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Carcinogenesis, Vol. 23, No. 1, 107-114, January 2002
© 2002 Oxford University Press

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION

Indomethacin induces differential expression of ß-catenin, {gamma}-catenin and T-cell factor target genes in human colorectal cancer cells

Gillian Hawcroft1,3, Mark D'Amico2, Chris Albanese2, Alexander F. Markham, Richard G. Pestell2 and Mark A. Hull1

1 Molecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds LS9 7TF, UK and
2 Department of Medicine and
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Indomethacin-induced G1 arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in ß-catenin protein levels. ß-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. ß-catenin, {gamma}-catenin and T-cell facter (TCF) target gene (cyclin D1, c-MYC and PPAR{delta}) expression was studied following indomethacin treatment of SW480 and HCT116 cells. Cyclin D1 was used as a model TCF target gene for analysis of ß-catenin–TCF-4 DNA binding and trans-activation. Indomethacin treatment was associated with a specific decrease in ß-catenin (but not {gamma}-catenin) expression. Resulting TCF target gene expression was gene specific (cyclin D1, decreased; c-MYC, increased; PPAR{delta}, no significant change). Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a ß-catenin–TCF-4–DNA complex. Indomethacin-induced G1 arrest and apoptosis is associated with specific ß-catenin down-regulation in human CRC cells in vitro. Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.

Abbreviations: APC, adenomatous polyposis coli; COX, cyclooxygenase; CRC, colorectal cancer; CREB, cAMP-responsive element binding protein; CTNNB1, human ß-catenin gene; EMSA, electromobility shift assay; FAP, familial adenomatous polyposis; G6PDH, glucose-6-phosphate dehydrogenase; GSK, glycogen synthase kinase; NSAIDs, non-steroidal anti-inflammatory drugs; PPAR, peroxisome proliferator-activated receptor; PK, protein kinase; TCF, T-cell factor; TK, thymidine kinase; WISP-1, WNT-1 induced secreted protein 1.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Data from colorectal carcinogenesis models in rodents, and multiple human epidemiological studies, suggest that several non-steroidal anti-inflammatory drugs (NSAIDs), including indomethacin, aspirin and sulindac, have anti-colorectal cancer (CRC) activity (1). Indomethacin has potent chemopreventative activity in the ApcMin/+ mouse model of familial adenomatous polyposis (FAP) (2) and in a similar fashion to the closely related NSAID sulindac, prevents or causes regression of rectal polyps in patients with FAP (3). In addition, indomethacin also has anti-neoplastic activity at later stages of colorectal carcinogenesis, decreasing human solid tumour growth in nude mice (4).

It is recognized that NSAIDs including indomethacin can inhibit both isoforms of cyclooxygenase (COX), COX-1 and COX-2 (1). COX-2 is believed to play an important role during murine intestinal tumorigenesis (5) and may represent a target for the anti-CRC activity of NSAIDs (1). More recently, genetic deletion of COX-1 has confirmed a role for this isoform, as well as COX-2, in intestinal tumorigenesis in the ApcMin/+ mouse (6). However, there is also compelling evidence that several NSAIDs may act via mechanisms which are independent of COX inhibition (1,7). For example, indomethacin can directly activate the transcription factor peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (8) which has been associated with growth inhibition of human CRC cell lines (9). In addition, sulindac sulfide has been shown to disrupt DNA binding by another transcription factor, PPAR{delta}, in human CRC cells (10).

In previous work, we have demonstrated that the NSAIDs indomethacin, aspirin and NS-398 induce growth arrest and apoptosis of human CRC cells in vitro (11). The anti-proliferative activity of indomethacin (but not aspirin or the selective COX-2 inhibitor, NS-398) was associated with a dose-dependent decrease in ß-catenin protein expression in several human CRC cell lines (11).

The vertebrate homologue of Drosophila armadillo,ß-catenin, plays a pivotal role in both cell–cell adhesion and WNT signal transduction (12). In normal epithelial cells, ß-catenin is either associated with E-cadherin at adherens junctions or is bound and phosphorylated by a protein complex which includes the adenomatous polyposis coli (APC) protein, glycogen synthase kinase-3ß (GSK-3ß) and conductin/axin, which targets cytosolic ß-catenin for degradation by the ubiquitin-proteasomal pathway. WNT signalling leads to GSK-3ß inhibition, reduced ß-catenin phosphorylation and accumulation of cytosolic ß-catenin (13,14). Mutations of APC (which occur in 85% of human sporadic colorectal adenomas) (15) or activating mutations of the CTNNB1 (ß-catenin) gene also lead to the accumulation of free, cytosolic ß-catenin (14).

Free ß-catenin can associate with members of the TCF (T-cell factor) family of transcription factors (TCF-4 in intestinal epithelial cells). The resulting ß-catenin–TCF complex (14) activates TCF target genes such as cyclin D1 (16), c-MYC (17), PPAR{delta} (10), gastrin (18) and matrilysin/MMP-7 (19). It has been demonstrated that mutant stable ß-catenin can constitutively activate TCF promoter elements and transform cultured cells in vitro (20,21). In vivo, transgenic overexpression of ß-catenin in intestinal epithelial cells leads to development of multiple intestinal adenomas (22). Therefore, ß-catenin can be considered as an oncogene.

The other vertebrate armadillo homologue, {gamma}-catenin (plakoglobin), shares many similarities with ß-catenin, including interactions with cadherins, APC and TCF (13). {gamma}-catenin also promotes cellular transformation in vitro (20,21). However, {gamma}-catenin is less sensitive to ubiquitin-proteasomal regulation than ß-catenin (13) and may differentially regulate known TCF target genes such as c-MYC compared with ß-catenin (21).

Dysregulation of WNT signalling and hence ß-catenin expression is believed to be central to the early stages of sporadic colorectal carcinogenesis in humans (12). Therefore, control of ß-catenin and/or control of downstream TCF target gene expression represents an ideal target(s) for colorectal cancer chemoprevention. Thus, we further investigated the effects of indomethacin on ß- and {gamma}-catenin expression as well as downstream ß-catenin/TCF (WNT) signalling in human CRC cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Cell culture
The human sporadic CRC cell lines SW480 and HCT116 (European Collection of Animal Cell Cultures, Porton Down, UK) as well as the breast cancer cell line SKBR3 (a gift from T.Nolan, University of Manchester, UK) were grown in RPMI medium plus Glutamax® supplemented with 10% (v/v) fetal bovine serum (FBS), 1000 U/ml penicillin and 500 U/ml streptomycin (all Life Technologies, Paisley, UK). All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO2. Cells were routinely sub-cultured using 0.25% (w/v) trypsin/EDTA solution (Life Technologies). Cells were harvested at 80–90% confluency for all experiments.

Drugs and antibodies
Indomethacin (Sigma, Poole, UK) and the selective COX-2 inhibitor, NS-398 (Cayman Chemical, Ann Arbor, MI) were prepared as 100 mM stock solutions in dimethyl sulphoxide (DMSO) obtained from Sigma. Control flasks or plates contained DMSO at an equivalent dilution to that in cultures containing NSAIDs. Lactacystin (Affiniti, Exeter, UK) was prepared as a 3 mM stock solution in sterile, distilled water. Mouse monoclonal anti-human ß-catenin antibodies 6F9 and C19220 were obtained from Sigma and BD Transduction Laboratories (Lexington, KT), respectively. Mouse monoclonal anti-human {gamma}-catenin antibody (C26220) was obtained from Transduction Laboratories. Mouse monoclonal anti-human ß-actin antibody (AC-15) was obtained from Sigma. Mouse monoclonal anti-human cyclin D1 (R-124) and rabbit polyclonal anti-human PKC-ß1 (C-16) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-human TCF-4 (6H5-3) antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-mouse and swine anti-rabbit horseradish peroxidase-conjugated secondary antibodies and goat anti-mouse TRITC-conjugated immunoglobulin were obtained from Dako (Cambridge, UK).

Western blot analysis
Cell monolayers were lysed in 50 mM Tris–HCl buffer (pH 7.2) containing 0.137 M sodium chloride, 1% Brij® 96 (Sigma), 0.2 mM 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF), 1 mM EDTA (Merck, Dorset, UK), 20 µM leupeptin and 1 µM pepstatin (all Sigma). Cell lysates were centrifuged at 15 000 g for 5 min at 4°C through QIAshredder columns (Qiagen, London, UK) and the supernatant used for electrophoresis. Protein concentration was determined using a BioRad DC Protein Assay (BioRad, Hemel Hempstead, UK). SDS–PAGE was performed on 20 µg total protein aliquots together with pre-stained molecular weight standards (Novex, San Diego, CA). Proteins were transferred to Hybond P polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, Amersham, UK) which were then blocked in 5% (w/v) dried skimmed milk powder in PBS for 1 h at 20°C. Membranes were probed with primary antibodies (anti-ß-catenin 6F9, 1/1000; anti-ß-actin, 1/1000; anti-cyclin D1, 1/1000; anti-PKC-ß1, 1/100 or anti-{gamma}-catenin, 1/1000) in PBS plus 5% (w/v) dried skimmed milk powder for 1 h at 20°C. Horseradish peroxidase-conjugated secondary antibodies were diluted 1/5000 in PBS plus 5% (w/v) dried skimmed milk powder and incubated with blots for 1 h at 20°C. Immunoreactive protein was detected using ECL chemiluminescence (Pierce, Chester, UK).

Indirect immunofluorescence
Cells grown on glass coverslips were treated for 24 h with indomethacin or an equivalent dilution of DMSO, under standard culture conditions as described above. Cells were fixed in 100% methanol at –20°C for 5 min and washed twice in PBS. Monolayers were incubated with primary antibody (anti-cyclin D1, 1/500; anti-{gamma}-catenin, 1/200) in PBS plus 1% (w/v) dried skimmed milk powder for 2 h at 20°C. Omission of the respective primary antibody was used as a negative control. Monolayers were incubated with TRITC-conjugated secondary antibody (1/100) in 1% dried skimmed milk in PBS for 1 h at 20°C. Coverslips were mounted in MOWIOL (Calbiochem, La Jolla, CA). Confocal microscopy was performed using a Leica TCS SP laser scanning confocal microscope.

Northern blot analysis
RNA was prepared from cultured cell lines grown in 150 cm2 flasks using RNeasy columns (Qiagen). Total RNA (20 µg) was resolved by electrophoresis in 1% agarose containing 6% formaldehyde and then transferred to ZetaProbe GT Genomic blotting membranes (BioRad). Northern blots were pre-hybridized for 1 h at 65°C in Church buffer (7% SDS, 0.5 M sodium phosphate, pH 7.2). [{alpha}-32P]dCTP labelled probes were added to fresh Church buffer and membranes hybridized overnight at 65°C. ß-actin cDNA probe was obtained from Clontech (Basingstoke, UK), ß-catenin (1 kb), cyclin D1 (1.2 kb), c-MYC (1 kb) and PPAR{delta} (250 bp) cDNA probes were amplified by RT–PCR from SW480 cell total RNA. Primers (Life Technologies) were used as follows: ß-catenin (Genbank accession no. Z19054); sense, dACGAGCTGCTATGTTCCCTG, antisense, d-AGGACAGTACGCACAAGAGC; cyclin D1 (Genbank accession no. M3554); sense, dTA-GGCATCTCTGTACTTTGC, anti-sense, dACCGAACTTAGGTTGAGTAC; c-MYC (Genbank accession no. E01841); sense, dAGAGAAGCTGGCCTCCTACC, antisense, dTCGAGGAGAGGAGAGAATCC and PPAR{delta} (Genbank accession no. AF187850); sense, dATCGAGACATTGTGGCAGGC, antisense, dAGCCTGGGGAAAAGGTGTGC. Membranes were washed under high stringency conditions at 65°C in 0.2x SSC/0.5% (w/v) SDS for 3x20 min. Membranes were stripped in 0.1% (w/v) SDS and reprobed. Densitometric quantification of replicate blots was performed using Quantity One software (BioRad) and adjusted for ß-actin signal intensity.

Transient transfection luciferase reporter assays
Cyclin D1 promoter constructs –1745CDLUC and –1745CDLUCTCFmut have been previously described (23). pRL-TK Renilla luciferase reporter gene plasmid (0.5 µg; Promega, Madison, WI) was always co-transfected to normalize for transfection efficiency. SW480 cells were transiently transfected using Lipofectamine (Gibco). Dual-luciferase reporter assays (Promega) were performed in triplicate as per the manufacturer's instructions.

Electrophoretic mobility-shift assay (EMSA)
SW480 cells were transfected with either empty pCGN vector, an expression vector encoding mutant N-terminal ß-catenin (ß-cat Y33), TCF-4 vector or a dominant negative TCF-4 (TCF-4{Delta}N) vector as previously described (16). The wild-type TCF/LEF site of the cyclin D1 promoter was synthesized as complementary oligodeoxyribonucleotide strands for EMSA (dCTCTGCCGGGCTTTGATCTTTGCTTAACAACA). The wild-type TCF/LEF binding sequence is underlined. Nuclear extracts were obtained and EMSAs were performed after loading 5 µg nuclear extract per lane. The specificity of DNA–protein interactions was established by competition and supershift experiments as described (16). For supershift analysis 0.5 µg anti-ß-catenin C19220, anti-TCF-4 or non-specific IgG was added to the pre-incubation mixture.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
We have previously described a dose-dependent decrease in ß-catenin protein expression [compared with glucose-6-phosphate dehydrogenase (G6PDH) protein expression which remained unchanged] in four human CRC cell lines following indomethacin treatment (100–600 µM) for 72 h (11). Indomethacin-induced apoptosis of human CRC cells, measured by fluorescence microscopy and flow cytometric analysis of DNA content, was apparent by 48 h and was maximal at 72 h (11). Therefore we determined the time course of reduction in ß-catenin protein levels associated with indomethacin treatment of SW480 and HCT116 cells. We used SW480 (mutant APC, wild-type CTNNB1, COX-2-negative) and HCT116 (wild-type APC, mutant CTNNB1, COX-2-negative) cells as these two CRC cell lines mirror most closely the genotype and phenotype of the majority of dysplastic epithelial cells in human sporadic colorectal adenomas (the putative target lesion for CRC chemoprevention) (24). Incubation with 600 µM indomethacin was associated with a significant time-dependent decrease in total immunoreactive ß-catenin levels compared with untreated SW480 and HCT116 cells (Figure 1AGo). A marked reduction in total ß-catenin levels was evident by 24 h in both cell lines. ß-actin protein levels remained unchanged. Therefore, reduction in ß-catenin levels preceded indomethacin-induced apoptosis of human CRC cells. Indomethacin was capable of ß-catenin protein down-regulation in cells with either mutant APC (SW480) or mutant ß-catenin (HCT116), which suggests that this occurred via a mechanism independent of APC-mediated ß-catenin degradation. Dihlmann et al. (25) have recently reported equivocal changes in ß-catenin protein in SW948 CRC cells treated with lower concentrations of indomethacin. However, it was unclear whether the concentrations of indomethacin used in this study induced apoptosis.



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  		Fig. 1. Indomethacin is associated with decreased ß-catenin protein and mRNA levels in SW480 and HCT116 cells. (A) Western blot analysis of ß-catenin (92 kDa) and ß-actin (42 kDa) in SW480 and HCT116 cells after incubation with indomethacin (600 µM) for 24–72 h. C, control; I, indomethacin-treated cells. (B) Northern blot analysis of ß-catenin (3.4 kb) and ß-actin (2.1 kb) mRNA in SW480 and HCT116 cells after 24–48 h incubation with indomethacin (600 µM). C, control; I, indomethacin-treated cells. The blot is representative of three separate experiments. (C) Corresponding densitometric quantitation of ß-catenin mRNA levels compared with the DMSO control, after indomethacin (600 µM) treatment in SW480 and HCT116 cells for 24–48 h. Triplicate experiments are expressed as the mean (+ the standard error of the mean) percentage of control ß-catenin mRNA after correction for ß-actin mRNA levels.

 
Northern blot analysis of ß-catenin mRNA expression also revealed a time-dependent decrease in transcript levels following indomethacin treatment (Figure 1BGo). ß-catenin mRNA levels were decreased ~2-fold in SW480 cells and were almost completely abolished in HCT116 cells after indomethacin treatment for 48 h (Figure 1CGo). As the decrease in ß-catenin protein levels in SW480 and HCT116 cells at 24 h appeared greater than the corresponding reduction in mRNA levels, we tested whether increased ubiquitin-proteasomal degradation contributed to the reduction of ß-catenin protein levels, using the proteasome inhibitor lactacystin. Lactacystin (100 µM) inhibited proteasomal degradation, as demonstrated by increased ß-catenin protein levels in SKBR3 cells (with an intact WNT signalling pathway) treated with lactacystin (Figure 2Go), which is in agreement with previously published data (26). In contrast, addition of lactacystin alone was not associated with increased ß-catenin protein levels in SW480 or HCT116 cells (Figure 2Go). However, in the presence of lactacystin, the indomethacin-induced reduction of ß-catenin levels was diminished in mutant APC-containing SW480 cells (Figure 2Go). This suggests that indomethacin may promote ubiquitin-proteasomal degradation of ß-catenin in SW480 cells in an APC-independent fashion. These data are in keeping with those of Thompson et al. (27) who have demonstrated that the NSAID metabolite sulindac sulfone promoted proteasomal degradation of ß-catenin in SW480 cells via a protein kinase G-dependent mechanism. In contrast, lactacystin did not abrogate indomethacin-induced ß-catenin down-regulation in HCT116 cells (Figure 2Go). Mutation of ß-catenin (Ser45) in HCT116 cells may explain the inability of these cells to utilize APC-independent mechanisms of ß-catenin phosphorylation leading to degradation.



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  		Fig. 2. Inhibition of proteasome activity partially reverses indomethacin-induced down-regulation of ß-catenin in SW480 cells. Western blot analysis of ß-catenin (92 kDa) and ß-actin (42 kDa) in SKBR3, SW480 and HCT116 cells after 24 h incubation with indomethacin (600 µM) and/or lactacystin (100 µM).

 
The concentration range of indomethacin which we used in this and our previous study (100–600 µM) was equivalent to that used in several other studies of the effects of NSAIDs on human CRC cells in vitro (7,10). These concentrations are unlikely to be achieved in human and rodent colorectal mucosa in vivo. However, similar anti-neoplastic properties and effects on ß-catenin expression have been demonstrated in vivo using anti-inflammatory doses of indomethacin and other NSAIDs. Brown et al. (28,29) have reported that indomethacin abrogated dimethylhydrazine (DMH)-induced colon carcinogenesis in rats which was associated with reduced nuclear translocation of ß-catenin. Anti-neoplastic activity in vivo as well as down-regulation of ß-catenin expression in human CRC cells in vitro has also been demonstrated for the closely related NSAID metabolites sulindac sulphide and sulphone (27,30,31).

In order to further determine whether the effects of indomethacin on ß-catenin were specific, the effect of indomethacin on the other armadillo homologue, {gamma}-catenin, was determined in SW480 and HCT116 cells. Subcellular localization of {gamma}-catenin was similar to that previously described for ß-catenin (11,1332). {gamma}-catenin was localized to the cytoplasm and nucleus of SW480 cells (Figure 3AGo). In contrast, prominent membranous {gamma}-catenin staining was observed in HCT116 cells with low cytoplasmic immunoreactivity (Figure 3AGo). Following indomethacin (600 µM) treatment, there was no difference in subcellular localization (Figure 3AGo) or total {gamma}-catenin protein levels (Figure 3BGo), in either SW480 or HCT116 cells. Therefore, despite the similarity in expression patterns and function of the two Armadillo homologues (13), indomethacin treatment of human CRC cells was not associated with down-regulation of {gamma}-catenin protein. It specifically down-regulated ß-catenin alone.



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  		Fig. 3. Indomethacin does not alter {gamma}-catenin protein expression in SW480 or HCT116 cells. (A) Indirect immunofluorescence detection of {gamma}-catenin in SW480 (i and ii) or HCT116 cells (iii and iv) treated with DMSO (i and iii) or indomethacin (600 µM) (ii and iv). Bar, 10 µm. (B) Western blot analysis of {gamma}-catenin (82 kDa) and ß-actin (42 kDa) in SW480 and HCT116 cells following 24 h incubation with indomethacin (600 µM). C, control; I, indomethacin-treated cells.

 
Further evidence that reduction in ß-catenin protein levels was a specific phenomenon and not due to non-specific translational repression was obtained from investigation of the effect of indomethacin on protein kinase C-ß1 (PKC-ß1) expression. Indomethacin-induced apoptosis of human gastric cancer cells has been reported to be mediated by down-regulation of PKC-ß1 expression (33). However, we did not detect any change in PKC-ß1 protein levels following indomethacin treatment of SW480 cells (Figure 4Go). PKC-ß1 was not detectable in HCT116 cells by Western blot analysis. These data not only suggest that PKC-ß1 is not a target for indomethacin in human CRC cells but also provide more evidence, along with the absence of change in ß-actin and G6PDH (11) protein levels, for the specificity of the effects of indomethacin on ß-catenin protein expression in human CRC cells.



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  		Fig. 4. Indomethacin does not alter PKC-ß1 protein levels in SW480 cells. Western blot analysis of PKC-ß1 (83 kDa) and ß-actin (42 kDa) in SW480 cells after 24 h incubation with indomethacin (600 µM). C, control; I, indomethacin-treated cells. The blot is representative of two separate experiments.

 
We have previously noted a reduction in nuclear ß-catenin following indomethacin treatment in SW480 cells and HCT116 cells (11). In order to determine whether down-regulation of nuclear ß-catenin levels was associated with changes in downstream TCF target gene expression, we analysed the transcript levels of three classical TCF target genes cyclin D1, c-MYC and PPAR{delta} which have all been demonstrated to play a role in colorectal carcinogenesis and could therefore represent targets for NSAID chemoprevention (34–36).

In both SW480 and HCT116 cells, indomethacin treatment was associated with decreased cyclin D1 mRNA levels at 24 and 48 h (Figure 5Go). However, indomethacin treatment resulted in a marked increase in c-MYC mRNA levels in both cell lines at 24 h, with a decrease at 48 h in HCT116 cells (Figure 5Go). Indomethacin-induced apoptosis of human gastric cancer cells in vitro has been demonstrated to be associated with increased c-MYC expression (37). In addition, the pro-apoptotic gene BAX, which is a transcriptional target for, and mediator of c-MYC-induced apoptosis (38), has been demonstrated to play a critical role in indomethacin-induced apoptosis of HCT116 cells (39). PPAR{delta} mRNA expression was increased slightly in SW480 cells but remained unchanged in HCT116 cells after indomethacin exposure (Figure 5Go). This is in keeping with data from He et al. (10), who have reported that the NSAID sulindac sulphide did not affect PPAR{delta} mRNA levels in either SW480 or HCT116 cells.



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  		Fig. 5. Differential effects of indomethacin on T-cell factor target gene expression in SW480 and HCT116 cells. Northern blot analysis of cyclin D1 (4.4 kb), c-MYC (1.2 kb), PPAR{delta} (3.3 kb) and ß-actin (2.1 kb) mRNA expression in SW480 and HCT116 cells following 24–48 h incubation with indomethacin (600 µM). C, control; I, indomethacin-treated cells. The blot is representative of three separate experiments.

 
As indomethacin induced G1 arrest of human CRC cells (11), we further analysed cyclin D1 as a model TCF target gene and potential target for the anti-neoplastic activity of indomethacin in human CRC cells. In keeping with the effects on cyclin D1 mRNA levels, cyclin D1 protein expression markedly decreased following indomethacin treatment of both SW480 and HCT116 cells (Figure 6A and BGo). Dihlmann et al. (25) have recently reported that indomethacin was also associated with a reduction of cyclin D1 levels in SW948 cells.



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  		Fig. 6. Indomethacin is associated with decreased cyclin D1 protein expression in SW480 and HCT116 cells. (A) Western blot analysis of cyclin D1 (34 kDa) and ß-actin (42 kDa) protein levels in SW480 or HCT116 cells following 24–72 h incubation with indomethacin (600 µM). C, control; I, indomethacin-treated cells. The blot is representative of three separate experiments. (B) Indirect immunofluorescence for cyclin D1 in SW480 (i and ii) or HCT116 (iii and iv) cells treated with DMSO (i and iii) or indomethacin (600 µM) (ii and iv), for 24 h. Bar, 10 µm.

 
Reduction of synthetic TCF reporter gene activity (pTOPflash) in SW480 cells by indomethacin has recently been described by Dihlmann et al. (25). Therefore, we investigated the effect of indomethacin on cyclin D1 promoter activity. Indomethacin treatment for 24 h was associated with a significant (91%) reduction in activity of the full length cyclin D1 promoter (–1745CDLUC) in SW480 cells (Figure 7AGo). In contrast, the selective COX-2 inhibitor NS-398, which induces a similar degree of growth arrest and apoptosis but without alteration in ß-catenin protein levels (11), was associated with only a 23% decrease in cyclin D1 promoter activity (Figure 7AGo). A single TCF site is required for TCF-dependent trans-activation of the cyclin D1 promoter and this has been shown to bind ß-catenin/TCF (16). Consistent with constitutively active WNT signalling in SW480 cells and the importance of this pathway in regulating cyclin D1 promoter activity, point mutation of the TCF site (–1745CDLUCTCFmut) reduced promoter activity by 75% (Figure 7BGo). However, –1745CDLUCTCFmut retained similar sensitivity to indomethacin as wild type –1745CDLUC suggesting that additional elements in the cyclin D1 promoter also contribute to indomethacin-mediated cyclin D1 repression (Figure 7BGo).



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  		Fig. 7. Indomethacin (but not NS-398) reduces cyclin D1 promoter activity in SW480 cells but is not dependent on a functional cyclin D1 TCF site. (A) –1745 cyclin D1 luciferase reporter (1 µg) was transiently co-transfected with a pRL-TK Renilla luciferase construct (0.5 µg) into SW480 cells treated with indomethacin (600 µM) or NS-398 (75 µM). The ratio of reporter luciferase activity to control Renilla luciferase activity was calculated from triplicate experiments and the data are expressed as the mean (+ the standard error of the mean) percentage activity compared with the appropriate DMSO control. (B) –1745 cyclin D1 luciferase reporter plasmids containing a wild-type (–1745CDLUC) or mutant TCF (–1745CDLUCTCFmut) binding site (1 µg) or control pA3LUC plasmid (1 µg) were transiently co-transfected with a pRL-TK Renilla luciferase construct (0.5 µg) into SW480 cells. Triplicate experiments are expressed as the mean (+ the standard error of the mean) of the ratio of reporter luciferase activity to control Renilla luciferase activity. In both (A) and (B) dual reporter luciferase assays were performed at 24 h after treatment with indomethacin (600 µM) or NS-398 (75 µm).

 
In order to determine whether indomethacin-induced ß-catenin down-regulation affected ß-catenin–TCF binding at the cyclin D1 promoter TCF site, we performed EMSA analysis using SW480 nuclear extracts. A cyclin D1 TCF oligonucleotide probe bound two complexes in control SW480-pCGN cells (Figure 8Go, lane 1, bands A and B) which were increased in SW480 cells expressing mutant stable ß-catenin (SW480-Y33) (Figure 8Go, lane 5, bands A and B). Addition of cold TCF competed with the labelled TCF probe (Figure 8Go, lane 12). Band A was supershifted by ß-catenin antibody (Figure 8Go, lanes 2 and 6). Indomethacin specifically inhibited formation of complexes A and B, in both SW480-pCGN and SW480-Y33 cells, respectively (Figure 8Go, lanes 3 and 7) with no ß-catenin supershift being apparent (Figure 8Go, lanes 4 and 8). In cells overexpressing wild-type TCF-4 (SW480-TCF-4) or a dominant negative mutant TCF (SW480-TCF-4{Delta}N), the cyclin D1 TCF probe also bound two complexes (Figure 8Go, lanes 9 and 14, bands A and B) which supershifted with TCF-4 antibody (Figure 8Go, lanes 10 and 15). However, ß-catenin antibody did not supershift band A in SW480-TCF-4{Delta}N cells (Figure 8Go, lane 16). Control IgG did not supershift bands A or B (Figure 8Go, lane 13). These results suggest that band A consists of a ß-catenin–TCF-4 DNA complex and band B consists of a complex containing TCF-4 but not ß-catenin. Indomethacin inhibited the formation of both ß-catenin/TCF-4-containing and TCF-4-containing complexes at the cyclin D1 TCF site (Figure 8Go, lanes 3 and 7), thus providing direct evidence that indomethacin-induced down-regulation of ß-catenin is associated with inhibition of cyclin D1 expression in a TCF-4 dependent manner in SW480 cells. This can not be explained by decreased TCF-4 levels as it has been demonstrated that TCF-4 protein levels are not affected by indomethacin treatment in these cells (25).



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  		Fig. 8. Indomethacin inhibits the formation of a ß-catenin–cyclin D1 TCF DNA complex. Electrophoretic mobility-shift assays were performed using nuclear extracts prepared from SW480 cells transfected with empty expression vector (pCGN) (lanes 1–4), ß-cat Y33 (lanes 5–8), TCF-4 (lanes 9–13), TCF-4{Delta}N (lanes 14–16) after 24 h indomethacin treatment (600 µM). SS indicates supershifted bands. Band A is a complex containing both TCF-4 and ß-catenin. Band B is a complex containing TCF-4 but not ß-catenin. Antibodies against ß-catenin and TCF-4 plus control IgG were added as indicated. Cold TCF oligonucleotide was used as a competitor.

 
Disruption of ß-catenin–TCF complex formation at the cyclin D1 promoter TCF site occurred (Figure 8Go) but did not fully explain down-regulation of cyclin D1 promoter activity by indomethacin (Figure 7BGo). One possibility is that indomethacin-induced down-regulation of ß-catenin may lead to cyclin D1 repression via a TCF-4-independent as well as a TCF-4-dependent mechanism. Evidence has recently emerged that ß-catenin can trans-activate the proto-oncogene WISP-1(Wnt-1 induced secreted protein 1) by a mechanism that requires the CREB (cAMP-responsive element binding protein) binding site but not the TCF site (40). WNT-independent mechanisms of action of indomethacin on the cyclin D1 promoter are also possible. For example, indomethacin can activate PPAR{gamma} at micromolar concentrations (8) and it has recently been shown that inhibition of MCF-7 breast cancer cell proliferation occurs through PPAR{gamma}-dependent repression of cyclin D1 (41). This suggests the possibility that PPAR{gamma} activation could contribute to down-regulation of cyclin D1 and thus anti-proliferative activity of indomethacin in human CRC cells. In other experiments, we have used a peroxisome proliferator-activated receptor response element-luciferase reporter gene and demonstrated that indomethacin indeed activates PPAR{gamma} in SW480 and HCT116 cells (Hawcroft,G., Hull,M.A., in preparation). This observation also provides confirmatory evidence that the transcriptional repression of cyclin D1 by indomethacin is specific and not explained by generalized transcriptional repression.

Our experiments were not designed to determine whether the anti-neoplastic properties of indomethacin occurred via COX inhibition and/or COX-independent mechanisms of action. However, both of the human CRC cell lines used in our studies expressed COX-1 but not COX-2 (11). Therefore, we can conclude that COX-2 inhibition is not required for ß-catenin down-regulation by indomethacin in human CRC cells. Experiments with transformed COX-null mouse embryonic fibroblasts have provided strong evidence that indomethacin (at concentrations equivalent to those used in our studies) induces apoptosis via a COX-independent mechanism in vitro (7). Derivatives of indomethacin have recently been designed which selectively inhibit COX-2 and have little COX-1 inhibitory activity (42,43). It will be interesting to see whether these indomethacin derivatives (with a much improved GI safety profile in vivo) (43) retain the anti-neoplastic properties of the parental compound.

In conclusion, the anti-neoplastic activity of indomethacin on human CRC cells in vitro was associated with a specific reduction in ß-catenin expression and differential expression of TCF target genes relevant to colorectal carcinogenesis. Indomethacin-induced down-regulation of cyclin D1 occurred by TCF-dependent and TCF-independent mechanisms. In keeping with prior knowledge of the complex pharmacological modes of action of indomethacin (COX-1 and COX-2 inhibition, PPAR{gamma} activation), this particular NSAID appears to modulate catenin expression and WNT signalling, and hence modulate genes critical for colorectal carcinogenesis, by more than one mechanism.


   Notes
 
3 To whom correspondence should be addressed Email: medgha{at}stjames.leeds.ac.uk Back


   Acknowledgments
 
We acknowledge the kind assistance of the Molecular Medicine Unit Confocal Imaging Group (University of Leeds) with the immunofluorescence studies. This research was supported by Yorkshire Cancer Research, The West Riding Research Trust (G.H.), NYS CO15706 (M.D.), ROL CA70897, ROICA75503, ROLCA77552, the Komen Foundation, Breast Cancer Alliance Inc. and Cancer Center Core National Institute of Health grant S-P30-CA13330-26 (R.G.P). M.A.H currently holds an MRC (UK) Clinician Scientist Fellowship.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received August 24, 2001; revised October 3, 2001; accepted October 15, 2001.


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