雷公藤红素通过调控ATF4/caspase-3/GSDME信号通路促进肝癌细胞焦亡

Tripterine promotes pyroptosis of liver cancer cells by regulating ATF4/caspase-3/GSDME signaling pathway

目的探究雷公藤红素(tripterine,TRI)抑制肝癌细胞(hepatocellular carcinoma,HCC)进展的新机制。方法利用不同浓度(0、0.5、2和8μmol·L-1)TRI作用于肝癌细胞,然后利用CCK-8、细胞克隆、Transwell和蛋白免疫印迹等实验方法评估细胞表型和可能机制;再利用siRNA将靶基因GSDME进行干扰,确定GSDME的作用;最后使用移植肿瘤模型验证TRI在体内抑制HCC细胞的生长的机制。结果TRI可抑制HCC细胞增殖、克隆和侵袭,促进细胞焦亡。免疫印迹结果显示,TRI处理后肝细胞癌HepG2或Hepa1-6中GSDME表达均上调,同时cleaved caspase-3和PARP也明显升高。敲除GSDME后可部分逆转TRI诱导的细胞焦亡。同时GSDME敲低后的细胞具有更强的克隆和迁移能力,且与单独的TRI治疗组相比,细胞凋亡率降低。在体内实验中,TRI抑制可HCC肿瘤生长,TRI+siGSDME组小鼠肿瘤生长速度比单独TRI治疗组快。此外,TRI刺激后HepG2和Hepa1-6细胞中p-eIF2a、ATF4均升高。免疫荧光结果显示,TRI刺激后HepG2和Hepa1-6细胞中ATF4阳性细胞数呈剂量依赖性增加。结论TRI抑制肝癌细胞生长和侵袭可能与其调控ATF4/caspase-3/GSDME信号通路促进肝癌细胞焦亡有关。

Aim To explore the new mechanism of triptolide(TRI)inhibiting the progression of hepatocellular carcinoma(HCC).Methods Different concentrations(0,0.5,2,and 8μmol·L-1)of TRI were administered to act on liver cancer cells,and then the cell phenotypes and possible mechanisms were explored using experimental methods such as CCK-8,cell cloning,Transwell,and protein immunoblotting;siRNA was used to interfere with the target gene GSDME and its role was determined.Finally,the mechanism of TRI inhibiting the growth of HCC cells in vivo was validated using a transplanted tumor model.Results TRI could inhibit the proliferation,cloning,and invasion of HCC cells,and promote cell apoptosis.Immunoblotting results showed that the expression of GSDME was significantly upregulated in HepG2 or Hepa1-6 hepatocellular carcinoma after TRI treatment,while the expression of cleaved caspase-3 and PARP also significantly increased.Knocking out GSDME could partially reverse TRI-induced cell apoptosis.At the same time,cells knocked down by GSDME had stronger cloning and migration abilities,and the apoptosis rate was reduced compared to the TRI treatment group alone.In vivo experiments showed that TRI inhibited HCC tumor growth,and the TRI+siGSDME group had a faster tumor growth rate than the TRI treatment group alone did.In addition,after TRI stimulation,p-eIF2a and ATF4 in HepG2 and Hepa1-6 cells significantly increased.The immunofluorescence results showed a dose-dependent increase in the number of ATF4 positive cells in HepG2 and Hepa1-6 cells after TRI stimulation.Conclusion The inhibitory effect of TRI on the growth and invasion of liver cancer cells may be related to its regulation of the ATF4/caspase-3/GSDME signaling pathway and promotion of liver cancer cell apoptosis.