【摘要】 目的:探讨重楼/七叶一枝花(Paris polyphylla)的醇提物单体pp-10诱导人胃癌BGC-823细胞凋亡和自噬及其分子机制。方法:采用MTT法和克隆形成抑制实验观察不同浓度的重楼单体pp-10对人胃癌BGC-823细胞的增殖抑制作用;Hoechst33342染色法检测pp-10作用于人胃癌BGC-823细胞后细胞核形态的改变;Annexin V-FITC/PI双染法检测细胞凋亡;Western blotting检测重楼单体pp-10对细胞凋亡和自噬相关蛋白Caspase-3、Caspase-9、(ADP-核糖)聚合酶(PARP)、Bax、Bcl-2、LC3、P62以及PI3K/Akt信号通路相关蛋白(Akt、p-Akt、m TOR、p-m TOR、P70s6k、p-P70s6k)表达的影响。结果:重楼单体pp-10能显著抑制BGC-823细胞的生长,作用呈时间-效应关系及剂量-效应关系;克隆形成抑制实验表明随着pp-10浓度的增加,细胞克隆形成逐渐减少,与对照组相比有显著差异;在荧光显微镜下观察可见其细胞核固缩、边聚、裂解等细胞凋亡形态学变化;流式细胞术检测显示,随着作用药物浓度的增高,其凋亡率逐渐升高;Western blotting结果表明,随着药物浓度的增加,线粒体相关凋亡信号通路蛋白Caspase9、Caspase3及PARP均出现酶切活化条带,细胞促凋亡蛋白Bax的表达水平增加,抗凋亡蛋白Bcl-2减少,自噬相关蛋白Ⅱ型LC3增加,P62蛋白减少,p-Akt蛋白的表达水平下降,Akt下游蛋白p-m Tor、p-p70S6K表达减少。结论:重楼单体pp-10通过抑制BGC-823细胞增殖,诱导细胞凋亡和自噬,与下调P13K/Akt信号通路有关。更多还原

【Abstract】 Objective: To investigate pp-10,a monomer purified from Paris Polyphylla,induced apoptosis of human gastric carcinoma BGC-823 cells and the possible mechanism behind this effect. Methods: The effects of pp-10 on the proliferation of BGC-823 Cells were evaluated by the MTT assays and colony formation assays. The cellular morphology changes of BGC-823 cells were observed by fluorescence microscopy after Hoechst 33342 staining. Cell apoptosis was detected by Annexin V-FITC / Propidium iodide( PI) double staining using flow cytometry. The expression of apoptotic and autophagy associated proteins as LC3,P62,Caspase-9,Caspase-3,the Caspase substrate poly( ADP-ribose) polymerase( PARP),Bcl-2 proteins family such as Bax,Bcl-2,PI3 k /Akt signaling pathway numbers such as Akt,p-Akt,m TOR,p-m TOR,P70s6 k and p-P70s6 k were detected by Western blotting. Results: Paris Polyphylla monomer pp-10 inhibited the proliferation of BGC-823 cells significantly in dose- and time- dependent manner( P < 0. 05). Cell clone formation inhibition assay demonstrated that cell clones decreased with the increase of drug concentrations. Apoptotic morphology such as cell shrinkage,nuclear condensation,nuclear fragmentation,chromatin condensation were also observed by staining with Hoechst33342 under fluorescence microscope. Flow cytometry analysis showed an increase of the percentage of apoptotic cells in a dose-dependent manner treated with pp-10 for 24 h. Active bands of mitochondria associated apoptotic signaling pathway proteins such as Caspase-3,Caspase-9 and PARP could be seen with western blotting analysis after BGC-823 cells were incubated with different concentrations of pp-10 for24 h. In addition,pp-10 upregulated the expression of Bax,a pro-apoptotic protein,down-regulated the expression of Bcl-2 protein,an anti-apoptotic protein,up-regulated the expression of LC3,down-regulated the expression of P62 protein,and also down-regulated PI3 K / Akt signal pathway as decreasing the phosphorylation of p-m Tor,p-p70S6 K,downstream of Akt. Conclusions: pp-10 inhibits cell proliferation and induces the apoptosis and autophagy of human Gastric carcinoma BGC-823 cells by activation of Bax,down-regulation of Bcl-2 and reduction of PI3 K / Akt signaling pathway.更多还原