Ursodeoxycholic acid suppresses the transcription of genes responsible for proliferation in the normal intestinal epithelial cells in vivo and in vitro

Introduction: Ursodeoxycholic acid (UDCA) prevents colitis-related colon cancer which potentially can be attributed to enhanced proliferation during tissue regeneration. We investigated therefore the effects of UDCA on growth of normal rodent intestinal epithelial cells and on the expression of proliferation-relevant genes.

Materials and methods: Two groups of six C57BL/6J mice were fed with standard diet with and without 0.4% UDCA for 3 weeks. Sections of the colon were stained for Ki-67 using Tec-3 antibody and % of Ki-67 expressing cells was determined. In parallel, total RNA from the isolated colonic epithelial cells was analysed using three Affymetrix 430A 2.0 chips per group. The proliferation-relevant genes affected by UDCA were identified and validated by real time PCR. Normal rat intestinal cell line, IEC-6 was used for in vitro experiments. MTT test was performed on cells treated with UDCA (0 to 800µM for 3 days). The expression of genes obtained from microarray experiment was checked in IEC-6 cells treated with 600µM UDCA for 3 days by real time PCR and by Western blotting. Cells were treated with 10µM U0126 for 3 days to inhibit ERK phosphorylation.

Results: UDCA reduced the number of Ki-67 expressing cells to 60% related to the non-treated group. The microarray results showed >1.5 fold suppression of proliferation-regulating genes like Tcf4, Klf5, Irs1, Flot2, Gbp2 and Ghr. In MTT test, IEC-6 cells showed 40% and 50% decrease in proliferation at 600µM and 800µM UDCA, respectively. Real-time PCR of the RNA isolated from IEC-6 cells showed 2 to 25-fold suppression of these six genes. Western blotting also showed decrease in protein expression. Additionally, 600µM UDCA treatment for 3 days decreased ERK phosphorylation in IEC-6 cells. After treatment with U0126, similar inhibition of ERK phosphorylation and of cell proliferation was observed as after treatment with UDCA.

Conclusion: Our results indicate that UDCA decreases proliferation of normal colonic epithelial cells both in vivo and in vitro. The suppression of several proliferation-promoting genes and the dephosphorylation of ERK might be responsible for the proliferation inhibition caused by UDCA and hence for its chemopreventive effects.