Rip2诱导人胰腺癌细胞自噬和凋亡的交互作用及机制研究  

Crosstalk between autophagy and apoptosis induced by Rip2 and its mechanisms in human pancreatic cancer cells

作　　者：周晗 杨文欣 王华东 胡巢凤 ZHOU Han;YANG Wen-xin;WANG Hua-dong;HU Chao-feng(Department of Pathophysiology,Key Laboratory of Pathophysiology,State Administration of Traditional Chinese Medicine of The People’s Republic of China,School of Medicine,Jinan University,Guangzhou 510632,China;Foshan Chancheng Central Hospital,Foshan 528031,China)

机构地区：[1]暨南大学基础医学院病理生理学系,国家中医药管理局病理生理学实验室,广东广州510632 [2]佛山市禅城区中心医院,广东佛山528031

出　　处：《中国病理生理杂志》2020年第7期1199-1206,共8页Chinese Journal of Pathophysiology

基　　金：广东省自然科学基金资助项目(No.S2012010008161)。

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。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-Ⅱwas 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 associ

关 键 词：PANC-1细胞 自噬 细胞凋亡 受体相互作用蛋白2 PI3K/Akt/mTOR信号通路 

分 类 号：R735.9[医药卫生—肿瘤] R730.23[医药卫生—临床医学]