Ursodeoxycholic acid and F₆-D₃ inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: Roles of cyclin D1 and E-cadherin

We have previously demonstrated that ursodeoxycholic acid (UDCA) and a fluorinated analogue of vitamin D₃, F₆-D₃, inhibited colonic carcinogenesis in the azoxymethane (AOM) model. Generalized colonic mucosal hyperproliferation and aberrant crypt foci (ACF) are intermediate biomarkers of colon cancer. Using these biomarkers, in this study we examined the anticarcinogenic mechanisms of these chemopreventive agents. Rats were maintained on AIN-76A chow or supplemented with 0.4% UDCA or F₆-D₃ (2.5 nmol/kg chow) and treated weekly with AOM 20 mg i.p./kg wt or saline x 2 weeks. F₆-D₃ was continued for an additional 2 weeks and UDCA for the duration of the study. At 40 weeks, animals received bromodeoxyuridine (BrdUrd) i.p. 2 h before sacrifice. A portion of each tumor was fixed in formalin and the remainder flash frozen. Colons were divided longitudinally and half-fixed in formalin and half in ethanol. The size and location of methylene bluestained ACF were recorded. Cell proliferation (BrdUrd labeling) and apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay) were measured in colonic crypts and tumors. Protein expression levels of several regulators of cell proliferation were analyzed by immunostaining and Western blotting. Colonic crypt cyclin D1 and E-cadherin mRNA levels were measured by real-time PCR. In saline injected controls, neither UDCA nor F₆-D₃ alone had any effect on cytokinetic parameters or on the expression of mitogenic regulators. AOM significantly increased the proliferation (percentage of BrdUrd-positive cells) of both ACF (23.1 ± 1.7%) and non-ACF crypts (17.6 ± 1.6%), compared with normal colonic crypts (4.5 ± 0.8%; P < 0.05). This hyperproliferation was accompanied by a 5-fold increase in cyclin D1 and >50% decrease in E-cadherin protein (P < 0.05) in ACF, both of which are predicted to be growth-enhancing alterations. UDCA and F₆-D₃ significantly (P < 0.05) inhibited AOM-induced crypt cell hyperproliferation, ACF development, and tumor burden. These chemopreventive agents also significantly blocked AOM-induced alterations in cyclin D1 and E-cadherin protein in ACF and tumors. In ACF, changes in mRNA levels of cyclin D1, but not E-cadherin, paralleled alterations in protein expression. Cyclooxygenase-2 and inducible nitric oxide synthase were increased in AOM tumors but not in ACF, and these changes were blocked by UDCA and F₆-D₃. UDCA and F₆-D₃ significantly inhibited ACF development and hyperproliferation, in part, by preventing carcinogen-induced alterations in cyclin D1 and E-cadherin. In established tumors, UDCA and F₆-D₃ also limited inductions of cyclooxygenase-2 and inducible nitric oxide synthase, which together with their effects on cyclin D1 and E-cadherin, contribute to their chemopreventive actions.