| Folate deficiency has been linked to multiple health problems including neural tube defects and cancer, especially colorectal cancer. Folate is an essential dietary vitamin that is required for DNA synthesis and cellular methylation reactions. The mechanistic links between these associations is not completely understood. Folate deficiency has been shown to affect genes associated with the Wnt pathway, including beta-catenin. E-cadherin is an important regulator of Wnt signaling through controlling the level of free beta-catenin. It has also been shown that E-cadherin is affected by folate deficiency. The purpose of this study was to determine if folate deficiency altered the levels of E-cadherin protein and RNA, beta-catenin protein, and E-cadherin and beta-catenin cellular localization in two colon epithelial cell lines. Caco-2 and FHC cells were grown for 15 and 30 days in folate sufficient and deficient medium. Cellular growth rates for both cell lines were reduced in folate deficient cells, although the cells continued to grow. Cell cycle phase distribution showed increases in S phase and G2/M phase in Caco-2 cells, whereas there was no change in FHC cells. E-cadherin and beta-catenin protein levels decreased slightly in folate deficient Caco-2 cells at days 15 and 30. This was associated with a decrease in E-cadherin transcripts at day 30. FHC cells showed similar results at day 15, but a small increase in E-cadherin protein at day 30 and no change in beta-catenin. Immunofluorescent staining of Caco-2 cells showed diffuse cytoplasmic staining of E-cadherin and beta-catenin in folate sufficient cells for both Caco-2 and FHC cells. Folate deficient Caco-2 cells showed no change in E-cadherin, but an increase in membrane associated beta-catenin. Folate deficient FHC cells showed an increase in nuclear localization of both E-cadherin and beta-catenin. The AKT pathway was also analyzed in Caco-2 cells to determine if alterations in this growth regulatory pathway was associated with folate deficiency. In Caco-2 cells, an increase in activated AKT was detected at day 30 in folate deficient cells, and other proteins in the AKT pathway (PDK1, PTEN, and c-Raf) showed slight decreases whereas a small increase was detected for GSK-3beta. Finally, we confirmed published findings that Caco-2 cells were adapting to folate deficiency by upregulating the folate transporters: folate receptor 1 (FolR-1) and reduced folate carrier (RFC). In general, those results suggest that alterations in E-cadherin and beta-catenin occur in folate deficient colon epithelial cells, although the cells can adapt and continue to proliferate. |
