[TNF-α activates PI3K/AKT pathway to promote proliferation of SW620^Lgr5+ colon cancer stem cells]

Objective To investigate the role of PI3K/AKT pathway in the proliferation of SW620^Lgr5+ colon cancer stem cells (CSCs) in inflammatory environment. Methods The expression level of Lgr5 in SW620, SW480, HT29 and HCT116 human colon cancer cells were analyzed by Western blot analysis. SW620 cells were selected to analyze the proportion of Lgr5⁺ cells by fluorescence activated cell sorting (FACS). The cells were cultured in serum-free medium (SFM) to form spheroid cells. Furthermore, Lgr5⁺ CSCs were isolated from the spheroid cells by FACS system. The biological characteristics of Lgr5⁺ CSCs were assessed by the colony formation assay and 5-FU chemotherapy sensitivity assay. The inflammatory microenvironment of Lgr5⁺ CSCs was established with TNF-α and the optimum conditions of TNF-α were analyzed using CCK-8 assay. CSCs were treated with PI3K/AKT pathway inhibitor MK2206. The experimental cells were divided into a blank control group, MK2206 group, TNF-α group and TNF-α combined with MK2206 group. The cell proliferation and apoptosis of each group were detected by colony formation assay and annexin V-FITC/PI double labeling assay. Finally, Western blot analysis was used to analyze the protein expression of AKT, phospho-AKT(p-AKT), GSK-3β and p-GSK-3β. Results The expression of Lgr5 in the SW620 cells was significantly higher than that in the other colon cancer cells. FACS showed 6.9% of SW620 cells were Lgr5⁺. After cultured in SFM, the proportion of Lgr5⁺ in SW620 spheroid cells increased to 34.5%. The proliferation ability and drug resistance were significantly enhanced in SW620^Lgr5+ CSCs compared with SW620 cells. The treatment of 1 ng/mL TNF-α for 24 hours promoted the most remarkably increase of the viability of SW620^Lgr5+ CSCs. Compared with the control group, TNF-α significantly increased the colony forming ability of SW620^Lgr5+ CSCs. MK2206 statistically decreased the colony forming ability of SW620^Lgr5+ CSCs, and increased its apoptosis rate. In addition, MK2206 significantly decreased the colony forming ability of SW620^Lgr5+ CSCs compared with the TNF-α treatment group. TNF-α treatment increased the phosphorylation of AKT and GSK-3β in SW620^Lgr5+ CSCs, but the phosphorylation was inhibited by MK2206. Conclusion TNF-α activates PI3K/AKT pathway to promote the proliferation of SW620^Lgr5+ CSCs.