拉罗替尼通过AMPK/mTOR信号通路调控结肠癌细胞自噬和抑制增殖与迁移的实验研究

Experimental Study of Larotrectinib Regulating Autophagy and Inhibiting Proliferation and Migration of Colon Cancer Cells Through AMPK/mTOR Signaling Pathway

目的探讨拉罗替尼(Larotrectinib,Lar)对结肠癌细胞自噬、增殖和迁移的影响及其分子机制。方法用不同浓度的Lar(0,100,200,400,800,1600和3200 nmol/L)作用于人结肠癌细胞COLO 205,HCT 116及人结肠黏膜上皮细胞CP-H040。采用CCK-8比色法检测Lar对COLO 205,HCT 116和CP-H040细胞存活率的影响。将COLO 205和HCT 116细胞随机分为对照组(Con组)、Lar组、氯喹组(hydroxychloroquine,CQ组)和Lar+CQ组,采用Transwell实验检测细胞侵袭能力;划痕实验检测细胞迁移能力;Ki67免疫荧光染色检测细胞增殖能力;实时荧光定量聚合酶链式反应检测Lar对结肠癌细胞上皮-间充质转化相关标志物mRNA表达的影响;腺病毒转染实验和透射电子显微镜检测细胞自噬情况;蛋白免疫印迹法检测腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(adenosine monophosphate-activated protein kinase/mammalian target of rapamycin,AMPK/mTOR)通路相关蛋白的表达。结果Lar可显著抑制COLO 205和HCT 116细胞的存活率,具有浓度依赖性(F=355.181,403.758,均P<0.001)。与Con组相比,Lar组结肠癌细胞侵袭细胞数、Ki67荧光强度和划痕愈合率均明显降低,E-钙黏附蛋白(E-cadherin)mRNA表达水平升高,波形纤维蛋白(Vimentin)和基质金属蛋白酶2(MMP2)mRNA表达降低,自噬小体和自噬流形成增多,微管相关蛋白轻链3(LC3)II/I和p-AMPK/AMPK比值升高,p62蛋白表达和p-mTOR/mTOR比值降低,差异具有统计学意义(t=4.399~54.214,均P<0.05)。与Lar组相比,Lar+CQ组细胞自噬小体形成减少,p62蛋白表达升高,差异具有统计学意义(t=2.755~24.784,均P<0.05)。结论Lar能够抑制结肠癌细胞增殖和迁移,其潜在机制与激活AMPK/mTOR信号通路从而诱发细胞内自噬有关。

Objective To investigate the effects of Larotrectinib(Lar)on autophagy,proliferation and migration of colon cancer cells and its molecular mechanism.Methods Human colon cancer cell lines COLO 205,HCT 116 and human colonic epithelial cell line CP-H040 were treated with different concentrations of Lar(0,100,200,400,800,1600 and 3200 nmol/L).CCK-8 assay was used to detect the cell viability of COLO 205,HCT 116 and CP-H040 cells.COLO 205 and HCT 116 cells were randomly divided into control group(Con group),Lar group,Chloroquine group(CQ group)and Lar+CQ group.Cell invasion was detected by Transwell assay.Scratch test was used to detect cell migration ability.Ki67 immunofluorescence staining was used to detect cell proliferation.Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of epithelialmesenchymal transition related markers in colon cancer cells.Autophagy was detected by adenovirus transfection experiment and transmission electron microscopy.Western blot was used to detect the expression of adenosine monophosphate-activated protein kinase/mammalian target of rapamycin(AMPK/mTOR)pathway related proteins.Results Lar significantly inhibited the viability of COLO 205 and HCT 116 cells in a concentration-dependent manner(F=355.181,403.758,all P<0.001).Compared with the Con group,the number of invasive cells,Ki67 fluorescence intensity and scratch healing rate of colon cancer cells in the Lar group were decreased,the expression of E-cadherin mRNA was increased,the expressions of Vimentin and MMP2 mRNA were decreased,the formation of autophagosomes and autophagic flow,the ratio of microtubule-associated protein light chain 3(LC3)II/I and p-AMPK/AMPK were increased,and the expression of p62 protein and p-mtor/mTOR ratio were decreased,with significant differences(t=4.399~54.214,all P<0.05).Compared with the Lar group,the formation of autophagosome was decreased and the expression of p62 protein was increased in the Lar+CQ group,and the difference was statistically significant(t=2.755~24.784,all P<0.05).Conclusion Lar can inhibit the proliferation and migration of colon cancer cells,and the underlying mechanism is related to activation of the AMPK/mTOR signaling pathway and thus inducts autophagy.