多拉菌素通过线粒体信号通路诱导食管癌细胞凋亡

Doramectin induced apoptosis of esophageal carcinoma cells through mitochondrial signaling pathway

目的:探讨多拉菌素(doramectin,DRM)在体内、体外对食管癌细胞增殖、凋亡的影响及其作用机制。方法:DRM处理Eca109和EC9706细胞,检测细胞在24,48,72 h存活率;药物处理48 h后,观察两种细胞的细胞形态变化,细胞迁移增殖能力;药物干预48 h后,检测Eca109细胞周期和凋亡的变化,B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Cytochrome-c、Caspase-3和Caspase-9蛋白表达水平以及凋亡相关基因Bax、Bcl-2、Caspase-3和Caspase-9变化情况;为进一步探究在体内DRM对食管癌是否有凋亡作用,建立裸鼠荷瘤模型,利用免疫组织化学法检测Caspase-3和Caspase-9表达情况。结果:DRM能抑制Eca109和EC9706细胞增殖,并以浓度依赖方式诱导其发生凋亡(P<0.01),同时抑制细胞迁移(P<0.05)。通过透射电子显微镜观察,经DRM处理后Eca109和EC9706细胞体积缩小、染色质浓缩并出现凋亡小体,而且Eca109细胞经40μmol·L^(-1) DRM处理后,其凋亡率(34.02±2.03)%显著高于对照组(2.98±1.57)%(P<0.001)。同时Eca109细胞周期被阻滞在G2/M期(P<0.01),Bax表达量上调而Bcl-2表达量下调,凋亡相关蛋白Cytochrome-c、Caspase-3和Caspase-9表达量均显著上调(P<0.05),免疫组化结果显示,多拉菌素在体内同样可以抑制食管癌细胞的增殖作用(P<0.05),并促进凋亡蛋白表达,但在转录组测序结果中,与对照组相比,凋亡相关基因Bax、Bcl-2、Caspase-3和Caspase-9,表达无显著性差异。结论:DRM在体内和体外均能诱导食管癌细胞发生凋亡,其机制可能与线粒体信号通路转录后调控或翻译后调控相关,DRM可能成为治疗食管癌的有效药物。

OBJECTIVE To explore the effect and mechanism of doramectin(DRM)on the proliferation and apoptosis of esophageal carcinoma cells in vitro and in vivo.METHODS Eca109 and EC9706 cells were treated with DRM for detecting the survival rate of cells at 24/48/72 h.After 48-hour treatment of DRM,the morphological changes,migration and proliferation of two types of cells were observed.After 48-hour drug intervention,the changes of Eca109 cell cycle and apoptosis,the expression levels of Bax,Bcl-2,Cytochrome-c,Caspase-3 and Caspase-9 and the changes of apoptosis-related genes Bax,Bcl-2,Caspase-3 and Caspase-9 were detected.For further exploring whether DRM had apoptotic effect on esophageal cancer in vivo,a tumor-bearing model of nude mice was established and the expression of Caspase-3 and Caspase-9 detected by immunohistochemistry.RESULTS DRM suppressed cellular proliferation and induced apoptosis in a concentration-dependent manner(P<0.01).And DRM suppressed cellular migration(P<0.05).Transmission electron microscopy revealed that cellular volume declined,chromatin became concentrated and apoptotic bodies appeared after DRM treatment.And the apoptotic rate of Eca109 cell after DRM treatment was significantly higher than that of control[(34.02±2.03)%vs(2.98±1.57)%,P<0.001].Furthermore,Eca109 cell cycle was blocked in G2/M stage(P<0.001).Bax expression was up-regulated while Bcl-2 expression down-regulated.The expression levels of Cytochrome-c,Caspase-3 and Caspase-9 of apoptosis-related proteins were significantly up-regulated(P<0.05).Immunohistochemistry indicated that DRM also suppressed the proliferation of esophageal cancer cells in vivo(P<0.05)and promoted apoptotic protein expression.Hpwever,based upon the transcriptome sequencing results,as compared with control group,the expressions of apoptosis-related genes Bax,Bcl-2,Caspase-3 and Caspase-9 showed no significant difference.CONCLUSION DRM can induce apoptosis of esophageal cancer cells both in vitro and in vivo.Its mechanism may be correlated with post-transcriptional or post-translational regulation of mitochondrial signaling pathway.DRM may become an effective drug for the treatment of esophageal cancer.