高三尖杉酯碱阻断mTOR信号通路抑制HT29人结直肠肿瘤细胞增殖并诱导其凋亡

Blockage of mTOR signaling pathway by homoharringtonine inhibits proliferation and induces apoptosis of HT29 human colorectal tumor cells

目的探讨高三尖杉酯碱(HHT)对HT29人结直肠肿瘤细胞增殖和凋亡调控的分子机制。方法用(0、0.0078125、0.015625、0.03125、0.0625、0.125、0.25、0.5、1、2、4、8)μg/m L HHT分别处理HT29细胞24、48、72 h。CCK-8法检测HT29细胞的增殖,集落形成实验检测细胞的集落形成能力;Hoechst33258染色观察染色质凝集情况,流式细胞术检测细胞凋亡;实时定量PCR检测BAX、Bcl2、胱天蛋白酶3(caspase-3)、caspase-9、哺乳动物雷帕霉素靶蛋白(mTOR)、磷脂酰肌醇3激酶(PI3K)、丙酮酸脱氢酶激酶(PDK1)、蛋白激酶B(AKT)、雷帕霉素靶蛋白调节相关蛋白(raptor)、雷帕霉素不敏感的伴侣蛋白(rictor)的mRNA水平;Western blot法检测BAX、Bcl2、胱天蛋白酶3前体(pro-caspase-3)、裂解型caspase-3(c-caspase-3)、pro-caspase-9、裂解型caspase-9(c-caspase-9)、多腺苷二磷酸核糖聚合酶(PARP)、裂解型PARP(c-PARP)、mTOR、raptor、rictor、PI3K、PDK1、AKT、p-AKT的蛋白水平。结果与对照组相比,HHT能抑制HT29细胞增殖,诱导细胞核破裂,染色质凝聚,凋亡小体形成;同时,HHT处理能够提高HT29细胞中BAX/Bcl2比值、caspase-3、caspase-9、raptor的mRNA水平,降低PI3K、AKT、rictor的mRNA水平;并上调HT29细胞中c-caspase-3、c-caspase-9、c-PARP、BAX、raptor蛋白水平,下调pro-caspase-3、pro-caspase-9、PARP、PI3K、PDK1、AKT、mTOR、rictor蛋白水平。结论 HHT能够通过阻断mTOR信号通路,进而抑制HT29人结直肠肿瘤细胞增殖并诱导其凋亡。

Objective To investigate the molecular mechanisms underlying the effect of homoharringtonine（ HHT） on the proliferation and apoptosis in HT29 human colorectal tumor cells. Methods HT29 cells were treated by 0,0. 007812,0. 015625,0. 03125,0. 0625,0. 125,0. 25,0. 5,1,2,4,8 μg/m L HHT for 24,48 and 72 hours. CCK-8 assay was used to assess the cell viability. Colony formation assay was performed to detect the cell proliferation ability. Flow cytometry was used to analyze cell apoptosis. Hoechst33258 fluorescent staining was used to observe the morphology of the cell nuclei. The real-time quantitative PCR was used to detect the mRNA expressions of BAX,Bcl2,caspase-3,caspase-9,mammalian target of rapamycin（ mTOR）,PI3 K,pyruvate dehydrogenase kinase-1（ PDK1）,protein kinase B（ AKT）,raptor,rictor.The protein levels of Bax,Bcl2,pro-caspase-3,cleaved caspase 3（ c-caspase-3）,pro-caspase-9,cleaved caspase-9（ c-caspase-9）,poly（ ADP-ribose） polymerase（ PARP）,cleaved PARP（ c-PARP）,mTOR,raptor,rictor,PI3 K,PDK1,AKT,p-AKT were detected by Western blotting. Results Compared with the control group,the proliferation of HT29 cells was inhibited when treated with HHT. Meanwhile,the nuclear fragmentation,chromatin condensation,and apoptotic body of the cells could be observed. Treatment of HHT could increase the mRNA expressions of BAX/Bcl2,caspase-3,caspase-9 and raptor,and decrease PI3 K,AKT and rictor in the HT29 cells. The protein levels of pro-caspase-3,pro-caspase-9,PARP,PI3 K,PDK1,AKT,mTOR,and rictor were down-regulated,and the c-caspase-3,c-caspase-9,c-PARP,BAX and raptor were up-regulated. Conclusion HHT has the function of inhibiting the HT29 cell proliferation and inducing its apoptosis by blockage of mTOR signaling pathway.