二甲双胍通过抑制线粒体氧化磷酸化降低结直肠癌干细胞的自我更新能力

Metformin inhibits self-renewal of colorectal cancer stem cells by inhibiting mitochondrial oxidative phosphorylation

目的探讨二甲双胍抑制结直肠癌干细胞自我更新的作用及机制。方法通过Wnt报告基因慢病毒从人原代结直肠癌类器官中分选出癌干细胞(Wnt+)和癌分化细胞(Wnt-);用0、5、10、20、30μmol/L二甲双胍分别处理Wnt+细胞和Wnt-细胞,检测其对体外细胞球体形成能力的影响,并筛选合适的药物浓度。以该浓度二甲双胍处理Wnt+细胞,与空白对照相比,克隆形成实验和小鼠体内成瘤有限稀释实验检测体外和体内自我更新能力的改变;流式细胞术检测Wnt表达比例及其强度的变化;qRT-PCR检测干性、分化和Wnt信号通路下游关键基因的mRNA表达水平;Seahorse能量代谢分析仪检测细胞氧耗率和细胞外酸化率;TMRE探针检测线粒体膜电位;MitoSOX探针检测活性氧(ROS)水平。分别使用10 mmol/L半乳糖、10μmol/L二甲双胍、5 mmol/L N-乙酰-L-半胱氨酸和10 mmol/L半乳糖+10μmol/L二甲双胍调控Wnt+细胞ROS水平并用MitoSOX探针检测验证,细胞球体形成实验检测各组自我更新能力,流式细胞术检测各组Wnt比例和Wnt信号通路活性的改变。采用10μmol/L二甲双胍和空白对照分别与Wnt+细胞共培养,TMRE探针、MitoSOX探针、免疫荧光和流式细胞术分别检测转染酵母NADH脱氢酶NDI1和空白对照质粒对线粒体膜电位水平、细胞内ROS水平、Wnt表达比例及其信号强度的影响。结果二甲双胍可以显著抑制Wnt+细胞自我更新形成细胞球体(P<0.01),呈浓度依赖性,对Wnt-细胞无显著抑制效应(P>0.05)。与对照组相比,10μmol/L二甲双胍显著抑制结直肠癌干细胞形成单克隆和移植瘤的能力(P<0.001);显著降低其Wnt+细胞的比例和Wnt通路活性(P<0.01);显著降低其干性和Wnt通路相关基因的mRNA水平,并升高分化相关基因的mRNA水平(P<0.05)。糖代谢相关试验显示二甲双胍显著降低Wnt+细胞氧耗率、线粒体膜电位水平和ROS水平,显著增强其细胞外酸化率(P<0.001)。半乳糖显著增强结直肠癌干细胞细胞球体形成能力、细胞内ROS水平和Wnt+细胞比例及其Wnt活性(P<0.01);加入二甲双胍能显著抑制以上效应,并削弱半乳糖的促进效应(P<0.05)。转染NDI1替代人线粒体复合体I可显著削弱二甲双胍降低结直肠癌干细胞比例和Wnt信号通路活性的效应(P<0.001)。结论二甲双胍通过抑制人线粒体复合体I降低结直肠癌干细胞的线粒体氧化磷酸化及细胞内ROS水平,从而抑制其Wnt信号通路降低其自我更新能力。

Objective To investigate the mechanism of metformin for inhibiting self-renewal of colorectal cancer stem cells(CSCs).Methods CSCs were sorted from Wnt reporter-transfected colorectal cancer patient-derived organoids(PDOs)by fluorescence-activated cell sorting(FACS)and treated with metformin.The changes in self-renewal of the cells were assessed using sphere formation,colony formation and limiting dilution assays.The mRNA expressions of genes related with stemness and differentiation and Wnt target genes was detected by qRT-PCR.Wnt activity was assessed using flow cytometry in the CSCs.Seahorse analysis was used to evaluate cellular oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)after metformin treatment.Mitochondrial membrane potential levels were detected with TMRE staining,and reactive oxygen species(ROS)levels were detected using MitoSOX staining.Galactose(10 mmol/L),metformin(10μmol/L),NAC(5 mmol/L),and galactose+metformin were used to modulate ROS levels in the CSCs,and sphere-formation assay and flow cytometry were used to assess the changes in self-renewal capacity and Wnt activity.The effect of lentiviral transfection of yeast NADH dehydrogenase NDI1 on TMRE staining,MitoSOX staining and Wnt activity in the CSCs were analyzed with flow cytometry.Results Metformin significantly decreased the capacities of CSCs to form spheres,colonies and xenografts and reduced Wnt activity in the cells(P<0.01).The mRNA levels of stemness-related genes and Wnt target genes decreased significantly while those of differentiation-related genes increased in metformin-treated CSCs(P<0.05),which also showed significantly decreased OCR,TMRE and ROS levels with enhanced ECAR(P<0.001).Galactose significantly increased sphere forming capacity,ROS levels and Wnt activity of the cells,and these effects were significantly inhibited by metformin(P<0.05).Transfection of the CSCs with NDI1 significantly attenuated the inhibitory effects of metformin on proportion of CSCs and Wnt signaling pathway activity.Conclusion Metformin reduces mitochondrial oxidative phosphorylation and ROS levels by inhibiting mitochondrial complex I,thereby suppressing Wnt signaling pathway to reduce self renewal ability of colorectal CSCs.