Short-chain fatty acids and colon cancer cells: the vitamin D receptor--butyrate connection.

Butyrate and its prodrug tributyrin, as well as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), have important physiological effects on proliferation and differentiation in a variety of malignant cells. The aim of this study was to elucidate the role of the vitamin D receptor (VDR) in butyrate-induced cell differentiation and cell cycle arrest in Caco-2 cells, a human colon cancer cell line. Cell differentiation was evaluated by analyzing the activity of alkaline phosphatase (AP). Protein of VDR, cyclins, cyclin-dependent kinases (cdks) and of cdk inhibitors was quantified by Western blot analysis, VDR-mRNA by PCR. Pre- and postconfluent cells were assessed for VDR binding activity. Cell cycle was analyzed by flow cytometry. Tributyrin significantly increased VDR-mRNA level (250% vs. control) and VDR binding activity. Butyrate also enhanced VDR protein content in the nucleus in a time- and dose-dependent manner and more potently than other short-chain fatty acids of a related structure. Both butyrate (640% vs. control) and 1,25-(OH)2D3 (350% vs. control) significantly stimulated differentiation, whereas combined treatment with butyrate and 1,25-(OH)2D3 resulted in a synergistic amplification of AP activity (1400% vs. control). In the presence of the VDR antagonist ZK 191732, butyrate-induced differentiation was completely abolished (150% vs. control). While butyrate alone increased p21Waf1/Cip1 expression and down-regulated cdk 6 and cyclin A, and combined exposure with 1,25-(OH)2D3 resulted in a synergistic enhancement of butyrate-induced changes, expressions did not change from control level after treatment with butyrate and ZK 191732. G1 cell cycle arrest induced by butyrate was also abolished after combined treatment with butyrate and ZK 191732. In conclusion, differentiation and cell cycle arrest of Caco-2 cells induced by butyrate are mediated by up-regulation of VDR, followed by a stimulation of the negative cell cycle regulator p21Waf1/Cip1 and by a down-regulation of cdk 6 and cyclin A, both involved in cell cycle progression.