Dai Lu, Zhao Xiaopeng, Ma Lu, Li Xin, Zuo Di, Li Yuanjie, Wei Bo, Sui Yu, Xu Fang
Ministry-of-Education Key Laboratory of Fertility Preservation and Maintenance, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
Ministry-of-Education Key Laboratory of Fertility Preservation and Maintenance, Ningxia Medical University, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2020 Jan;36(1):33-41.
Objective To investigate the role of PI3K/AKT pathway in the proliferation of SW620 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 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 CSCs. Compared with the control group, TNF-α significantly increased the colony forming ability of SW620 CSCs. MK2206 statistically decreased the colony forming ability of SW620 CSCs, and increased its apoptosis rate. In addition, MK2206 significantly decreased the colony forming ability of SW620 CSCs compared with the TNF-α treatment group. TNF-α treatment increased the phosphorylation of AKT and GSK-3β in SW620 CSCs, but the phosphorylation was inhibited by MK2206. Conclusion TNF-α activates PI3K/AKT pathway to promote the proliferation of SW620 CSCs.
目的 探讨PI3K/AKT信号通路在炎症环境下对SW620结肠癌干细胞（CSCs）增殖的作用。方法 采用蛋白质免疫印迹法分析Lgr5在人结肠癌SW620、SW480、HT29及HCT116细胞中的表达水平。选取SW620细胞，采用荧光激活细胞分选技术（FACS）分析Lgr5阳性细胞比例。将细胞接种于无血清培养基（SFM）中培养形成球状体细胞。进一步通过FACS系统从球状体细胞中分离出Lgr5 CSCs。采用集落形成实验和5-氟尿嘧啶化疗敏感性实验评估Lgr5 CSCs的生物学特性。用肿瘤坏死因子-α（TNF-α）构建Lgr5 CSCs的炎症微环境，并采用CCK-8实验分析TNF-α的最佳作用条件。用PI3K/AKT信号通路抑制剂MK2206处理CSCs。实验细胞分为空白对照组、MK2206组、TNF-α组和TNF-α+MK2206组。采用集落形成实验和膜联蛋白V-FITC/PI双染法检测各组细胞增殖及凋亡情况。最后，采用蛋白质免疫印迹法分析AKT、磷酸化AKT（p-AKT）、糖原合成酶激酶-3β（GSK-3β）及磷酸化GSK-3β（p-GSK-3β）的蛋白表达。结果 SW620细胞中Lgr5的表达明显高于其他结肠癌细胞。FACS检测显示，SW620细胞中Lgr5阳性细胞比例为6.9%。在SFM中培养后，SW620球状体细胞中Lgr5阳性细胞比例增至34.5%。与SW620细胞相比，SW620 CSCs的增殖能力和耐药性明显增强。1 ng/mL TNF-α处理24小时对SW620 CSCs活力的促进作用最为显著。与对照组相比，TNF-α显著提高了SW620 CSCs的集落形成能力。MK2206可使SW620 CSCs的集落形成能力明显降低，并增加其凋亡率。此外，与TNF-α处理组相比，MK2206使SW620 CSCs的集落形成能力显著降低。TNF-α处理可使SW620 CSCs中AKT和GSK-3β的磷酸化水平升高，但MK2206可抑制这种磷酸化。结论 TNF-α激活PI3K/AKT信号通路促进SW620 CSCs增殖。
