Ulcerative Colitis-associated Colon Cancer In Animal Models And Mechanisms Of Carcinogenesis

Background Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) of the large bowel, the etiology of which is not completely understood, Patients with UC have been associated with a high risk of developing colorectal cancer (CRC). It is hard work to obtain cases in a short time, so it is necessary to set up an animal model of ulcerative colitis neoplasia. For ulcerative colitis-related CRC, several animal models have been reported. However, the model using DSS needs a long period to induce colitis-related CRC, and the incidence of induced tumors is relatively low[1,2]. It is reported that AOM combined with one cycle of2%DSS can establish an efficient animal model in12weeks[3]. It is believed that β-catenin which are key proteins in Wnt signal pathway play an important role in the pathogenesis of the UC-associated dysplasia-carcinoma sequence. The neoplastic transformation in UC is thought to be different from the canonical adenoma-carcinoma sequence in sporadic CRC. Loss of APC function, considered to be a very common early event in sporadic CRC pathogenesis, is much less frequent and usually occurs late in the UC-associated dysplasia-carcinoma sequence[4]. Conversely, P53mutations in sporadic CRC usually occur late in the adenoma-carcinoma sequence[5], whereas in patients with UC, P53mutation occur early and are often detected in mucosa that is non-dysplastic or indefinite for dysplasia[6]. In our study, we present here a newly developed ulcerative colitis-related colorectal cancer (UCRCC) mouse model in which colonic tumors developed within9weeks, when mice were given a low dose of AOM followed by3cycles of4-day exposure to DSS (3%-2%-2%) in drinking water. We investigated the roles of APC, β-catenin, P53and K-ras played in the pathogenesis of the UC-associated colorectal cancer.Objective We try to establish an animal model of ulcerative colitis-related colorectal carcinogenesis, The expression of APC, β-catenin, P53and the mutation of k-ras were determined and compared between the pathogenesis of ulcerative colitis and UC-associated colorectal cancer.Methods and materials Male C57BL mice aged4-5weeks were used in this study. They were quarantined for the first7days, and then randomized by body weight into four groups such as AOM+DSS group (Group A,20), DSS group (Group B,12), AOM group (Group C,12) and control group (Group D,12). Group A was given a single intraperitoneal injection of AOM (10mg/kg body weight). Starting1week after the injection, animals received3%DSS (MW36-50kDa) in the drinking water for4days, followed by17days of regular water. This cycle was repeated twice (four days of2%DSS). Groups B and C were given DSS or AOM alone, respectively. Group D was untreated. After each cycle,4mice in each group were sacrificed. All mice were killed ten days after the last cycle by ether overdose. The large bowels were flushed with saline, and were cut open longitudinally along the main axis, and then washed with saline. Proximal and distal tissue were cut and fixed in10%buffered formalin for at least24h. Histological examination was performed on paraffin-embedded sections after hematoxylin and eosin (H&E) staining. Masses were also fixed if can be observed by naked eyes. Immunohistochemistry (IHC) was performed to study the expression of the β-catenin, APC and P53in the colon mucosa of the specimens, using antibodies of anti-β-catenin (Abcom), anti-APC, and anti-p53(Santa Cruz). Tissue DNA was extracted and PCR-sequecing were used to detect the mutation of K-ras in the specimens.Results1. Animal model:Fifty-six male C57BL mice were randomized into3experimental and control groups. During the experiment bloody stools and diarrhea were observed at the6th day in group A and B, and were alleviated in the following14days. The mean of body weight in groups A (AOM+DSS) and B (DSS alone) were significantly smaller than that of group D (untreated)(P<0.05). After3cycles of DSS, masses can be found in the5/8mice (62.5%) which were in the distal colon and middle next and none were found in the proximal colon. No tumor was found in the other2experimental groups.2. P53and β-catenin were negative in normal colon mucosa. The expression levels of β-catenin had significant difference between UC-associated colorectal cancer and ulcerative colitis mucosa(P value0.029). P53was detected in only2dysplasia mucosa after2cycles of DSS in group A and was negative in tumor tissues. And there was no statistical difference of APC expression between ulcerative colitis related colorectal cancer and ulcerative colitis tissues. No K-ras mutation was detected.Conclusion1. The results indicate that3cycles of4-day administration of DSS (MW36-50kDa) combined with an initiation with a low dose of AOM (10mg/Kg body weight) can induce tumors in the distal and middle colons of male C57BL mice, and the procedures may provide an efficient mouse model for investigating colitis-related colon carcinogenesis.2.β-catenin which is key component of Wnt pathway showed a significant difference between UC and UCRCC. It suggests that Wnt/β-catenin signal pathway may be involved in the molecular mechanism of the neoplastic transformation in UCRCC.3. P53mutation may occur early in the carcinogenesis of ulcerative colitis and the pahtogenesis in animal model can be different from that in human beings.4. The effect of K-ras pathway in the carcinogenesis may be relatively weak.