内质网应激蛋白ATF4调控自噬在人胶质瘤细胞替奈唑胺耐药性中的作用及机制

A variety of anticancer agents could induce endoplasmic reticulum（ER） stress, and some adaptive responses involved in this process such as unfolded protein response (UPR) and autophagy were thought to be closely related to chemotherapy resistance .The ER stress protein ATF4 not only is the cross talk of the survival and death of the cell, but also involved in the regulation of ER stress signaling networks; however, the molecular link between these two is still not very clear. As studies focusing on the function of ATF4 at the level of the ER goes far, the research and development of ATF4 which acts the switch role in the survival and death of the glioma cell. Our experiment aims to study the effects of the AFT4 on ER stress,autophagy and apoptosis in temozolomide resistant human glioma U251 cell lines. Experimental protocols in molecular biology and bioinformatics are used to explore the roles for ER stress and autophagy under the regulation of the AFT4,and to clarify the relevant mechanisms for ATF4 regulation of ER stress signaling networks, thereby providing certain theoretical and experimental basis for the role of ER stress protein ATF4 which regulated autophagy in glioma cell lines with temozolomide resistance.

目前认为多种抗肿瘤药物能够诱导细胞内质网应激,其活化的未折叠蛋白反应和自噬等细胞应答反应与肿瘤化疗药物耐受密切相关。我们前期工作已经发现：内质网应激蛋白ATF4在于多种调控细胞生存或死亡信号途径中起串话作用，并引起了内质网应激和自噬相关蛋白表达的变化。因而我们假设：ATF4在决定细胞存活与死亡中扮演开关的角色。一般情况下，替莫唑胺除造成DNA损伤外，还使ATF4通过上调NOXA促进肿瘤细胞凋亡；而耐药细胞中，ATF4转而活化自噬，通过自噬的内稳作用促进肿瘤细胞存活，进而对替莫唑胺耐药。本课题拟以人胶质瘤U251细胞为实验对象，观察ATF4调控替奈唑胺耐药细胞诱导的内质网应激、自噬及凋亡的变化，探讨内质网应激和自噬在耐药过程中的作用；并使用分子生物学与生物信息学等实验方法，进一步明确胶质瘤替奈唑胺耐药的发生机制及分子调节网络，阐述ATF4调控自噬在人胶质瘤细胞替奈唑胺耐药性中的作用及机制。

目前的研究表明，肿瘤的耐药性与内质网应激和自噬等细胞应答反应密切相关。作为内质网应激蛋白的ATF4在决定细胞生存与死亡中起到开关作用。本研究采用U251细胞为实验对象，探讨替奈唑胺（TMZ）作用下ATF4对自噬及凋亡的调控作用。结果发现：（1）TMZ作用下，U251细胞发生了ERS，并参与自噬及凋亡的调控；（2）TMZ作用下，ERS诱导了自噬的发生，缓解ERS，起到了抗凋亡作用。（3）使用eIF2α抑制剂Salubrinal后，增强了TMZ对U251的杀伤效果，NOXA表达升高，证明ATF4通过调控NOXA参与了凋亡过程。(4)抑制自噬可以增强TMZ对U251/TMZ的杀伤效果。这些研究结果显示，TMZ作用于胶质瘤细胞时，发生ERS，ATF4是ERS调控下游事件的重要蛋白，同时介导了自噬及凋亡相关通路的变化，抑制自噬可以增强TMZ的杀伤效果，证明ATF4调控的自噬是脑胶质瘤耐药发生的一个重要原因。