Carcinogenesis vol.30 no.7 pp.1217–1224, 2009doi:10.1093/carcin/bgp113Advance Access publication May 6, 2009

5-Aminosalicylic acid inhibits colitis-associated but not sporadic colorectal neoplasia ina novel conditional Apc mouse model

Pim J.Koelink, Els C.Robanus-Maandag1, Peter Devilee1,Daniel W.Hommes, Cornelis B.H.W.Lamers and HeinW.Verspaget�

Department of Gastroenterology-Hepatology and 1Department of HumanGenetics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden,The Netherlands

�To whom correspondence should be addressed. Department ofGastroenterology-Hepatology, Leiden University Medical Center, C4-P, POBox 9600, 2300 RC Leiden, The Netherlands. Tel: þ31 71 526 2680; Fax: þ3171 524 8115;Email: H.W.Verspaget@lumc.nl

Genetic predisposition, life-style habits and inflammatory boweldiseases (IBD)-related colitis are a main risk factor for colorectalcancer (CRC). 5-Aminosalicylic acid (5-ASA, mesalazine) isa mainstay therapy in IBD and believed to reduce the risk fordeveloping CRC. We aimed to determine the ability of 5-ASAenemas to inhibit the development of sporadic and colitis-relatedneoplasia in mice. FabplCre;Apc15lox/1 mice, which spontaneouslydevelop sporadic colorectal tumours, were treated at 5 weeks ofage with 5-ASA or placebo enemas for 3 weeks and examined forcolorectal tumourigenesis at 8 weeks of age. Colitis-related tu-mour development was investigated in these mice by administra-tion of dextran sodium sulphate, inducing intestinal inflammationand accelerating colorectal tumourigenesis, combined with treat-ment of 5-ASA or placebo enemas during and/or after colitis in-duction. 5-ASA significantly reduced colitis-accelerated neoplasiadevelopment by 50%, from 19.4 ± 2.7 to 9.4 ± 2.4 (mean tumournumbers ± SEM, P 5 0.02), in the distal part of the large intestinecovered by the enema. 5-ASA was only effective when given dur-ing and/or after the intestinal inflammatory period. 5-ASA did notreduce, however, sporadic neoplasia development in the Fabpl-Cre;Apc15lox/1 mice. 5-ASA tended to reduce proliferation ofepithelial cells in the colitis-associated colorectal tumours butnot in the sporadic colorectal tumours. In conclusion, 5-ASAmed-ication inhibits the development of colitis-associated tumours inFabplCre;Apc15lox/1 mice when administered during and/or afterthe induction of inflammation. 5-ASA does not reduce, however,sporadic tumour development in this mouse model.

Introduction

Inflammation and carcinogenesis are pathological consequences ofinjury and repair at a cellular and molecular level. Several studiessuggest inflammation as a risk factor for the development of cancer,including colorectal cancer (CRC) (1). Individuals suffering fromeither of the two main chronic forms of inflammatory bowel disease(IBD), ulcerative colitis (UC) and Crohn’s disease, are at increasedrisk of developing CRC (2,3). 5-Aminosalicylic acid (5-ASA) isa non-steroid anti-inflammatory drug, widely used in the maintenanceof remission and treatment of mild inflammatory exacerbations inIBD. Most epidemiological studies have shown that the chronic useof 5-ASA in IBD has chemopreventive effects on the development ofCRC, although some studies failed to show this, as described in a re-cent meta-analysis by Velayos et al. (4). To study prospectivelywhether 5-ASA has chemopreventive actions in patients is hardly

feasible, due to long duration of such a study, the high patient numbersneeded, and ethical considerations. Therefore, for this kind of re-search, animal models are used that mimic the human disease, al-though most models lack similarities.

The ApcMin/þ mouse model carries a germ line mutation in oneallele of the adenomatous polyposis coli (Apc) tumour suppressorgene. ApcMin/þ mice develop multiple intestinal neoplasia (min),mainly in the small intestine, due to loss of function of the intactApcþ allele (5). Apc is a crucial negative regulator of the canonicalWnt pathway, forming a complex with axins and glycogen synthase3b kinase, targeting b-catenin for degradation. Loss of functional Apcprevents formation of the complex, resulting in accumulation ofb-catenin. Upon translocation to the nucleus and association withthe TCF-4 transcription factor, b-catenin induces transcriptional acti-vation of many genes involved in cell adhesion, migration and pro-liferation (6). The ApcMin/þ mouse model mimics the familialadenomatous polyposis syndrome in humans, an inherited form ofCRC, caused by a germ line Apc mutation and characterized by de-velopment of 100–1000 adenomas in the large intestine. Most CRCs,however, are sporadic but they share early Apc mutations in 80% ofthe cases.

Reports on the effects of 5-ASA on the development of intestinalpolyps in ApcMin/þ mice have been conflicting. McGregor et al. (7)showed that 5-ASA reduced the number of polyps, whereas Ritlandet al. (8) found no reduction in polyp numbers. The ApcMin/þ mousemodel is limited for studying the chemopreventive effect of 5-ASA onthe development of CRC because these mice develop only a smallfraction of lesions in the colon compared with the small intestine, andintestinal tumourigenesis is most probably already initiated in the first2 weeks of life in these mice.

Recently, we generated a novel tissue-specific, sporadic CRCmouse model, FabplCre;Apc15lox/þ. Compared with ApcMin/þ mice,FabplCre;Apc15lox/þ mice develop relatively few (,50) intestinal tu-mours, at a more advanced age, almost exclusively located in thedistal part of the large intestine, similar to the human disease coun-terpart (E.C.Robanus-Maandag, manuscript in preparation).

Awidely used and thoroughly described colitis model is the dextransodium sulphate (DSS) model. DSS dissolved in the drinking water ofrodents induces intestinal inflammation, including crypt damage andcrypt loss. When DSS is applied in a low concentration (2% wt/vol)for a few days, followed by a period of normal drinking water, theinflammatory period is followed by a healing and repair period, re-sembling exacerbation and remission stages in human UC (9,10).DSS-induced colitis in the ApcMin/þ mouse model was shown to ac-celerate the development of colorectal tumours and to be useful asa colitis-associated intestinal cancer model (11,12).

In the present study, we determined the effects of local 5-ASAtreatment on the development of colorectal tumours in (i) Fabpl-Cre;Apc15lox/þ mice, which develop distally located tumours, tomimic human sporadic CRC development and (ii) FabplCre;Apc15lox/þ mice exposed to DSS, to study colitis-associated develop-ment of neoplasia. 5-ASA was found to effectively reduce the de-velopment of colitis-associated tumours, whereas it was ineffectivein inhibiting sporadic tumour development.

Material and methods

Mice

All animal studies were approved by the ethical committee for animal studiesof the Leiden University Medical Center and complied with national lawsrelating to the conduct of animal experiments. FabplCre;Apc15lox/þ C57Bl/6mice were generated by mating FabplCre C57Bl/6 mice (line Fabpl4x at �132Cre(13), kind gift of J.Gordon) with Apc15lox/15lox C57Bl/6 mice and genotypedby polymerase chain reaction (E.C.Robanus-Maandag, manuscript in

Abbreviations: Apc, adenomatous polyposis coli; 5-ASA, 5-aminosalicylicacid; CRC, colorectal cancer; DAI, disease activity index; DSS, dextran so-dium sulphate; IBD, inflammatory bowel disease; min, multiple intestinal neo-plasia; PBS, phosphate-buffered saline; RT, room temperature; UC, ulcerativecolitis.

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preparation). FabplCre;Apc15lox/þ mice were weaned at 4 weeks of age andhoused individually under standard housing conditions with drinking water andfood available ad libitum.

Colitis induction

Colonic inflammation was induced in 5-week-old mice, with a median bodyweight of 17.5 g (range 13.0–22.0), by administration of 2% (wt/vol) DSS (MPBiomedicals, Aurora, OH; MW 5 36 000–50 000) in tap water to the mice adlibitum. The solution was refreshed every other day, and the consumption wasmonitored daily by weighing the drinking bottle. In order to induce inflamma-tion followed by a recovery period, the 2% DSS solution was given for 5 daysfollowed by 16 days of tap water (Figure 1A). Mice were killed at the end of theexperiment at 8 weeks of age or before the end of the experiment if theyexhibited .20% weight loss, rectal bleeding, anaemia and lethargy. Theselatter mice were excluded from the results of the neoplasia scoring.

5-ASA medication

Salofalk 4gr 5-ASA/60 gr enemas (Tramedico BV, Weesp, The Netherlands)were given daily by rectal injection of �200 ll of enema solution in animalsthat were anesthesized with isoflurane/O2. The control animals were treatedwith 0.9% NaCl ‘placebo enema’. In the first experiment, the effect of 5-ASAwas tested on DSS-induced colitis-associated neoplasia development in Fabpl-Cre;Apc15lox/þ mice at 5 weeks of age, starting 2 days before DSS adminis-tration, in order to be effective before the colitis induction in the preventionprotocol and to adjust the animals to the enema treatment protocol, up to theend of the 2 week recovery period (DSS/5-ASA and DSS/Placebo groups,experiment 1, Figure 1A). Similarly, FabplCre;Apc15lox/þ mice were treatedat 5 weeks of age with either 5-ASA or placebo enemas for 3 weeks withoutDSS administration, to determine the effect of 5-ASA on sporadic colorectaltumour development (5-ASA and Placebo groups, experiment 1, Figure 1A).In the second experiment, the FabplCre;Apc15lox/þ mice were treated

simultaneously with DSS and 5-ASA or placebo medication during the DSS-induced colitis (DSSþ5-ASA and DSSþPlacebo groups) or with 5-ASA onlyin the subsequent 2 week recovery period (5-ASA-post-DSS group, experiment2, Figure 1B).

Assessment of colitis

The presence of blood in faeces, stool consistency and general appearancewere recorded daily for each animal. Together, these factors constitute thedisease activity index (DAI, range 0–6, fig 1C), as adapted from Cooperet al. (9). The daily-recorded body weight was not included in the DAI.

Histopathology

The large intestine (from caecum to anus) of killed mice was isolated and thelength was measured in a relaxed position without stretching. The large in-testine was opened longitudinally, cleaned in phosphate-buffered saline (PBS),spread onto filter paper (luminal side up), fixed in buffered 4% formalin(pH 7.2) for 24 h at room temperature (RT) and stored in 70% ethanol at4�C. The neoplastic lesions were macroscopically counted under a dissectionmicroscope (with an up to �40 magnification) in a blinded fashion. The in-testinal tissues were embedded in paraffin (Swiss roll technique), sectioned at5 lm and stained with heamatoxylin and eosin, according to standard procedures.The sections were graded, in a blinded fashion, for the residual inflammatoryinfiltrate, with a range from 0 to 4 as to the amount and depth of infiltration:0 5 no infiltrate, 1 5 focal infiltrate crypt base, 2 5 infiltrate in lamina propria,3 5 infiltrate throughout the lamina propria and mucosal thickening and 4 5infiltrate extending into the submucosa. In addition, the lymphoid activity basedon the number of lymph follicles was scored from 0 5 none to 4 5 more thanthree follicles. In combination a score of 0–8 could be obtained, a modificationfrom the DSS-colitis microscopic activity index as reported before (14).

Immunohistochemistry

Paraffin sections were deparaffinized, endogenous peroxidase activity wasblocked in 0.3% H2O2 (Merck, Darmstadt, Germany) in methanol for 30min RT and rehydrated in a graded ethanol series to PBS. For antigen retrieval,the slides were boiled in 10 mM citrate buffer (pH 6.0) for 10 min in a micro-wave oven. The slides were cooled down to RT, rinsed with PBS (3�) andblocked with 5% goat serum (Dakocytomation, Glostrup, Denmark) in 1%bovine serum albumin in PBS for 20 min at RT. Sections were incubated withthe primary antibody in 1% bovine serum albumin/PBS: Ki67 (1:1000, Mono-san San Bio BV, Uden, The Netherlands) 1 h at RT, active caspase-3 (1:200,Cell Signaling Technology, Danvers, MA) or b-catenin (1:3200, BD Trans-duction Laboratories, Lexington, KY) overnight at 4�C. The slides were rinsedwith PBS (3�) and incubated with biotinylated secondary antibodies (1:200,Dakocytomation) in 1% bovine serum albumin/PBS for 45 min at RT. Theslides were rinsed with PBS (3�) and the biotinylated proteins were visualizedby incubation with streptavidin–biotin complex/horseradish peroxidase(Dakocytomation) for 45 min at RT, followed by 0.015% H2O2/0.05% diami-nobenzidene (Sigma, Schneldorf, Germany)/0.01 M Tris–HCL pH 7.6 for 10min at RT. Sections were counterstained with Mayers’ haematoxylin (Merck),dehydrated and mounted in entallan (Merck). Negative controls without pri-mary antibodies were included. The Ki67 index was determined in five repre-sentative areas of neoplastic tissue and in five normal appearing crypts ata �400 magnification and expressed as the percentage of Ki67-positive epi-thelial cells. Photomicrographs were taken with a Nikon Elipse E800 micro-scope equipped with a Nikon DXM 200 digital camera.

Statistical analysis

Statistical analysis of the changes in body weight, DAI, microscopic activityindex, length of the large intestine, the number of neoplastic lesions and the(immuno) histology scores were performed using the Student’s t-test. Differ-ences were considered significant when P � 0.05.

Results

Increased DAI and loss of body weight due to DSS

The typical colitis symptoms, recorded as the DAI, increased dramat-ically in FabplCre;Apc15lox/þ mice during and shortly after the 5 dayDSS period in both DSS/5-ASA and DSS/Placebo groups (Figure 2A).In addition, both DSS-treated groups displayed a gradual loss of bodyweight at the end and shortly after the DSS period in contrast to thetwo non-DSS-treated groups (Figure 2B). The DSS-treated animalsrecovered from this severe colitis during the next two weeks, asindicated by a decrease in DAI and gain of body weight. In general,5-ASA treatment by enema tended to a faster/better recovery of theDSS-treated mice (Figure 2A and B). This difference could not be

Fig. 1. Experimental protocols. (A) FabplCre;Apc15lox/þ mice were treatedwith placebo or 5-ASA enemas from 5 to 8 weeks of age without DSSadministration (Placebo and 5-ASA panel, respectively) or in combinationwith 2% DSS administration for 5 days at 5 weeks of age (DSS/Placebo andDSS/5-ASA panel, respectively). (B) FabplCre;Apc15lox/þ mice were treatedwith placebo or 5-ASA enemas for 7 days at 5 weeks of age during 2% DSSadministration for 5 days (DSSþPlacebo and DSSþ5-ASA panel,respectively) or with 5-ASA enemas after the DSS administration from 6 to 8weeks of age (5-ASA-post-DSS panel). (C) Table to determine the DAI. TheDAI is defined as the total score for all three parameters (stool consistency,blood loss and appearance), range 0–6.

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attributed to a difference in consumption of DSS-supplemented drink-ing water (Figure 2C).

DSS-induced colitis accelerates large intestinal tumourigenesis

Due to the DSS-induced colitis, the length of the large intestine of theDSS/Placebo group was significantly decreased compared with that ofnon-treated mice on average from 9.3 to 7.5 cm (P , 0.0001, Figure3A and B). The DSS-induced colitis strongly accelerated intestinaltumourigenesis as indicated by the increased average number of largeintestinal tumours from 5.2 tumours in non-treated mice to 28.8 tu-mours in DSS/Placebo mice (P , 0.0001, Figure 3C).

5-ASA suppresses colitis-accelerated large intestinal tumourigenesis

5-ASA treatment tended to increase the length of the large intestine ofFabplCre;Apc15lox/þ mice treated with DSS (on average from 7.5 cmto 7.9 cm, P 5 0.17, Figure 3B). More importantly, however, 5-ASAtreatment significantly reduced the average number of colitis-accel-erated large intestinal tumours by 37.4% from 28.8 tumours in theDSS/Placebo group to 18.0 tumours in the DSS/5-ASA group(P 5 0.03, Figure 3C).

In contrast, 5-ASA treatment was unable to reduce the averagenumber of sporadic tumours in the large intestine (5.2 and 4.8 tumoursin the 5-ASA group and Placebo group, respectively, P 5 0.93,Figure 3C).

5-ASA exerts tumour-inhibiting effect during recovery period

To determine whether 5-ASA exerted its preventive effect due to anti-inflammatory actions on the DSS-induced colitis or due to effectsduring the recovery period, mice were treated with 5-ASA or placeboenemas during the DSS administration period and 2 days thereafter(DSSþ5-ASA group and DSSþPlacebo group) and with 5-ASA en-emas only during the 2 weeks of subsequent colitis recovery (5-ASA-post-DSS group, experiment 2, Figure 1B). The DAI and the bodyweight changes of these DSS-treated mice showed similar curves asthose of the corresponding DSS-treated animals of experiment 1 (datanot shown). Importantly, 5-ASA treatment during the DSS adminis-tration was not able to reduce the average number of tumours (29.5and 29.2 tumours in the DSSþPlacebo group and DSSþ5-ASAgroup, respectively, P 5 0.96, Figure 3C), whereas 5-ASA treatmentduring the recovery period tended to reduce the average tumour

Fig. 2. Clinical signs of colitis. (A) DAI of the DSS/Placebo and DSS/5-ASA panels during the 3 weeks of DSS treatment and placebo or 5-ASA enemas, startingat 5 weeks of age. (B) Body weight curves of the Placebo, 5-ASA, DSS/Placebo and DSS/5-ASA panels during the 3 weeks of treatments. (C) The amount of DSS-supplemented water consumed by the DSS/Placebo and DSS/5-ASA panels during the 5 days of DSS administration. All mean þ SEM.

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numbers (20.8 and 28.8 tumours in the 5-ASA-post-DSS and DSS/Placebo groups, respectively, P 5 0.07 and 29.2 tumours in theDSSþ5-ASA group, P 5 0.19 versus 5-ASA-post-DSS).

5-ASA exerts tumour-inhibiting effect in the distal large intestine

We examined the tumour-inhibiting effect of the 5-ASA treatment inmore detail. First, the area covered by the 5-ASA enemas was de-termined in the DSS/5-ASA, 5-ASA-post-DSS and DSSþ5-ASAgroups. Mice were killed shortly after rectal installation of the5-ASA enemas on the last day of the experiment and the distancecovered by the 5-ASA enemas was measured. The 5-ASA enemasreached to �3 cm from the anus (Figure 4A), which was defined as thetreatment area. 5-ASA significantly reduced the number of tumours inthe treatment area by 51.3% in the DSS/5-ASA group as comparedwith the DSS/Placebo group (on average 9.4 and 19.4 tumours, re-spectively, P 5 0.02, Figure 4B). The reduction concerned the de-velopment of both small (,2 mm) and larger (�2 mm) sized tumours(both, P 5 0.02), leaving 15.3% of the tumours �2 mm in the DSS/5-ASA group compared with 23.9% of the tumours �2 mm in the

DSS/Placebo group. The large intestine proximal to the 5-ASA-trea-ted area clearly showed no reduction in total tumour number, or in sizeof tumours (Figure 4C), and served as internal control, nicely con-firming the tumour-inhibiting effect of the 5-ASA enema. Post-DSStreatment with 5-ASA also showed a consistent tendency towardsa decrease in the number and size of tumours in the 5-ASA-treatedarea (P 5 0.14–0.21, Figure 4B), leaving the more proximal largeintestine unaffected (Figure 4C).

The effect of 5-ASA on histopathology, b-catenin expression, cellularproliferation and apoptosis

Histopathology of the distal large intestines of the DSS groups alsoshowed many neoplastic lesions and a more severe inflammatory in-filtrate (P , 0.001), compared with that of non-treated FabplCre;Apc15lox/þ mice (Figure 5A–D). Histopathological evaluation of theDSS/5-ASA group revealed less inflammatory infiltration, althoughnot statistically significant, compared with DSS/placebo (Figure 5B,P 5 0.19) but not in the 5-ASA post-DSS group. The microscopicevaluations confirmed the reduced tumour numbers (Figure 5A) but

Fig. 3. 5-ASA suppresses colitis-accelerated large intestinal tumourigenesis during the recovery period. (A and B) DSS reduced the length of the large intestine.Individual measurements are indicated by closed circles and mean values are indicated by horizontal lines. (C) DSS increased the number of tumours. 5-ASAsuppressed DSS-induced, colitis-accelerated tumourigenesis, but not sporadic tumourigenesis of the large intestine. 5-ASA exerted its effect during the recoveryperiod. Individual tumour numbers are shown for males (closed circles) and females (open boxes). Mean tumour numbers are indicated by horizontal lines.Significant P-values �0.05 are shown in bold.

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an exact histological quantification of the neoplastic lesions was notperformed.

Immunohistochemistry showed nuclear accumulation of b-cateninin all tumours analyzed (illustrated in Figure 5D and Figure 6A,Dand G), indicating that the Wnt pathway was up-regulated due tofunctional loss of the Apcþ allele in all tumours. We did not detectany obvious differences in intensity or localization, i.e. nuclear versusmembranous/cytoplasmic, of b-catenin between the different groups.

The extent of proliferation, as determined by immunohistochemis-try for Ki67, of both neoplastic cells and normal intestinal epithelialcells was similar for the non-treated, DSS/Placebo and DSS/5-ASAgroups, showing a higher percentage of proliferating neoplastic cells(all groups P , 0.003) than that of normal epithelial cells (Figure6B,E,H,K and J). Quantification illustrated that 5-ASA tended to re-duce the proliferation of neoplastic cells in the DSS/5-ASA groupwhen compared with the DSS/Placebo group (on average from 55.6to 52.7%, P 5 0.09, Figure 6J), but 5-ASA did not reduce prolifera-tion of normal epithelial cells in these two groups (P 5 0.47, Figure6J). The tendency to reduce proliferation of neoplastic cells by 5-ASAwas not found in the groups without DSS treatment (P 5 0.73, Figure6J). Overall, proliferation of neoplastic and normal epithelial cells inthe DSS-treated groups was very similar to that in the non-DSS-treated groups.

Finally, we investigated the extent of apoptosis by immunohisto-chemical detection of active caspase-3, an apoptotic protease. Apo-ptotic cells were mainly found at the top of normal crypts (Figure 6L)and incidentally in the lamina propria, whereas a heterogeneous dis-tribution of apoptotic cells was seen in tumour tissue (Figure 6C,F andI,). Quantification of the number of caspase-3-positive apoptotic cells

within the epithelial lining and in the submucosa, excluding cryptabscesses and extruded cells, in the distal 3 cm of the colons revealedthat 5-ASA did not significantly affect overall apoptosis in the DSS/5-ASA group as compared with the DSS/Placebo group, with a mean ±SEM of 26.1 ± 6.8 versus 18.3 ± 1.7 of apoptotic cells per centimetermuscularis mucosa, respectively.

Discussion

The use of the FabplCre;Apc15lox/þ mouse model in the present studyprovided a unique opportunity to evaluate the chemopreventive effectof 5-ASA on both sporadic and IBD-associated neoplasia. Our resultsshow that 5-ASA is able to reduce IBD-associated tumour develop-ment by �50% but not that of sporadic tumours. 5-ASA medication isprescribed to IBD patients as 5-ASA prodrugs, like sulphasalazine,specially coated (pH released) tablets or enemas to circumvent 5-ASAacetylation by cells in the upper digestive tract (15,16) or 5-ASAoxidation (17). In animal studies, 5-ASA is usually given orally assupplement of the drinking water or food (7,8,18–21). However, thisadministration seems not to be the most ideal treatment in animalmodels since it does not really mimic 5-ASA medication in humans.Therefore, we have chosen to treat the mice by rectal installation ofa 5-ASA enema solution that is also used to treat IBD patients andallows a good distribution and high luminal concentration of the drug(22,23). Rectal administration of 5-ASA was not effective in suppress-ing the acute clinical signs of DSS-colitis, i.e. the DAI and bodyweight loss, during the induction phase. 5-ASA was nevertheless ableto significantly reduce the number and size of colorectal tumours inthat part of the intestine covered by the enemas, the tumourigenesis of

Fig. 4. 5-ASA affects the distal large intestine covered by the enema. (A) The enema treatment area was determined in killed animals shortly after enematreatment on the last day of the experiment. Individual measurements are indicated by closed circles and the mean value is indicated by the horizontal line. (B)5-ASA reduces the number of both small (,2 mm) and larger (�2 mm) tumours in the most distal 3 cm of the large intestine, defined as the 5-ASA treatment area,of the DSS/5-ASA and 5-ASA-post-DSS panels. (C) No reduction in total tumour number or in size of tumours in the large intestine proximal to the 5-ASA-treatedarea. Bars (mean þ SEM) indicate total tumour numbers (black), tumours ,2 mm (grey) and tumours �2 mm (white) (B and C). Significant P-values �0.05 areshown in bold (B and C).

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which was accelerated due to the DSS-induced colitis, mimickingcolitis-associated colorectal carcinogenesis. Our observation that thetumour development in the proximal part of the colon is not affectedby the 5-ASA enemas also illustrate that 5-ASA does primarily actlocally and not systemically. Interestingly, 5-ASA treatment duringthe DSS-induced inflammation period, mimicking an inflammatoryexacerbation in UC, was not able to reduce the development of co-lorectal tumours indicating that 5-ASA does not exert its cancer-preventive effects due to anti-inflammatory actions. In contrast, itwas the 5-ASA treatment during the subsequent recovery period mim-icking the remission stage in UC, which showed a reduction in thenumber of tumours, although borderline not significantly, but not inthe intestinal inflammatory infiltrate indicating tumour preventive

effects of 5-ASA during the recovery period. Two recent reportsshowed that 5-ASA is also able to reduce colitis-associated neoplasiaby �50% in the DSS/azoxymethane model. Clapper et al. (20) de-scribed a suppressive effect of 5-ASA on the colitis-induced colorec-tal carcinogenesis when 5-ASA was administered before, during andafter three DSS periods. Surprisingly, in this report only the lowest ofthree different dosages of 5-ASA supplemented to the drinking water,simultaneously with DSS during the DSS periods, was effective.Ikeda et al. (21) found a reduction in large intestinal polyp numbersand size when 5-ASA-supplemented to the food was administered inthe remission stage, after two cycles of DSS-induced colitis. In bothreports, however, it was not exactly clear which 5-ASA dosage theanimals received because the animals were not housed individually

Fig. 5. Histopathology of sporadic tumours and of IBD-associated tumours treated with 5-ASA or otherwise. (A) Heamatoxylin and eosin stained sections of thedistal large intestine of a non-treated, DSS/Placebo-treated and DSS/5-ASA-treated FabplCre;Apc15lox/þ mouse (original magnification �20). Neoplastic lesions(arrows) and infiltration of inflammatory cells (arrowhead) are indicated. The distal parts of the intestines are orientated at the right side of the picture. (B)Intestinal inflammatory infiltration of the different treatment groups, as scored by the microscopic activity index (mean þ SEM), significant P-value �0.05 shownin bold. (C) A higher magnification (�400) of the squared region of the DSS/Placebo heamatoxylin and eosin slide in (A), illustrating neoplasia as characterized bygoblet cell loss, crypt distortion and enhanced nuclear b-catenin staining in (D).

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and the dosages are only based on the assumption that each animal hasthe same consumption of 5-ASA supplemented to the water and thefood. However, due to the induced colitis each individual animal altersits eating or drinking pattern, which might influence the dosage intakeconsiderably. In Clapper et al. (20), the mean different dosages of5-ASA display different effects, indicating that the exact dosage peranimal is very important. Moreover, both reports lacked an internalcontrol and did not investigate the effect on sporadic tumour devel-opment without colitis induction. In the present study, we show thatlocal administration of 5-ASA specifically represses colitis-associatedneoplasia in the treatment area, when applied chronically during andafter acute intestinal inflammation. This corresponds to human epi-demiological studies showing a chemopreventive effect on colitis-associated CRC by chronic medication of 5-ASA (4). One of themechanisms of action of 5-ASA could be to reduce the proliferationof neoplastic cells as seen in our colitis-associated CRC model, in linewith the report from Ikeda et al. (21).

In contrast to several in vitro and in vivo studies on the effect of5-ASA on CRC cells (24–27), we found a somewhat higher but nosignificant increase of apoptosis in the intestinal tissues due to 5-ASAadministration. We used active caspase-3 immunohistochemistry to

investigate apoptosis, which worked well but hampered the exactidentification and counting of the percentage of apoptotic cells, be-cause of additional staining of other (non-epithelial) apoptotic cellsand cellular debris in the crypts and of extruded cells. Detection of thecaspase-degraded product of cytokeratin 18 by M30 immunohisto-chemistry, widely used on human tissues and easy to quantify epithe-lial apoptosis, unfortunately did not work in our hands (data notshown) since it is probably not specific for mouse. 5-ASA mightalso induce cell death via non-caspase mechanisms (P.J.Koelink andH.W.Verspaget, unpublished results).

5-ASA was not able to prevent the development of sporadic tu-mours in the FabplCre;Apc15lox/þ mice. Apparently, 5-ASA cannotprevent colorectal tumourigenesis that is solely driven by the Apcmutation and thereby mimicking sporadic CRC development. More-over, 5-ASA did not affect the proliferation of neoplastic cells in thesesporadic CRCs, in line with the absence of a preventive effect. Theseobservations correspond to the data from Ritland et al. (8), describingno effect on the development of adenomas in the ApcMin/þ mousemodel. The only study performed in humans, addressing whether5-ASA prevents sporadic CRC, examined the recurrence rate of spo-radic colorectal adenomas in patients who underwent polypectomy

Fig. 6. Effect of 5-ASA on b-catenin expression, proliferation and apoptosis. Immunohistochemical analysis of neoplastic lesions in the distal large intestine fromnon-treated (A–C), DSS/Placebo-treated (D–F) and DSS/5-ASA-treated (G–I) FabplCre;Apc15lox/þ mice and normal crypts from non-treated mice (K and L) forb-catenin (A,D and G), for Ki67 to determine the extent of proliferation (B,E,H and K), and for active caspase-3 to determine the extent of apoptosis (C,F,G and L).Magnification �400. (J) Quantification of proliferation in the distal large intestines of different mouse panels. Percentages of normal (grey bars) and neoplasticcells (black bars) and P-values are shown (mean þ SEM).

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(28). This study revealed that oral 5-ASA was not able to reduce theadenoma recurrence rate and was therefore unlikely to prevent spo-radic CRC, in line with the results presented here.

As far as we know, this is the first report that used a mouse model tostudy the effect of 5-ASA enema treatment on the development of bothsporadic and colitis-associated colorectal neoplasia. One could argue thatthe used FabplCre;Apc15lox/þ mouse model in combination withDSS-induced colitis is a too robust model, in which colorectal tumouri-genesis is so strongly accelerated, that it does not really mimic colitis-associated CRC in human IBD patients, which takes decades to develop.One could also argue that due to the robustness of the model a smallchemopreventive effect is unlikely to be found. The fact that we did findan effect of 5-ASA shows the contrary: the FabplCre;Apc15lox/þ mouse incombination with DSS is a very useful model to investigate the chemo-preventive effects of certain agents on colitis-associated carcinogenesisrather quickly and can also be used to study prevention of sporadic CRC.

In conclusion, in the present study, we observed that chronicallyadministered 5-ASA inhibited colitis-associated neoplasia in a mousemodel, whereas it did not prevent the development of sporadic tu-mours, very much in line with the available data from human studies.

Funding

Tramedico BV, The Netherlands.

Acknowledgements

We thank Junita Jankie and Johanna van der Zon for technical assistance andRiccardo Fodde for initiating the mouse model.

Conflict of Interest Statement: None declared.

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Received January 22, 2009; revised April 28, 2009; accepted April 29, 2009

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