【摘要】 目的:探讨受体相互作用蛋白2(Rip2)诱导人胰腺癌细胞自噬和凋亡的交互作用及其机制。方法:用jetPRIME将pEGFP-C2空质粒和pEGFP-Rip2重组质粒分别转染至人胰腺癌Panc-1细胞。用自噬抑制剂3-甲基腺嘌呤(3-MA)作用于过表达Rip2的细胞,流式细胞术检测细胞凋亡率;Western blot检测凋亡相关蛋白的表达;比色法检测caspase-8、-9、-3的活性。此外,用caspases抑制剂Z-VAD-FMK作用于过表达Rip2的细胞,Western blot检测自噬相关蛋白及PI3K/Akt/mTOR通路相关蛋白的表达;透射电子显微镜观察自噬体的数量及形态。结果:(1)pEGFP-Rip2组细胞凋亡率显著高于对照组和pEGFP-C2组(P<0. 05),而pEGFP-Rip2+3-MA组细胞凋亡率进一步升高(P<0. 05);与pEGFP-Rip2组比较,pEGFP-Rip2+3-MA组细胞Fas、Bax和胞浆细胞色素c(Cyt-c)蛋白表达水平显著升高(P<0. 05),Bcl-2蛋白表达水平则显著下降(P<0. 05);pEGFP-Rip2+3-MA组细胞caspase-8、-9、-3的活性显著高于pEGFP-Rip2组(P<0. 05)。(2)pEGFP-Rip2+Z-VAD-FMK组细胞beclin-1和LC3-Ⅱ蛋白表达水平显著高于pEGFP-Rip2组(P<0. 05),细胞内自噬体的数量亦显著多于pEGFP-Rip2组(P<0. 05);此外,与pEGFP-Rip2组比较,pEGFP-Rip2+Z-VAD-FMK组细胞p-Akt和p-mTOR蛋白表达水平显著降低(P<0. 05),而mTOR及Akt蛋白水平无显著改变。结论:抑制胰腺癌细胞自噬可促进Rip2诱导的细胞凋亡,其机制可能与进一步激活内、外源性凋亡途径有关。抑制胰腺癌细胞凋亡可促进Rip2诱导的细胞自噬,其机制可能与进一步抑制PI3K/Akt/mTOR信号通路的活化有关。因此,Rip2诱导的胰腺癌细胞自噬和凋亡之间可能具有交互拮抗作用。更多还原

【Abstract】 AIM:To investigate the crosstalk between autophagy and apoptosis caused by receptor-interacting protein 2(Rip2)and its underling mechanisms in human pancreatic cancer cells.METHODS:Plasmids(pEGFP-C2 and pEGFP-Rip2)were transfected into human pancreatic cancer Panc-1 cells by jetPRIME method. The Panc-1 cells transfected with pEGFP-Rip2 were treated with 3-methyladenine(3-MA),an autophagy inhibitor. The apoptotic rate was analyzed by flow cytometry. The levels of apoptosis-associated proteins were measured by Western blot. The activity of caspase-8,-9 and-3 was examined by colorimetric method. Moreover,the Panc-1 cells transfected with pEGFP-Rip2 were treated with Z-VAD-FMK,a broad inhibitor of caspases. Subsequently,the levels of autophagy-and PI3 K/Akt/mTOR signaling pathway-related proteins were assessed by Western blot. The autophagosomes were observed under transmission electron microscope.RESULTS:(1)The apoptotic rate in pEGFP-Rip2 group markedly increased as compared with control group and pEGFP-C2 group,while the apoptotic rate in pEGFP-Rip2+3-MA group was further elevated compared with pEGFP-Rip2 group(P<0. 05). Meanwhile,the protein levels of Fas,Bax and cytoplasmic cytochrome c(Cyt-c)were significantly increased,and the protein expression of Bcl-2 was markedly reduced in pEGFP-Rip2+3-MA group as compared with pEGFP-Rip2 group(P<0. 05). The activity of caspase-8,-9 and-3 in pEGFP-Rip2+3-MA group was higher than that in pEGFP-Rip2 group.(2)The protein expression of beclin-1 and LC3-Ⅱ was significantly increased and more accumulated autophagosomes were observed under transmission electron microscope in pEGFP-Rip2+Z-VAD-FMK group as compared with pEGFP-Rip2 group. Furthermore,the protein levels of p-mTOR and p-Akt in pEGFP-Rip2+Z-VAD-FMK group were markedly reduced compared with pEGFP-Rip2 group,while no significant difference of mTOR and Akt protein expression was found between the 2 groups.CONCLUSION:Inhibition of autophagy promotes apoptosis induced by Rip2 in the pancreatic cancer cells. Its mechanism may be associated with the further activation of the intrinsic and extrinsic apoptotic pathways. Suppression of apoptosis accelerates autophagy induced by Rip2 in the pancreatic cancer cells,and the mechanism may be related to the further down-regulation of PI3 K/Akt/mTOR signaling pathways. There is a mutual antagonistic effect between autophagy and apoptosis caused by Rip2 in pancreatic cancer cells.更多还原