Mucin‐depleted foci show strong activation of inflammatory markers in 1,2‐dimethylhydrazine‐induced carcinogenesis and are promoted by the inflammatory agent sodium dextran sulfate

Mucin‐depleted foci (MDF), formed by dysplastic crypts devoid of mucins, have been identified in the colon of carcinogen‐treated rodents and in humans at high risk for colon cancer. The lack of the protective layer of mucus may cause inflammation which has been linked to colon carcinogenesis, therefore, the expression of markers such as cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (i‐NOS) and macrophage infiltration was studied with immunohistochemistry (IH) in MDF harvested from F344 rats treated with the colon carcinogen 1,2‐dimethylhydrazine (DMH). The same determinations were performed in aberrant crypt foci (ACF) and, at a later time point, in tumours. A dramatic increase in COX‐2, i‐NOS and macrophage infiltration was observed in MDF but ACF showed a moderate increase compared with the paired normal mucosa. Tumours were positive for all the markers. RT‐PCR experiments demonstrated that i‐NOS RNA expression was increased in a set of MDF confirming the results obtained with immunohistochemistry. In an inflammation‐cancer experimental model [mice treated with azoxymethane (AOM) and dextran sodium sulphate (DSS)], we observed that DSS‐induced inflammation promoted MDF in a dose‐dependent manner, whereas ACF were not affected. In conclusion, we report here for the first time a strong activation of the inflammatory process in MDF, which may contribute to the further progression of MDF to tumours. © 2009 UICC     

Sustained proliferation and resistance to apoptosis after a cytotoxic insult are early alterations in rat colon carcinogenesis

To study the early alterations in carcinogenesis, we determined apoptosis and proliferation in rat mucin depleted foci (MDF), precancerous lesions in the colon under basal conditions and 24 h after treatment with 1,2-dimethylhydrazine (DMH), which induces apoptosis in the colon. Spontaneous apoptosis in MDF was higher than in normal mucosa (Apoptotic Index was 1.61 ± 0.30 and 0.21 ± 0.02 in MDF and normal mucosa, respectively, mean ± SE, p < 0.05). DMH (30 and 75 mg/kg) increased apoptosis in both normal mucosa and MDF (up to 20 times higher compared to basal levels in normal mucosa, but only two times in MDF). MDF had a higher and deregulated pattern of proliferation along the crypt compared to normal mucosa. After DMH, proliferation in normal mucosa was significantly depressed, but it did not vary in MDF. Survivin-Birc5 regulating apoptosis and proliferation was significantly over-expressed (RT-qPCR and immunohistochemistry experiments) in MDF vs. normal mucosa, but did not vary in response to DMH. The expression of the pro-apoptotic protein Bak did not vary in normal mucosa and MDF. Since inflammation is present in MDF, which may hamper apoptosis, we studied the effect of pre-treatment with aspirin (600 ppm in the diet for 10 days). No significant effects of aspirin were observed. In conclusion, MDF had a higher spontaneous apoptosis and proliferation coupled with a reduced response to apoptotic stimuli from cytotoxic compounds. Survivin over-expression in MDF indicates that this is an early event in colon carcinogenesis and suggests that down-regulation of Survivin may represent a strategy for cancer prevention.     

Mucin-depleted foci have beta-catenin gene mutations, altered expression of its protein, and are dose- and time-dependent in the colon of 1,2-dimethylhydrazine-treated rats

Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.     

JTE-522, a selective cyclooxygenase-2 inhibitor, inhibits induction but not growth and invasion of 1,2-dimethylhydrazine-induced tubular adenocarcinomas of colon in rats

We have previously demonstrated that JTE-522, a selective cyclooxygenase-2 (COX-2) inhibitor, inhibited development of aberrant crypt foci (ACF) in rats, a putative preneoplastic lesion in colon, and suggested its inhibitory potential in rat colon carcinogenesis. To evaluate the chemopreventive properties of JTE-522, the present study was design to evaluate the inhibitory effects of JTE-522 on rat colon tumorigenesis induced by 1,2-dimethylhydrazine (DMH). Rats at 6 weeks of age were divided into 4 groups. One week after the start of the experiment, all rats received DMH by s.c. injection at a dose of 40 mg/kg body weight once a week for 4 successive weeks. As the initiation and postinitiation treatment groups, groups 1-3 were fed diets containing 0, 50, or 150 ppm JTE-522, respectively, from the start of the study to the end. As the postinitiation treatment group, group 4 was given 150 ppm JTE-522 from 1 week after the last DMH injection to the end of the study. Forty weeks after the start of the experiment, administration of 150 ppm JTE-522 during both initiation and postinitiation stages significantly inhibited the incidences of tubular adenocarcinomas and total carcinomas, as well as total tumors in the colon. The inhibitory effect of JTE-522 was most prominent for tubular adenocarcinomas, but was not observed in the nontubular carcinomas (signet-ring cell and mucinous carcinomas). Almost equal inhibitory effects on tubular adenocarcinomas were also observed in the rats given 150 ppm JTE-522 during the postinitiation stage, suggesting that its major anticancer action is at the postinitiation phase. However, JTE-522 had no effect on the size or invasive extent of tubular adenocarcinomas. Furthermore, microarray analyses revealed that JTE-522 had no effect on gene expression levels in DMH-induced tubular adenocarcinomas. These findings suggest that JTE-522 possesses chemopreventive activity against induction but not progression of tubular adenocarcinomas in rat colon. In view of the significant inhibitory effects of JTE-522 on ACF, its major anticancer action may occur in the postinitiation stage but before the malignant conversion stage of DMH-induced colon carcinogenesis.