塞拉菌素促进人结肠癌细胞自噬体积累抑制其增殖的研究

Selamectin Inhibits the Proliferation of Human Colon Cancer Cells by Promoting the Accumulation of Autophagosome

目的研究塞拉菌素(Sela)对人结肠癌细胞SW480自噬的影响及蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在其中的调控作用。方法采用四甲基偶氮唑盐比色法(MTT)检测Sela对SW480细胞的半抑制浓度IC50;克隆形成方法检测Sela对SW480细胞的长期增殖能力抑制作用;Sela与不同死亡抑制剂、自噬抑制剂联合处理作用于SW480细胞,MTT法检测Sela对SW480细胞活力的影响;pEGFP-LC3质粒及pmCherry-EGFP-LC3串联质粒转染SW480细胞,荧光显微镜观察检测Sela诱导下自噬体和自噬溶酶体的形成情况;蛋白质印迹技术测定自噬相关蛋白LC3、p62以及Akt/mTOR通路相关蛋白的表达情况。结果与对照组相比,Sela处理后SW480细胞活力明显下降,且呈浓度依赖性,IC50为18.3μmol/L;克隆形成实验结果表明,与对照组相比,Sela处理对SW480细胞增殖具有长效抑制作用,差异有统计学意义(P<0.001);随着Sela作用浓度的升高,SW480细胞内的LC3-Ⅱ和p62含量均逐渐上升,自噬体不断累积及自噬溶酶体形成减少;自噬抑制剂Wortmanin与Sela联合处理,减少了LC3-Ⅱ的转换,明显恢复了Sela对SW480细胞的生长抑制作用;而羟氯喹则增加了SW480细胞内LC3-Ⅱ的累积,明显加剧了Sela对SW480细胞的生长抑制。Sela作用后,SW480细胞中p-Akt,p-mTOR以及下游p-p70S6k的蛋白表达量明显下调。结论Sela具有抑制结肠癌SW480细胞的作用,其潜在作用机制可能与抑制Akt/mTOR信号通路从而诱发SW480细胞内自噬体的积累有关。

Objective To determine whether selamectin (Sela) affects autophagy in human colon cancer cells SW480 as well as the role of protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway.Methods The methyl thiazolyl tetrazolium (MTT) assay was performed to examine the half-inhibitory concentrations(IC_(50)) of Sela on SW480 cells.Colony formation assay was used to assess the long-term inhibitory effects of Sela on SW480 cells.The effect of Sela on SW480 cells in combination with different death inhibitors and autophagy inhibitors were determined by MTT assay.The formation of autophagosomes and autophagolysosomes induced by Sela was examined by fluorescence microscopy in SW480 cells transfected with pEGFP-LC3 and pmCherry-EGFP-LC3 tandem plasmids.The autophagyrelated proteins LC3,p62 and Akt/mTOR pathway-associated proteins were detected by Western Bloting.Results The viability of SW480 cells was significantly reduced after Sela treatment in a concentration-dependent manner,with an IC_(50) of18.3μmol/L.Colone formation assay results indicated that compared with the control group,Sela treatment had a long-term inhibitory effect on SW480 cells,and the difference was statistically significant (P<0.001).The levels of LC3-Ⅱand p62in SW480 cells gradually increased,autophagosomes accumulated and the formation of autolysosomes decreased in a dosedependent manner.The co-treatment of Sela with autophagy inhibitor Wortmanin reduced the conversion of LC3-Ⅱand significantly restored the growth inhibitory effect of Sela on SW480 cells,while HCQ increased the accumulation of LC3-Ⅱand significantly exacerbated the growth inhibition of SW480 cells by Sela.The levels of p-Akt,p-mTOR and downstream p-p70S6k were significantly down-regulated in SW480 cells after Sela treatment.Conclusion Sela has a inhibitory effect on colon cancer SW480 cells,and the underlying mechanism may be related to the inhibition of the Akt/mTOR signaling pathway and thus inducing the accumulation of autophagosomes in SW480 cells.