TNF-α通过激活PI3K/AKT通路促进SW620^Lgr5+结肠癌干细胞增殖

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

目的探讨炎症环境下磷脂酰肌醇3激酶/蛋白激酶B (PI3K/AKT)通路对SW620^Lgr5+结肠癌干细胞(CSC)增殖的影响。方法以人结肠癌SW620、 SW480、 HT29、 HCT116细胞为材料, Western blot法检测四株细胞中干细胞标志物富含亮氨酸重复序列的G蛋白偶联受体5(Lgr5)的表达水平;选用Lgr5表达量最高的SW620细胞,采用荧光激活细胞分选技术(FACS)分析Lgr5^+细胞的比例;无血清悬浮培养得到结肠癌球细胞, FACS分选获得结肠癌Lgr5^+ CSC,集落形成实验、 5-氟尿嘧啶(5-FU)耐药实验鉴定分选得到的结肠癌Lgr5^+CSC生物学特征的变化;肿瘤坏死因子α(TNF-α)处理Lgr5^+ CSC建立炎症模型, CCK-8法筛选TNF-α的最适作用浓度及最佳作用时间;采用PI3K/AKT通路抑制剂MK2206处理Lgr5^+ CSC,分为空白对照组、 MK2206组、 TNF-α组、 MK2206联合TNF-α组;集落形成实验检测细胞增殖情况,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(AnnexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡情况;Western blot法检测AKT、磷酸化的AKT(p-AKT)、糖原合酶激酶3β(GSK-3β)、磷酸化的GSK-3β(p-GSK-3β)蛋白水平。结果 SW620细胞中Lgr5的蛋白表达量显著高于其他细胞系;FACS分析得到SW620细胞中Lgr5^+细胞比例为6.9%,无血清培养基悬浮培养后,分选得到SW620^Lgr5+ CSC的比例为34.5%;与SW620细胞相比, SW620^Lgr5+ CSC的增殖能力与耐药性显著提高;1 ng/mL TNF-α作用24 h时,SW620^Lgr5+ CSC具有最高的细胞活力;与对照组相比, TNF-α显著增加SW620^Lgr5+ CSC的集落形成能力,但MK2206显著性降低SW620^Lgr5+ CSC的集落形成能力,增加SW620^Lgr5+ CSC的凋亡率;同时MK2206显著降低TNF-α诱导的SW620^Lgr5+ CSC的集落形成能力,增加其凋亡比例。TNF-α处理增加SW620^Lgr5+ CSC中AKT和GSK-3β的磷酸化水平,但MK2206可抑制TNF-α诱导的AKT和GSK-3β的磷酸化水平。结论 TNF-α通过激活PI3K/AKT信号通路促进SW620^Lgr5+ CSC增殖。

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.