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Bile acids in the intestinal lumen contribute to the homeostatic regulation of proliferation and death of the colonic epithelial cells: Deoxycholic acid (DCA) appears to enhance and ursodeoxycholic acid (UDCA) to attenuate the process of chemically induced carcinogenesis. We studied the effects of UDCA on colitis-related colorectal carcinogenesis. Three groups of 25 mice were given 0.7% dextran sulphate in drinking water for 7 days and pure water for 10 days and were fed a standard diet containing double iron concentration. In 2 groups, the diet was supplemented with 0.2% cholic acid (CA), the precursor of DCA, or with 0.4% UDCA. After 15 cycles, the histology, the expression of MUC2, β-catenin, p27 and p16 and the fecal water concentration of DCA and UDCA were investigated. All animals showed colitis with similar severity and histologic as well as immunophenotypic alterations, resembling those of human colitis. Among the animals fed the nonsupplemented diet, 46% developed colorectal adenocarcinomas and 54% anal-rectal squamous cell carcinomas. The prevalence of dysplasia and carcinomas did not change significantly in the animals given CA. Among the mice fed with UDCA, none developed adenocarcinomas and 20% squamous carcinomas. Dysplastic lesions were found in 88%, 67% and 40% of each group, respectively. The prevalence of dysplasia as well as of carcinoma showed an inverse relationship to the UDCA concentration in the fecal water. These data indicate that UDCA suppresses colitis-associated carcinogenesis. This model is suitable for investigation of the mechanism of the anticarcinogenic effect of UDCA in vivo. © 2005 Wiley-Liss, Inc.
Ulcerative colitis (UC) in patients and in experimental animals is associated with a high risk of intraepithelial neoplasia and colonic carcinoma. The cumulative risk for colorectal cancer in a patient with UC is 2% at 10 years, 8% at 20 years and 18% at 30 years of disease duration.1 Since the occurrence of dysplasia can be a strong indication for prophylactic proctocolectomy,2 the inhibition or slowing down of the colitis-associated carcinogenesis would essentially reduce the number of necessary operations.
Factors contributing to colon carcinogenesis are high epithelial proliferation rate in the inflamed region and the resulting increased accumulation of mutations as well as the disturbed regulation of cell death and proliferation. Intestinal bile acids contribute to the homeostatic regulation of proliferation and apoptosis of the colonic epithelium. Deoxycholic acid (DCA), whose fecal water concentration in patients with colitis is increased,3 stimulates the proliferation of the colonic epithelial cells4 and promotes colon carcinogenesis in an azoxymethane (AOM) rat model.5 The addition of ursodeoxycholic acid (UDCA) to the diet decreases the prevalence of AOM-induced rat colonic tumors6 as well as the prevalence of tumors in MIN mice.7 These data indicate that DCA enhances the chemical colon carcinogenesis in the animal model, while UDCA inhibits the chemical as well as the wnt-pathway-driven colon carcinogenesis.
A chemopreventive effect of UDCA was recently demonstrated also in patients with primary sclerosing cholangitis (PSC), which accompanies 2–7% cases of ulcerative colitis (UC) and increases the risk for developing colon cancer 3- to 5-fold.8, 9 Two retrospective studies showed that patients with PSC and UC, who were treated with UDCA to improve the liver function, have a significantly lower risk of colitis-associated cancer than the non-treated group.10, 11 Whether UDCA treatment has also a protective effect in patients with UC alone, in whom the treatment with UDCA is not a standard therapy, has not been investigated.
The lack of data on this important subject is due to the very slow development of colitis-associated carcinomas and to the dysplasia-related preventive colectomy, which in most cases is performed prior to carcinoma development. This makes an animal model necessary for the investigation of the mechanism of action of UDCA in vivo.
In different colitis models, colon carcinomas develop at different speeds. The DSS-induced intermittent inflammation leads to a slow development of carcinomas within 8 months.12 This time can be reduced to 5 months if colitis-associated colon carcinogenesis is accelerated by a single injection of the carcinogens AOM13 or dimethylhydrazine (DMH),14 both of which are known to activate the wnt pathway.14, 15
The objective of the present work was to test the effects of UDCA on carcinogenesis induced solely by colitis and to compare them with the concentration of bile acids in the colonic fecal water. For this purpose, the mice with colitis were given either UDCA at a dose previously shown to inhibit chemical carcinogenesis6 or–as a control–CA, which is metabolised to DCA and was previously shown to enhance chemical carcinogenesis.6, 16 The results clearly show the chemopreventive effect of UDCA, while the increase of fecal DCA concentration had no significant effect. Our findings indicate that the present model is suitable for investigation of the UDCA-mediated tumour suppression mechanism under controlled in vivo conditions.
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- Material and method
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The present work demonstrates for the first time that the development of the murine colitis-associated colorectal adenocarcinomas can be inhibited by oral treatment with UDCA. It supports the previous retrospective studies on patients with PSC, who showed a lower risk of developing colon cancer when treated with UDCA, and offers a model for a detailed investigation of the mechanism of UDCA action.
The present model of DSS colitis-associated colon carcinogenesis exhibits several features of the etiology, tumor type, distribution and gene expression, similar to that of human colitis-associated carcinomas. The development of tumors in this model is slow, caused by the chronic inflammation alone, with no additional activation of signaling pathways by a high dose of a chemical carcinogen. According to the original report, the 2-fold iron-enriched diet enhances colitis and increases carcinoma prevalence.12 The frequent occurrence of adenocarcinomas of mucinous type observed in the present work is also found among human tumours associated with ulcerative or radiation colitis (30–50%) (reviewed in ref. 26). The prevalence of squamous carcinomas (54%) was, however, higher than in patients with colitis, in whom squamous carcinomas of the anal region are rare.27 These data indicate that in the present model, the prevalence of carcinoma types varies from that in the human colitis.
The altered localisation of β-catenin and the suppression of p16 and p27, observed in our study suggests a similar gene alteration profile as previously reported in colitis-associated carcinomas in humans,19, 21, 22 and we were able to demonstrate downregulation of p16 on the protein level.
Furthermore, the tumours in the present model appear to exhibit morphogenesis analogous to that of the human colitis-associated neoplasms. About 70% of the human sporadic colon carcinomas are assumed to develop from the adenoma-carcinoma sequence and 30% de novo, without an polypoid precursor.28, 29 In colitis, polypoid growth of adenomas is rare and the transition steps from flat colitis-associated intramucosal dysplasia to invasive carcinoma are not sharply delineated.
In the present model, adenomas most frequently had a sessile or pedunculated polypoid form and a tubular growth pattern. Their prevalence did not correlate with the prevalence of carcinomas. Furthermore, the expression of p16 and p27 proteins in the inflamed tissue showed a pattern similar to the adenocarcinomas rather than to the adenomas. The prevalence of dysplasia was, however, decreasing with the increase of UDCA concentration in fecal water. Together, these data are compatible with the hypothesis of a frequent transition from the inflamed and regenerating mucosa to intramucosal dysplasia and then to adenocarcinoma, without a polypoid adenoma intermediate.
On the other hand, the activation of the wnt pathway, evident through the translocation of β-catenin from the membrane to the cytosol and to the nucleus, was common in carcinomas and adenomas but was not detected in the epithelium of the inflamed tissue. This may be due to the comparatively small number of epithelial cells accessible to evaluation in the inflamed lesions.
The squamous carcinomas always retained the membranous β-catenin localisation. Together, these data indicate that β-catenin activation was not associated with the development of squamous carcinomas and its potential contribution to the development of adenocarcinomas could have occurred at a postinflammatory stage.
The cellular and molecular targets of the chemopreventive action of UDCA remain obscure. UDCA was previously shown to attenuate acute inflammation in TNBS colitis.30 In the present work, it did not affect the chronic inflammation, i.e. the modulation of inflammation was not the basis of the chemopreventive effect.
In fact, UDCA was shown to inhibit inflammation-independent tumor development in MIN mice,31 as well as in rats treated with AOM5, 6, 32, 33 or DMH,34 all of which are associated with the activation of the wnt pathway.34, 35 It is therefore possible, that the chemoprotection observed in the present work was related to inhibition of a downstream target of this pathway. This hypothesis is supported by a less efficient suppression of squamous carcinomas, in which the wnt pathway is not activated.
The inflammation-related suppression of the cyclin dependent kinase inhibitor p27 was reversed by UDCA treatment. In the dysplastic areas, the expression of p27 and the effects of UDCA were not consistent; this is compatible with the hypothesis that the dysplasias would further develop along different pathways. The reduced p27 expression was previously reported to enhance proliferation of gastrointestinal tumors in DMH-treated and in MIN mice and to cooperate with the activated wnt pathway.36 Whether a similar mechanism is affected by UDCA treatment during ulcerative colitis warrants further study.
The supplementation of the diet with the bile acids resulted, probably due to their bitter taste, in a decreased food intake and weight loss. This unspecific effect was observed in both supplemented groups, while the chemoprevention was specific for UDCA.
The lack of procarcinogenic action of CA was not expected, since the addition of 0.2% of CA to the diet has been previously shown to increase the prevalence of AOM-induced aberrant crypt foci37 as well as MNU-induced colon tumours.16 Other authors, however, observed the significant increase of AOM-induced tumours only at 0.4% but not 0.2% CA.6 These data suggests that the cocarcinogenic effect of 0.2% CA is weak and if the DSS treatment is a less potent activator of carcinogenic pathways than AOM or MNU, the applied CA dose was not sufficient to enhance inflammation-induced carcinogenesis. (Fig. 4b).
UDCA was suggested to prevent chemical carcinogenesis by decreasing the proportion of the cocarcinogenic DCA in the fecal water.25 In the present work, we did not observe any correlation between the decrease of DCA/UDCA concentration ratio and tumour prevalence (data not shown), which further underpins the difference between the chemical and the colitis-associated carcinogenesis.
In conclusion, the present model demonstrates the complete inhibition of colitis-associated colon adenocarcinomas by UDCA and offers a suitable tool for an in-depth analysis of the mechanistic aspects of anticarcinogenic effects of UDCA in vivo.