[TNF-α activates Wnt signaling pathway to promote the invasion of human colon cancer stem cells].

Abstract

Objective To explore the effect of Wnt/β-catenin signal pathway on the invasion of human colon cancer stem cells in the inflammatory environment. Methods The CD44⁺CD133⁺ human colon cancer stem cells were sorted from HT29 human colon cancer cell line by fluorescence-activating cell sorting (FACS), and these stem cells were identified by flow cytometry and single cell cloning formation assay. The inflammatory cell model was established by tumor necrosis factor-α (TNF-α) treating CD44⁺CD133⁺ HT29 cells and the optimal dose and reaction time of TNF-α were confirmed by MTT assay. DKK1 as an inhibitor of the Wnt signaling pathway was used in the inflammatory cell model. The experiment cells were divided into control group, TNF-α treatment group and TNF-α combined with DKK1 group. The cell proliferation was determined by MTT assay, and the expression of Wnt signaling pathway-related proteins including β-catenin, cyclin D1, c-Myc and epithelial mesenchymal transition (EMT)-related proteins (E-cadherin, vimentin) were detected by Western blot analysis. The invasive ability of cancer stem cells was detected by Transwell^TM assay. Results The CD44⁺CD133⁺ human colon cancer stem cells were successfully obtained from HT29 cells. Compared with the control group, the relative survival rate and invasive ability of CD44⁺CD133⁺ HT29 cells increased after treated with 10 ng/mL TNF-α for 48 hours, and the expression of E-cadherin was downregulated and vimentin was upregulated after CD44⁺CD133⁺ HT29 cells were treated with TNF-α. Compared with the TNF-α treatment group, the relative survival rate of CD44⁺CD133⁺ HT29 cells was reduced after DKK1 treatment. The number of transmembrane cells and the expression of Wnt signaling pathway-related proteins including β-catenin, cyclin D1 and c-Myc decreased after DKK1 treatment, and E-cadherin expression was upregulated and vimentin expression was reduced after CD44⁺CD133⁺ HT29 cells were treated with DKK1. Conclusion TNF-α promotes the invasion of colon cancer stem cells by activating the Wnt signaling pathway.