Comparative differential expression of genes modulated by vitamin D in HT29 human colon cancer cells

2118

Previous studies from our laboratory have shown that a synthetic analog of vitamin D, 1α-hydroxyvitamin D5, at non-calcemic concentrations is as effective as 1,25(OH)2D3 in inhibiting cell proliferation of colon cancer cells. Recent studies have demonstrated that the enzyme 25-hydroxyvitamin-1-alpha-hydroxylase [1α(OH)ase] responsible for the hydroxylation of the pro-hormone [25-hydroxyvitamin D3; 25(OH)D3] to the active metabolite is not only present in the kidney, but also expressed in the colon, breast and other tissues. This has resulted in evaluating 25(OH)D3 as a potential chemopreventive agent for colon cancer. Using HT-29 cells, we showed that 25(OH)D3 inhibits cell proliferation and induces cell differentiation. In order to compare the molecular signature of these vitamin D molecules in colon cancer, HT-29 cells were treated with vehicle (ETOH), 1α,25(OH)2D3 (100 nM), 25(OH)D3 (1 μM), or 1(OH)D5 (1 μM) for 24 hrs. The samples were hybridized to the Human Genome U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA). In GeneSpring v7.2 (Agilent Technologies, Santa Clara, CA) .cell files were preprocessed using Robust Multichip Average (RMA) and genes were normalized to the mean expression of the control sample. Canonical pathways were analyzed through the use of Ingenuity Pathway Analysis (IPA; Ingenuity Systems, www.ingenuity.com). Genes from the dataset that met the fold change cutoff of ≥ 2.0 and were associated with a canonical pathway in the IPA were considered for further analysis. Results showed that treatment with 1α,25(OH)2D3, 25(OH)D3, or 1(OH)D5 as compared to control altered expression of mRNA levels of 72, 77, and 39 genes respectively and involved three major pathways. In addition to the genes involved in cell cycle, beta-catenin signaling, apoptosis and differentiation pathways, genes implicated in inflammation, TGFβ mediated changes and Toll-like receptor pathways were also differentially expressed. Specifically, Chemokine [C-X-C motif] receptor 4 (C-X-CR4), G-protein-coupled receptor kinase 5 (GRK5), antigen CD14 (CD14), toll-like receptor 4 (TLR4), Inhibin beta C (INHBC), and transforming growth factor beta receptor II (TGFB2) were identified as major genes modulated by all the vitamin D agents as compared to the control. Data from this study reveal that vitamin D compounds share signature expression profiles which may serve as selective targets for the chemopreventive effects. (Supported by NIH Grants K01 CA103861 and R01 CA121157)