卡格列净激活cGAS-STING通路抑制肝癌细胞增殖

Canagliflozin inhibits proliferation of liver cancer cells by activating cGAS-STING pathway

目的探究卡格列净对肝癌细胞的影响及调控机制。方法卡格列净处理人肝癌细胞HepG2、Huh7和HCCM以及人正常肝细胞MIHA细胞后,通过细胞克隆形成实验和CCK-8实验检测细胞增殖能力,流式细胞术检测细胞和线粒体内活性氧(ROS)水平和线粒体膜电位变化,Western blot法检测增殖相关蛋白和cGAS-STING通路相关蛋白表达情况,RT-qPCR法检测线粒体DNA(mtDNA)释放水平和炎症因子mRNA表达水平。结果与人正常肝细胞MIHA相比,卡格列净对HepG2细胞增殖的抑制作用更显著(P<0.05,半抑制浓度分别为>40μmol·L^(-1)和15μmol·L^(-1))。与对照组相比,卡格列净组肝癌细胞增殖数目、增殖相关蛋白的表达均降低,呈浓度依赖性,细胞内和线粒体ROS水平升高,线粒体膜电位降低,胞质mtDNA释放水平、cGAS-STING通路相关蛋白表达和炎症因子mRNA水平均升高(均P<0.05)。与卡格列净组相比,N-乙酰-L-半胱氨酸(NAC)+卡格列净组细胞内和线粒体ROS水平、胞质mtDNA释放水平,cGAS-STING通路相关蛋白的表达和炎症因子的mRNA水平均降低(均P<0.05)。与卡格列净组相比,cGAS抑制剂RU.521+卡格列净组肝癌细胞增殖活力和增殖相关蛋白的表达均显著增加(均P<0.05)。结论卡格列净诱导肝癌细胞氧化应激和线粒体功能受损,导致mtDNA外泄至胞质中,进而激活cGAS-STING通路,引起细胞内炎症因子水平升高,最终抑制肝癌细胞增殖。

AIM To investigate the effect of canagliflozin on liver cancer cells and its regulatory mechanism.METHODS The effects of canagliflozin on the proliferation of liver cancer cell HepG2,Huh7 and HCCM were detected by cell clone formation assay and CCK-8 assay.Flow cytometry was used to measure changes in reactive oxygen species(ROS)levels,both intracellular and mitochondrial ROS levels,as well as alterations in mitochondrial membrane potential.Western blot was employed to assess the expression of proteins related with proliferation and cGAS-STING pathway.Mitochondrial DNA(mtDNA)release and inflammatory factor mRNA expression level were detected by RT-qPCR.RESULTS Compared with human normal liver cell MIHA,the inhibitory effect of canagliflozin on HepG2 cell proliferation was more significant(P<0.05,semi-inhibitory concentrations were>40μmol·L^(-1)and 15μmol·L^(-1),respectively).Compared with the control group,the clone number of liver cancer cells and the expression of proliferation-related proteins in the canagliflozin group were decreased in a concentration dependent manner(P<0.05).Additionally,the intracellular and mitochondrial ROS levels were increased,the mitochondrial membrane potential was decreased,and the cytoplasmic mtDNA release level,cGAS-STING pathway-related protein expression and pro-inflammatory factor mRNA levels were increased(all P<0.05).Compared with the canagliflozin group,the intracellular and mitochondrial ROS levels,cytoplasmic mtDNA release levels,cGAS-STING pathway-related protein expression and pro-inflammatory factor mRNA levels in the N-acetyl-L-cysteine(NAC)+canagliflozin group were decreased(all P<0.05).Compared with the canagliflozin group,the proliferation activity and the expression of proliferation-related proteins in the cGAS inhibitor RU.521+canagliflozin group were significantly increased(all P<0.05).CONCLUSION Canagliflozin induces oxidative stress in liver cancer cells,leading to the accumulation of ROS within cells and mitochondria,impairment of mitochondrial function,leakage of mtDNA into the cytoplasm,activation of the cGAS-STING pathway,and subsequent release of intracellular pro-inflammatory factors.These processes collectively contribute to the modulation of liver cancer cells proliferation.