舒尼替尼通过抑制 Akt／mTOR 信号通路诱导肾癌细胞自噬

目的：研究舒尼替尼引起的肾癌细胞出现细胞自噬的机制。方法：以肾癌细胞系ACHN细胞为细胞模型，利用3-(4，5-二甲基)-5-(3-羧甲基苯环)-2-(4-硫基苯)-2H 四唑盐复合物［3-(4，5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium，innersalt，MTS］检测法观察舒尼替尼对ACHN 细胞活性的影响；应用RNA干扰技术敲降自噬相关蛋白Beclin1和微管相关蛋白1轻链3融合蛋白（microtubule associated protein 1 light chain 3 fusion protein, LC3）检测自噬与舒尼替尼引起的细胞凋亡。使用电子显微镜和荧光显微镜观察在舒尼替尼作用下自噬体的形成；蛋白质免疫印迹检测舒尼替尼对LC3-Ⅱ的积累，自噬相关信号通路蛋白激酶B(protein kinase B, PKB/Akt)、哺乳动物雷帕霉素受体( mammalian target of rapamycin, mTOR) 及聚腺苷二磷酸核糖聚合酶( poly ADP-ribose polymerase，PARP) 切割的变化和过量表达，以及敲降Akt检测诱导自噬的变化。结果: 舒尼替尼能显著抑制ACHN的细胞活性，这种作用具有时间和浓度依赖性；敲降自噬相关蛋白Beclin1和LC3减少自噬可改变舒尼替尼引起PARP的切割；透射和荧光显微镜观察结果表明，舒尼替尼引起细胞自噬体明显增加；蛋白质免疫印迹结果显示舒尼替尼增加自噬同时减少了Akt/mTOR的磷酸化。过量表达持续活化的Akt抑制了该化合物引起的自噬，而敲降Akt可促进自噬发生。mTOR抑制剂雷帕霉素能上调舒尼替尼引起的自噬并促进细胞活性的丢失。结论：舒尼替尼通过抑制Akt/mTOR信号通路促进肾癌细胞ACHN的自噬，其诱导的自噬与凋亡有关。

Sunitinib induces autophagy via suppressing Akt/mTOR pathway in renal cell carci-noma

Objective:To determine the mechanism of sunitinib-induced autophagy in renal cell carci-noma cells.Methods:MTS assay was applied to detect the cell viability alteration under the treatment of sunitinib (2,8 μmol /L).The sunitinib-induced autophagy as well as cell apoptosis was measured and compared after knocking down autophagy-related protein Beclin1 and microtubule associated protein 1 light chain 3 fusion protein (LC3)by RNA interference.The transmission electron microscope was used to observe the formation of autophagosomes in ACHN cells.The fluorescence microscope was used to mo-nitor distribution and aggregation of endogenous LC3-Ⅱ.The expressions of protein such as LC3-Ⅱ,the autophagic regulation molecules protein kinase B /mammalian target of rapamycin (Akt/mTOR)and the symbol of apoptosis poly ADP-ribose polymerase (PARP)were capable to be detected by immunoblotting assay.Results:Sunitinib was able to significantly trigger cell viability loss in the renal carcinoma cell ACHN,which was both in a concentration-dependent and time-dependent manner (P <0.05 ).After reducing the autophagy by knocking down Beclin1 and LC3,the number of cleavage of PARP was in-creased remarkably,whereas there was nearly not any cleavage in the mock group.By the transmission electron microscope,there were more autophagic vacuoles in ACHN cells after being administrated with sunitininb compared with the control.And the nuclear-to-cytosol translocation as well as aggregation of LC3-Ⅱ was presented after sunitinib treatment by the fluorescence microscope,which was the proof of the enhanced autophagy.According to the immunoblotting,sunitinib was able to increase the accumula-tion of LC3-Ⅱ.At the same time,the result of sunitinib combined with chloroquine,a drug which blocked the fusion of autophagosomes and lysosomes,demonstrated that the increasing amount of LC3-Ⅱwas due to the enhanced autophagy flux by sunitinib treatment in ACHN cells.However,phosphorylation of Akt as well as mTOR was decreased at the same time.The rapamycin (mTOR inhibitor)or knocking down Akt subunits could change the sunitinib-induced LC3-Ⅱ accumulation,whereas overexpression of Akt subunits decreased the autophagic flux,indicating that Akt/mTOR was the target of sunitinib in auto-phagy.Conclusion:Sunitinib induced autophagy via suppressing Akt/mTOR pathway,and the auto-phagy was involved in apopotosis.