仑伐替尼通过下调IGF1R/Mek/Erk信号通路治疗瑞戈非尼耐药的肝细胞癌

Lenvatinib down-regulates IGF1R/Mek/Erk signaling pathway in the treatment of regorafenib-resistant hepatocellular carcinoma

目的探讨仑伐替尼对瑞戈非尼耐药肝癌细胞的治疗疗效及其作用机制。方法采用细胞计数试剂盒8(CCK-8)和克隆形成实验观察仑伐替尼对肝癌细胞生长的抑制作用,流式细胞术检测仑伐替尼处理后瑞戈非尼耐药肝细胞癌细胞的凋亡情况,Western blot和免疫组化染色检测相关蛋白表达水平变化,小鼠皮下成瘤实验观察仑伐替尼对瑞戈非尼耐药肝癌细胞体内成瘤能力的抑制效果。结果CCK-8和克隆形成实验显示,仑伐替尼能够抑制肝癌瑞戈非尼耐药细胞的增殖能力;仑伐替尼组HepG2、SMMC7721和瑞戈非尼耐药的HepG2、SMMC7721细胞克隆数[分别为(120.67±11.06)个、(53.00±11.14)个、(55.00±9.54)个和(78.67±14.64)个]均低于对照组[分别为(478.00±24.52)个、(566.00±27.87)个、(333.67±7.02)个和(210.00±12.77)个,均P<0.05]。流式细胞术检测显示,仑伐替尼能够促进肝癌瑞戈非尼耐药细胞凋亡,仑伐替尼组HepG2、SMMC7721和瑞戈非尼耐药的HepG2、SMMC7721细胞凋亡率[分别为(12.30±0.70)%、(9.83±0.38)%、(15.90±1.32)%和(10.60±0.00)%]均高于对照组[分别为(7.50±0.87)%、(5.00±1.21)%、(8.10±1.61)%和(7.05±0.78)%,均P<0.05]。仑伐替尼处理后凋亡相关蛋白比值提示细胞凋亡能力增加。动物实验显示仑伐替尼治疗能够在体内抑制肝癌瑞戈非尼耐药细胞的生长。免疫组化及Western blot结果显示,仑伐替尼能够下调瑞戈非尼耐药细胞中异常活化的IGF1R/Mek/Erk信号通路。结论仑伐替尼能够逆转肝细胞癌瑞戈非尼耐药,其机制可能是通过下调IGF1R/Mek/Erk信号通路实现。

Objective To investigate the therapeutic effect and mechanism of lenvatinib on regorafenib-resistant hepatocellular carcinoma cells.Methods CCK-8 and clone formation assay were used to observe the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma cells.Flow cytometry was used to detect the apoptosis of regorafenib-resistant hepatocellular carcinoma cells treated with lenvatinib.The expression levels of related proteins were detected by western blot and immunohistochemical staining.The inhibitory effect of lenvatinib on the tumor formation ability of regorafenib-resistant hepatocellular carcinoma cells in vivo was observed by subcutaneous tumor formation experiment in mice.Results CCK-8 and clone formation assay showed that lenvatinib could inhibit the proliferation of regorafenib-resistant hepatocellular carcinoma cells.The number of clones of HepG2,SMMC7721 and regorafenib-resistant HepG2,SMMC7721 cells in lenvatinib group(120.67±11.06,53.00±11.14,55.00±9.54,78.67±14.64)were all lower than those in control group(478.00±24.52,566.00±27.87,333.67±7.02,210.00±12.77,all P<0.05).Flow cytometry showed that lenvatinib could promote apoptosis of regorafenib-resistant hepatocellular carcinoma cells,the apoptosis rates of HepG2,SMMC7721 and regorafenib-resistant HepG2,SMMC7721 cells in lenvatinib group[(12.30±0.70)%,(9.83±0.38)%,(15.90±1.32)%,(10.60±0.00)%]were all higher than those in control group[(7.50±0.87)%,(5.00±1.21)%,(8.10±1.61)%,(7.05±0.78)%,all P<0.05].The apoptosis-related protein levels suggested that apoptosis was increased in the treatment of lenvatinib.The animal study showed that lenvatinib can inhibit the growth of regorafenib-resistant cells in vivo.Immunohistochemistry and western blot results showed that lenvatinib could down-regulate the abnormally activated IGF1R/Mek/Erk signaling pathway in regorafenib-resistant cells.Conclusion Lenvatinib can reverse regorafenib resistance in hepatocellular carcinoma,possibly by down-regulating IGF1R/Mek/Erk signaling pathway.