DNA array analysis of the effects of aspirin on colon cancer cells: involvement of Rac1

James C. H. Hardwick1, Marije van Santen, Gijs R. van den Brink, Sander J. H. van Deventer and Maikel P. Peppelenbosch

Department of Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands

1 To whom correspondence should be addressed Email: j.c.hardwick{at}amc.uva.nl


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aspirin and other non-steroidal anti-inflammatory drugs show efficacy in the prevention of colon cancer. The mechanism by which they do this is unclear. We used a commercially available DNA microarray to study changes in gene expression in 1176 cancer related genes in the HT29 colon cancer cell line induced by aspirin. Overall we find more genes that are significantly induced than are repressed. The pattern of gene expression changes is different at high concentrations of aspirin (5 mM) than at lower levels (500 and 50 µM). Genes involved in DNA damage signaling, nucleotide metabolism and the stress response are induced, and cell cycle related genes repressed. The small GTPase Rac1 is highly induced and this was confirmed by immunoblotting. We show using immunohistochemistry that Rac1 is expressed in mature colonocytes at the intercrypt table in human and mouse colon tissue. These results support the previous findings that aspirin has different actions at high concentrations than at low concentrations and further show the use of DNA array technology in the investigation of drug mechanisms of action. Furthermore, they point towards a role for Rac1 in the action of aspirin in colon cancer.

Abbreviations: COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs; PBS, phosphate buffered saline


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-steroidal anti-inflammatory drugs (NSAIDs) are effective in preventing colon cancer (1). Currently available compounds, however, have side-effect profiles that make them unsuitable for widespread use in the prevention of this commonly fatal disease. Understanding the mechanisms of action of NSAIDs in preventing colon cancer is central to the development of safer and more effective treatments for the chemoprevention of colon cancer. Despite much research effort there is still much controversy over the mechanisms involved in the chemopreventative actions of NSAIDs (2). Most research has focused on the first described molecular target of NSAIDs, prostaglandin synthetase or cyclooxygenase (COX). Two COX isoforms have been described. COX-1 is constitutively expressed in almost all tissues and may perform housekeeping functions. COX-2 is an immediate early gene, undergoing rapid transcriptional up-regulation in response to tissue injury. COX-2 is induced in colon cancer and may contribute to tumour growth by producing prostaglandins that inhibit apoptosis (3) and induce the formation of new blood vessels (4). Inhibiting COX-2 pharmacologically with COX-2 specific or non-specific COX inhibiting compounds reduces colon cancer growth both in animal models (5) and in an inherited form of colon cancer, familial adenomatous polyposis (6). Knocking out COX-2 genetically in mice also lowers their susceptibility to colon cancer (7).

However, there is increasing evidence that COX-independent actions of NSAIDs are important both for their anti-inflammatory and their chemopreventative actions. NSAID-related compounds with no COX-inhibitory activity retain their antitumour activity (8) and cancer cells lacking COX-2 enzyme are still sensitive to these compounds (9). At the same time new molecular targets for NSAIDs continue to be identified. Among these the inhibition of NF-{kappa}B (10) and PPAR{delta} (11) provide alternative explanations for the tumour suppressive actions of NSAIDs. Many other possible COX-independent targets of NSAIDs in colon cancer have been suggested (reviewed in ref. 12). One of the criticisms of the COX-independent theories is that they require far higher drug concentrations than those needed to inhibit COX, concentrations that may not be achieved in vivo (2).

A relatively new method of drug target validation and the identification of alternative drug targets is use of DNA microarrays (13). Here we used a commercially available array to study the effects of aspirin on a large panel of cancer related genes. We hoped to find genes or groups of genes that were significantly up or down regulated by the treatment of colon cancer cell lines with aspirin, and thus to determine targets of aspirin relevant to neoplasia in colon cancer cells. We also addressed the question of drug concentration by performing parallel arrays where different concentrations of drug were used. Genes were classified according to function to look for patterns in the changes in gene expression induced by aspirin.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
The HT 29 colon cancer cell line was obtained from the ATCC, and cultured in Dulbecco's Modified Eagles Medium (Gibco, Paisley, Scotland) with 4.5 g/l glucose and L-glutamine. This was supplemented with penicillin (50 U/ml) and streptomycin (50 µg/ml) and, where serum was used, with 10% fetal calf serum (Gibco). Cells were grown in monolayers in a humidified atmosphere containing 5% CO2.

DNA array
Confluent HT29 cells were treated by adding aspirin or vehicle (DMSO) for 24 h. At 24 h clear effects have been seen on the cell cycle while apoptosis is not yet found (14). Cells were then washed in ice-cold phosphate buffered saline (PBS) x3. Total RNA was extracted using the ATLAS Pure Total RNA labelling system (Clontech, Palo Alto, CA) according to the manufacturer's instructions. Briefly, cells were lysed and total RNA extracted with three rounds of phenol:chloroform extraction. The resulting RNA was then treated with DNase and analysed on a denaturing agarose gel. Poly A+ Enrichment was performed using biotinylated oligo(dT) and avidin coated magnetic beads with a magnetic particle separator. Probes were made using the Atlas Human Cancer 1.2 array kit (Clontech) according to the manufacturer's instructions. cDNA probes were transcribed using a mix of primers specific for each of the genes on the array and labelled with [{alpha}-32P]dATP. Probes were purified by removing unincorporated 32P-labelled nucleotides and small cDNA fragments using an extraction column. Nylon cDNA expression array membranes were pre-hybridized with sheared salmon tested DNA and then hybridized with the probes overnight. Membranes were then sealed in plastic wrap and exposed for 7 days to a phosphoimaging screen and then developed in a phosphoimager and analysed using DNA array analysis software. A complete list of the genes and gene categories can be found at http://atlasinfo.clontech.com/genelists/huCa1.2.xls.

Signals were normalized between arrays using a correction factor calculated from the average expression of nine housekeeping genes. Spots were disregarded if neither treatment nor control levels were two times the average background level. Gene expression was considered significantly altered if the change was more than twice the standard deviation of the signal of the housekeeping genes. The change in gene expression is expressed as a multiple of the control (DMSO) value for that gene. The full array experiment from cell culture to phosphoimager was repeated twice for each condition and the membrane stripped according to the manufacturer's instructions.

Immunoblotting
Treated cells were washed in ice cold PBS and scraped into 250 µl of lysis buffer (Cell Signalling, Beverly, MA) with the addition of 1 mM Pefabloc (Sigma, St Louis, MO). The lysates were sonicated and then centrifuged at 20 g for 10 min at 4°C and protein concentration measured with the BCA protein assay kit (Pierce Chemical, Rockford, IL). Sample buffer (125 mM Tris–HCl, pH 6.8; 4% SDS; 2% ß-mercaptoethanol; 20% glycerol, 1 mg bromphenol blue) was added so as to equalize protein concentrations. Seventy-five micrograms of protein per lane was loaded onto SDS–PAGE and blotted onto PVDF membrane (Millipore, Billerica, MA). The blots were blocked with 2% low fat milk powder in TBST (Tris-buffered saline with 1% Triton) for 1 h at room temperature and washed 3x 10 min in TBST before overnight incubation at 4°C with primary antibody in TBST with 2% milk. Blots were then washed 3x 10 min in TBST and incubated for 1 h at room temperature in 1/2000 horse radish peroxidase (HRP) conjugated secondary antibody in block buffer. After a final 3x 10 min wash in TBST, blots were incubated for 5 min in Lumilite plus (Boehringer-Mannheim, Mannheim, Germany) and then chemiluminescence detected and quantified using a Lumi-Imager (Boehringer-Mannheim).

Immunohistochemistry
Sections (4 mm) were prepared from the formalin-fixed, paraffin-embedded tissue and mounted on slides coated with polylysin. Sections were dewaxed and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched with 1.5% H2O2 in PBS for 30 min and then washed in PBS. Antigen retrieval was performed by boiling slides for 10 min in 0.01 M sodium citrate pH 6.0. Non-specific binding sites were blocked with TENG-T [10 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.25% gelatin, 0.05% (v/v) Tween 20, pH 8.0] for 30 min, and then washed (3x 5 min in PBS). Slides were incubated with the primary antibodies overnight at 4°C in PBS with 0.1% Triton and 1% bovine serum albumin. After washing (3x 10 min in PBS), slides were then incubated with biotinylated secondary antibodies at room temperature for 1 h in PBS with 10% human serum. Slides were washed (3x 5 min in PBS), incubated with streptavidin–biotin–horseradish peroxidase (Dako, Glostrup, Denmark) for 1 h, washed again (3x 5 min in PBS), and peroxidase activity was detected with ‘Fast DAB’ (Sigma). Finally, sections were counterstained with Mayer's haematoxylin, dehydrated and mounted in ‘Entellan’ (Merck, Darmstadt, Germany) under cover slips.

Antibodies
Rac1 mouse monoclonal antibody IgG2b clone 23A8 was from Upstate (Lake Placid, NY) and was used for both immunoblotting and immunohistochemistry. Rac1 mouse monoclonal antibody IgG2b clone 102 was from Transduction Laboratories (Lexington, KY) and was used to confirm immunohistochemistry results (not shown). Cip1/WAF1 mouse monoclonal antibody IgG2a clone 70 was from Transduction Laboratories. HRP conjugated rabbit anti-mouse antibodies were from Cell Signalling, and biotinylated rabbit anti-mouse antibodies were from Dako.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
More genes are induced by aspirin treatment than repressed
Of the 1176 genes whose expression was analysed, 149 were significantly up-regulated and 51 significantly down-regulated when average gene expression was analysed over the three doses of aspirin used. Induction or repression is expressed as a multiple of the DMSO control value for that gene. For example, the expression of prostate differentiation factor after aspirin treatment using three different concentrations is on average nearly four times higher than the control value. The 10 most up regulated and 10 most down regulated genes using 5 mM aspirin are shown in Tables I and II.


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Table I. A list of the 10 genes most highly induced by 5 mM aspirin treatment, together with their GeneBank accession numbers and the mean fold induction

 

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Table II. A list of the 10 genes most highly repressed by 5 mM aspirin treatment, together with their GeneBank accession numbers and the mean fold repression

 
To look at the effect of aspirin dosage on gene expression we compared the results obtained for each gene in each condition to obtain a correlation coefficient for the various comparisons. We found that increasing the aspirin dose by a factor of 10 from 0.05 to 0.5 mM leads to very little difference in gene expression, a further factor of 10 increase, from 0.5 to 5 mM, results in a very different gene expression pattern. This is interesting as it agrees with data regarding the minimum effective dose required to influence many of the new non-COX targets of NSAIDs. These all require aspirin doses in the 1–10 mM range (12). Aspirin mediated effects such as induction of apoptosis (14,15) and inhibition of angiogenesis (16,17) also need doses in this millimolar range, doses that are at least 100-fold higher than those needed to inhibit prostaglandin synthesis (18). Our results confirm that aspirin has different actions in the millimolar range than at doses below this. The differences seen involve large numbers of genes and hence our representation of this using the correlation coefficient and graphically in Figure 1. In the two dot plot graphs we have plotted the mean fold induction and repression values for two different aspirin concentrations against each other. Thus, expression levels of each gene obtained using one concentration of aspirin divided by the DMSO controls were plotted against the expression levels obtained with a different aspirin concentration divided by the DMSO controls. The greater the similarity between the gene expression patterns the more linear the dot plot graph. In Table III we show a small selection of the genes that are exclusively altered by high dose (5 mM) aspirin treatment.



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Fig. 1. The list of values obtained for gene expression under the various treatments were compared to give a correlation coefficient between –1 and 1, representing the degree to which gene expression between two treatments is linearly related. While there was a good correlation in gene expression pattern between 0.05 and 0.5 mM aspirin treatments, the gene expression pattern obtained with 5 mM aspirin was very different to that of 0.5 mM aspirin treatment.

 

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Table III. A selection of the genes highly affected using 5 mM aspirin treatment, which are not affected using the other two aspirin concentrations

 
A number of these genes are interesting in that they are already described as being aspirin targets and even as requiring millimolar doses of aspirin to effect these changes. p21 and heat shock protein are reviewed by Tegeder et al. The effects of aspirin on NF-{kappa}B are also reviewed and largely ascribed to inhibition of I{kappa}B kinase ß. This also requires millimolar concentrations of aspirin. Here we show reduced transcription of p105, which could provide a further explanation. NF-{kappa}B is relevant to colorectal cancer as we (19) and others have shown (20).

Aspirin induces genes involved with DNA damage, nucleotide metabolism and stress response
To analyse whether there was a pattern to the up-regulated genes we analysed them by functional category to see if any categories were significantly over represented. For example, of the 1176 genes, seven code for ribosomal proteins. Six of these genes are among the 149 genes significantly up-regulated by aspirin. If 149 genes were selected at random from 1176 then on average they would contain 0.88 genes from the ribosomal protein gene category. As shown in Table III, genes involved with DNA damage signalling, stress response and nucleotide metabolism were among the most significantly affected categories.

Aspirin represses genes involved with the cell cycle
We further analysed whether there was a pattern to the significantly down-regulated genes. Fifty-one of these were categorized and categories significantly over-represented amongst the down-regulated genes are displayed in Table IV. Interestingly both of these are cell cycle related. The effects of aspirin and other NSAIDs on various cell cycle proteins are well documented (21,22).


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Table IV. A list of the gene categories significantly over represented among the genes induced by aspirin treatment

 

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Table V. A list of the gene categories significantly over represented among the genes repressed by aspirin treatment

 
This also fits well with some of the up-regulated genes such as p21 Cip1/WAF1, a cyclin-dependant kinase inhibitor that induces cell cycle arrest. This appears high in the list of most up-regulated genes by 5 mM aspirin. This we checked by immunoblotting HT29 cells treated with aspirin as shown in Figure 2. This confirms that cell cycle inhibition is one of the major effects of aspirin in colon cancer cells, and also confirms the reliability of the array results.



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Fig. 2. Immunoblot for p21 Cip1/WAF1 in HT29 cells treated with aspirin 5 mM for various times shown in hours. Seventy-five micrograms of total protein was loaded per lane.

 
Rac1 is induced by aspirin treatment
The gene most highly induced by aspirin treatment is prostate differentiation factor (PDF) a gene otherwise known as macrophage inhibitory cytokine or NSAID activated gene (NAG), which as the latter name suggests, has already been described as being up regulated by aspirin treatment (23). Another interesting gene that is highly induced by aspirin is Rac1, a molecule involved with differentiation of intestinal epithelial cells (24). This up regulation was also shown by immunoblotting aspirin-treated HT29 cells. Rac1 expression was induced in a time- and concentration-dependant manner as shown in Figure 3.



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Fig. 3. HT29 cells were treated with aspirin 5 mM for various times (A), or with various concentrations of aspirin for 24 h (B). Seventy-five micrograms of total protein was loaded per lane and blotted for Rac1. A time- and concentration-dependant increase in Rac1 expression is seen.

 
Rac1 is expressed most highly by mature colonocytes in human and mouse colon
One possible way to determine the function of Rac1 in the colon is to study its expression pattern by immunohistochemistry. As shown in Figure 4, Rac1 is expressed most highly at the intercrypt table in both mouse and human colonic epithelium. Thus, Rac1 expression increases as the colonocytes differentiate and is highest in mature colonocytes about to undergo apoptosis. This would support in vivo evidence that suggests that Rac1 is involved in differentiation of the colonic epithelium.



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Fig. 4. Immunohistochemistry in sections of normal mouse (A) and human (B) colon showing Rac1 expression (Brown) predominantly at the intercrypt tables. Strong staining for Rac1 is also seen in the stroma in the human colon. Control staining where the primary antibody was omitted or an isotype control antibody was used showed no staining (not shown). The same staining pattern was also seen with a Rac1 monoclonal antibody from a different manufacturer (not shown).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The exciting prospect of being able to prevent colon cancer with NSAIDs has led to intense interest in their working mechanism in preventing colon cancer. Since the discovery that NSAIDs inhibit COX (25), most of the work has concentrated on this as the working mechanism. However, in the last few years a number of new molecular targets of NSAIDs have been reported and several of these have been suggested to provide alternative working mechanisms for NSAIDs in colon cancer (12). One of the criticisms of many of these COX-independent mechanisms is that they require far higher doses of NSAIDs than required for COX inhibition, concentrations that may be difficult to achieve in vivo (2). In this study we set out to determine the changes in gene expression induced by aspirin at three different concentrations in the colon cancer cell line HT29 using a commercially available DNA microarray consisting of 1176 genes selected for their known or suspected roles in cancer. We analysed both the average gene expression obtained with the three aspirin doses and also the differences seen between the different doses.

Trials that address the issue of aspirin dosage and chemoprevention of colorectal cancer seem to indicate that aspirin at low dose (81 mg/day) is as effective as higher dosages (325 mg/day) (26). Epidemiological studies also show a statistically significant reduced risk for the development of colorectal cancer in those taking approximately 75 mg aspirin/day (27). It seems that even a low number of aspirin tablets (16 x 325 mg) per month taken regularly over 10 years reduces the risk of colorectal cancer development (1). Clinical evidence from the use of aspirin in other diseases suggests that for use as an anti-inflammatory drug far higher doses are required than when used for pain relief (28,29). Ultimately in vitro concentrations of aspirin are difficult to equate to in vivo concentrations. While plasma levels of up to 1 mM have been obtained in subjects taking 900 mg of aspirin/day (30), this may underestimate the concentration to which the intestinal mucosa is exposed as the drug is taken orally.

To look for general patterns within the complex changes induced in this large number of genes, we have grouped genes into functional groups and looked to see whether the affected genes fell into these groups more frequently than would be expected by chance. We also compared the pattern of gene expression found at each of the three aspirin concentrations to see to what extent they overlapped. Finally, we selected one of the most highly up-regulated genes and investigated this in more depth.

The most highly up-regulated gene has been identified previously by subtractive hybridization as the gene most highly up-regulated by NSAIDs. PDF, otherwise known as NAG or macrophage inhibitory cytokine, is highly up regulated by NSAID treatment. It is a member of the TGF-beta family and has proapoptotic properties (23,31). Our finding that this is the most highly up-regulated gene confirms the reliability of our array technique. We further checked the reliability of our results by analysing protein levels by the immunoblotting of a number of highly affected genes. One interesting gene never studied previously in this context is Rac1. Rac1 is a small GTPase and part of the Rho family that form part of the oncogenic RAS signal transduction pathway. In the intestine Rac1 plays an important role in intestinal epithelial differentiation. Introduction of a constitutively active form of Rac1 into mice under the control of an exclusively intestinal promoter leads to precocious differentiation of the intestinal epithelial cells (24). Recent studies in vitro have shown that Rac1 is essential for Cadherin-mediated cell–cell adhesion (32). Cadherins can also behave as morphogens influencing the differentiation and maturation of cells (33,34) and are lost at the ‘invasive front’ of a tumour (35).

In the adult colonic epithelium pluripotent stem cells residing at the bottom of the crypts give rise to daughter cells that migrate up the crypt to the epithelial surface where they undergo apoptosis and/or are shed into the gut lumen (3638). Along this migratory path the cells differentiate. Thus, mature, non-proliferating cells, about to undergo apoptosis are found at the villus tip and this we show is also the site of highest Rac1 expression in humans and mice. This provides supporting evidence that Rac1 may be involved with colonocyte maturation and apoptosis. Thus, our findings support the view that the chemopreventative effect of NSAIDs may be at least partly due to promotion of differentiation (39), and that this may be due to its effects on RAC1.

In conclusion, we find evidence that aspirin at 5 mM concentration has very different effects on cells than at two lower concentrations, 50 and 500 µM, a finding that supports clinical evidence that aspirin at low and high dose has different effects. We find that aspirin predominantly induces genes involved with DNA damage signalling, nucleotide metabolism and the stress response. On a single gene level a member of the TGF-beta family is the most highly induced gene and Rac1 is also highly induced. Rac1 plays an important role in cell–cell adhesion and its induction by aspirin may therefore underlie the beneficial effects of aspirin in colon cancer progression.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Thun,M.J., Namboodiri,M.M. and Heath,C.W.,Jr (1991) Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med., 325, 1593–1596.[Abstract]
  2. Marx,J. (2001) Cancer research. Anti-inflammatories inhibit cancer growth—but how? Science, 291, 581–582.[Free Full Text]
  3. Sheng,H., Shao,J., Morrow,J.D., Beauchamp,R.D. and DuBois,R.N. (1998) Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res., 58, 362–366.[Abstract]
  4. Seno,H., Oshima,M., Ishikawa,T.O., Oshima,H., Takaku,K., Chiba,T., Narumiya,S. and Taketo,M.M. (2002) Cyclooxygenase 2- and prostaglandin E (2) receptor EP (2)-dependent angiogenesis in Apc (Delta716) mouse intestinal polyps. Cancer Res., 62, 506–511.[Abstract/Free Full Text]
  5. Jacoby,R.F., Seibert,K., Cole,C.E., Kelloff,G. and Lubet,R.A. (2000) The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res., 60, 5040–5044.[Abstract/Free Full Text]
  6. Steinbach,G., Lynch,P.M., Phillips,R.K. et al. (2000) The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med., 342, 1946–1952.[Abstract/Free Full Text]
  7. Oshima,M., Dinchuk,J.E., Kargman,S.L., Oshima,H., Hancock,B., Kwong,E., Trzaskos,J.M., Evans,J.F. and Taketo,M.M. (1996) Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87, 803–809.[ISI][Medline]
  8. Charalambous,D. and O'Brien,P.E. (1996) Inhibition of colon cancer precursors in the rat by sulindac sulphone is not dependent on inhibition of prostaglandin synthesis. J. Gastroenterol. Hepatol., 11, 307–310.[ISI][Medline]
  9. Hanif,R., Pittas,A., Feng,Y., Koutsos,M.I., Qiao,L., Staiano-Coico,L., Shiff,S.I. and Rigas,B. (1996) Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. Biochem. Pharmacol., 52, 237–245.[CrossRef][ISI][Medline]
  10. Kopp,E. and Ghosh,S. (1994) Inhibition of NF-kappa B by sodium salicylate and aspirin. Science, 265, 956–959.[ISI][Medline]
  11. He,T.C., Chan,T.A., Vogelstein,B. and Kinzler,K.W. (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell, 99, 335–345.[ISI][Medline]
  12. Tegeder,I., Pfeilschifter,J. and Geisslinger,G. (2001) Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J., 15, 2057–2072.[Abstract/Free Full Text]
  13. Marton,M.J., DeRisi,J.L., Bennett,H.A. et al. (1998) Drug target validation and identification of secondary drug target effects using DNA microarrays. Nat. Med., 4, 1293–1301.[CrossRef][ISI][Medline]
  14. Qiao,L., Hanif,R., Sphicas,E., Shiff,S.J. and Rigas,B. (1998) Effect of aspirin on induction of apoptosis in HT-29 human colon adenocarcinoma cells. Biochem. Pharmacol., 55, 53–64.[CrossRef][ISI][Medline]
  15. Barnes,C.J., Cameron,I.L., Hardman,W.E. and Lee,M. (1998) Non-steroidal anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis. Br. J. Cancer, 77, 573–580.[ISI][Medline]
  16. Jones,M.K., Wang,H., Peskar,B.M., Levin,E., Itani,R.M., Sarfeh,I.J. and Tarnawski,A.S. (1999) Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing. Nat. Med., 5, 1418–1423.[CrossRef][ISI][Medline]
  17. Tsujii,M., Kawano,S., Tsuji,S., Sawaoka,H., Hori,M. and DuBois,R.N. (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 93, 705–716.[ISI][Medline]
  18. Kawai,S., Nishida,S., Kato,M., Furumaya,Y., Okamoto,R., Koshino,T. and Mizushima,Y. (1998) Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal anti-inflammatory drugs using human platelets and synovial cells. Eur. J. Pharmacol., 347, 87–94.[CrossRef][ISI][Medline]
  19. Hardwick,J.C., van den Brink,G.R., Offerhaus,G.J., van Deventer,S.J. and Peppelenbosch,M.P. (2001) NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps. Oncogene, 20, 819–827.[CrossRef][ISI][Medline]
  20. Karin,M., Cao,Y., Greten,F.R. and Li,Z.W. (2002) NF-kappaB in cancer: from innocent bystander to major culprit. Nat. Rev. Cancer, 2, 301–310.[CrossRef][ISI][Medline]
  21. DuBois,R.N., Shao,J., Tsujii,M., Sheng,H. and Beauchamp,R.D. (1996) G1 delay in cells overexpressing prostaglandin endoperoxide synthase-2. Cancer Res., 56, 733–737.[Abstract]
  22. Law,B.K., Waltner-Law,M.E., Entingh,A.J., Chytil,A., Aakre,M.E., Norgaard,P. and Moses,H.L. (2000) Salicylate-induced growth arrest is associated with inhibition of p70s6k and down-regulation of c-myc, cyclin D1, cyclin A and proliferating cell nuclear antigen. J. Biol. Chem., 275, 38261–38267.[Abstract/Free Full Text]
  23. Baek,S.J., Kim,K.S., Nixon,J.B., Wilson,L.C. and Eling,T.E. (2001) Cyclooxygenase inhibitors regulate the expression of a TGF-beta superfamily member that has proapoptotic and antitumorigenic activities. Mol. Pharmacol., 59, 901–908.[Abstract/Free Full Text]
  24. Stappenbeck,T.S. and Gordon,J.I. (2000) Rac1 mutations produce aberrant epithelial differentiation in the developing and adult mouse small intestine. Development, 127, 2629–2642.[Abstract/Free Full Text]
  25. Vane,J.R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New Biol., 231, 232–235.[ISI][Medline]
  26. Baron,J.A., Cole,B.F. and Sandler,R.S. (2003) A randomized trial of aspirin to prevent colorectal adenomas. N. Engl. J. Med., 348, 891–899.[Abstract/Free Full Text]
  27. Giovannucci,E., Egan,K.M., Hunter,D.J., Stampfer,M.J., Colditz,G.A., Willett,W.C. and Speizer,F.E. (1995) Aspirin and the risk of colorectal cancer in women [see comments]. N. Engl. J. Med., 333, 609–614.[Abstract/Free Full Text]
  28. Preston,S.J., Arnold,M.H., Beller,E.M., Brooks,P.M. and Buchanan,W.W. (1989) Comparative analgesic and anti-inflammatory properties of sodium salicylate and acetylsalicylic acid (aspirin) in rheumatoid arthritis. Br. J. Clin. Pharmacol., 27, 607–611.[ISI][Medline]
  29. April,P., Abeles,M., Baraf,H. et al. (1990) Does the acetyl group of aspirin contribute to the antiinflammatory efficacy of salicylic acid in the treatment of rheumatoid arthritis? Semin. Arthritis Rheum., 19, 20–28.[ISI][Medline]
  30. Stark,L.A., Din,F.V., Zwacka,R.M. and Dunlop,M.G. (2001) Aspirin-induced activation of the NF-kappaB signaling pathway: a novel mechanism for aspirin-mediated apoptosis in colon cancer cells. FASEB J., 15, 1273–1275.[Abstract/Free Full Text]
  31. Kim,K.S., Baek,S.J., Flake,G.P., Loftin,C.D., Calvo,B.F. and Eling,T.E. (2002) Expression and regulation of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in human and mouse tissue. Gastroenterology, 122, 1388–1398.[CrossRef][ISI][Medline]
  32. Braga,V.M., Machesky,L.M., Hall,A. and Hotchin,N.A. (1997) The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol., 137, 1421–1431.[Abstract/Free Full Text]
  33. Takeichi,M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251, 1451–1455.[ISI][Medline]
  34. Takeichi,M. (1993) Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell Biol., 5, 806–811.[Medline]
  35. Brabletz,T., Jung,A., Reu,S., Porzner,M., Hlubek,F., Kunz-Schughart,L.A., Knuechel,R. and Kirchner,T. (2001) Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc. Natl Acad. Sci. USA, 98, 10356–10361.[Abstract/Free Full Text]
  36. Stappenbeck,T.S., Wong,M.H., Saam,J.R., Mysorekar,I.U. and Gordon,J.I. (1998) Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium. Curr. Opin. Cell Biol., 10, 702–709.[CrossRef][ISI][Medline]
  37. Montgomery,R.K., Mulberg,A.E. and Grand,R.J. (1999) Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology, 116, 702–731.[ISI][Medline]
  38. Hall,P.A., Coates,P.J., Ansari,B. and Hopwood,D. (1994) Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J. Cell Sci., 107 (Pt 12), 3569–3577.[Abstract/Free Full Text]
  39. Ricchi,P., Pignata,S., Di Popolo,A., Memoli,A., Apicella,A., Zarrilli,R. and Acquaviva,A.M. (1997) Effect of aspirin on cell proliferation and differentiation of colon adenocarcinoma Caco-2 cells. Int. J. Cancer, 73, 880–884.[CrossRef][ISI][Medline]
Received January 2, 2004; revised January 27, 2004; accepted February 9, 2004.