5-FU通过靶向Wnt/β-catenin信号通路对结直肠癌干细胞活性的影响

Effect of 5-FU on the activity of colorectal cancer stem cells by targeting Wnt/β-catenin signaling pathway

目的 基于Wnt/β-catenin信号通路探讨5-氟尿嘧啶(5-FU)对结直肠癌干细胞(CRC-CSC)活性的影响。方法 流式细胞术分选CD133标记的CRC-CSC,以不同浓度5-FU(6.25、12.50、25.00μmol/L)处理细胞,对照组以等体积培养液处理。药物作用48 h后,CCK-8法检测细胞增殖,划痕实验检测细胞迁移,流式细胞术检测细胞凋亡,平板克隆实验和球形成实验检测细胞克隆能力及悬浮球形成能力,蛋白免疫印迹法检测Wnt/β-catenin信号通路表达。结果 与对照组比较,高剂量5-FU处理后CRC-CSC细胞增殖能力(24 h、48 h两组分别为0.31±0.03vs. 0.53±0.05;0.37±0.03 vs. 0.61±0.05)和迁移能力(24 h、48 h两组分别为9.63±0.72 vs. 38.69±0.88;16.21±1.17 vs.58.69±1.32)显著增加(P<0.05),细胞凋亡率(24 h、48 h两组分别为46.91±0.94 vs. 18.46±0.88;55.22±1.24 vs. 28.42±1.18)显著降低(P<0.05);48 h时高剂量5-FU组细胞克隆数(50.32±10.15 vs. 102.62±11.36)和悬浮球形成数(29.15±8.42 vs. 96.87±9.33)显著增加(P<0.05),Cyclin D1(0.17±0.03 vs. 0.95±0.02)、C-Myc(0.27±0.03 vs. 1.15±0.12)和β-catenin(0.46±0.03 vs. 0.85±0.02)蛋白相对表达量显著升高,且呈浓度依赖性(P<0.05)。结论 5-FU可通过靶向激活Wnt/β-catenin信号通路,诱导CRC-CSC细胞活化,提高其克隆和悬浮球形成能力,从而促进肿瘤的复发和转移。

Objective cells(CRC-CSC)based on Wnt/β-catenin signaling pathway.Methods cytometry and treated with different concentrations of 5-FU(6.25,12.50,25.00 μmol/L). Cells in control group were treated with same amount of medium. After 48 hours of treatment,cell proliferation was detected by CCK-8 assay,cell migration was detected by scratch assay,cell apoptosis was detected by flow cytometry,cells were tested using flat colony formation assay and the cell sphere formation assay,and the expression of Wnt/β-catenin signaling pathway related proteins was detected by Western blotting.ResultsCompared with the control group,the proliferation ability(0.31±0.03 vs. 0.53±0.05;0.37±0.03 vs. 0.61±0.05)and migration ability(9.63±0.72 vs. 38.69±0.88;16.21±1.17 vs. 58.69±1.32)of CRC-CSC cells increased significantly after high-dose 5-FU treatment(P<0.05),and the apoptosis rate(46.91±0.94 vs. 18.46±0.88;55.22±1.24 vs. 28.42±1.18)decreased significantly(P<0.05),The number of cell clones(50.32±10.15 vs. 102.62±11.36)and the number of suspended spheres(29.15±8.42 vs. 96.87±9.33)increased significantly(P<0.05),Cyclin D1(0.17±0.03 vs. 0.95±0.02),C-myc(0.27±0.03 vs. 1.15±0.12)and β-The relative expression of catenin(0.46±0.03 vs. 0.85±0.02)protein increased significantly in a concentration dependent manner(P<0.05).Conclusion5-FU can induce the activation of CRCCSC cells through targeted activation of the Wnt/β-catenin signaling pathway,and improve clone formation and sphere formation rate,thus promoting tumor recurrence and metastasis.