecular mechanisms by which celecoxib exerts its anticarcinogenic effects in human colon carcinoma cells independently of COX-2 inhibition. We were able to show that celecoxib treatment causes dephosphorylation and activation of GSK-3ß, which then phosphorylates ß-catenin, thereby triggering its translocation from the cell membrane into the cytoplasm and the nuclei. The activation of GSK-3ß may be directly associated with celecoxib's ability to inhibit the PDK-1 and Akt-Kinase, as described in the literature. The translocated nuclear ß-catenin was shown to lack DNA binding activity. Both celecoxib-induced caspase-3 and the proteasomal pathway then degradate-free cytoplasmatic ß-catenin. C, -catenin; BC, ß-catenin; GSK-3ß, glycogen synthase kinase-3ß; lef-1, lymphoid enhancer factor-1; TCF, T cell-factor.

4. Rofecoxib and R-flurbiprofen had no effect on ß-catenin expression in Caco-2 cells
To finally answer the question of celecoxib’s specificity to cause ß-catenin degradation, we treated Caco-2 cells with two other NSAIDs differing in their ability to induce apoptosis or inhibit cyclooxygenase-2. The NSAID R-flurbiprofen was described to show strong antiproliferative effects in vivo and in vitro but lacks COX-2 inhibitory activity. Treatment of Caco-2 cells with 1000 µM R-flurbiprofen for up to 24 h did not affect the protein level of ß-catenin but caused PARP-cleavage after 16 and 24 h of incubation. Rofecoxib was shown to have only a weak antiproliferative effect in various cell lines, but is one of the most selective COX-2 inhibitors available. In line with these findings no induction of apoptosis, as determined by the lack of PARP-cleavage or down-regulation of ß-catenin level was observed after treatment of Caco-2 cells with up to 100 µM rofecoxib. These results clearly indicate that celecoxib mediated ß-catenin degradation is neither an unspecific effect owing to induction of apoptosis in general nor a consequence of COX-2 inhibition, but seems to be specific for celecoxib.

CONCLUSIONS AND SIGNIFICANCE

In conclusion, we have obtained evidence that targeting the ß-catenin/APC signaling pathway may be a novel approach to explain the COX-2-independent anticarcinogenic effects of celecoxib. The importance of these findings for the in vivo use of celecoxib becomes apparent considering that strong nuclear or cytoplasmatic ß-catenin staining in colorectal cancer tissues correlates with more invasive tumor growth, a higher susceptibility of disease recurrence after surgery, and a lower survival rate.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3274fje;

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