BH3-only蛋白模拟物S1通过内质网应激信号网络逆转人卵巢癌细胞耐药机制的研究

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 anti-apoptotic protein Bcl-2 not only is related to drug resistance, but also involved in the regulation of endoplasmic reticulum stress signaling networks; however, the molecular link between these two is still not very clear. As studies focusing on the function of Bcl-2 at the level of the ER goes far, the research and development of BH3-only protein mimetics which shows target inhibition of the anti-apoptotic protein Bcl-2 especially provide us with new possibilities. Our experiment aims to comparatively study the effects of the BH3-only protein mimetics S1 and the control group ABT737 on endoplasmic reticulum stress,autophagy and apoptosis in cisplatin- resistant human ovarian cancer cell lines. Experimental protocols in molecular biology and bioinformatics are used to explore the roles for endoplasmic reticulum stress and autophagy in the anti-tumor process of the BH3-only protein mimetics,and to clarify the relevant mechanisms for Bcl-2 regulation of endoplasmic reticulum stress signaling networks, thereby providing certain theoretical and experimental basis for the role of the BH3-only protein mimetics S1 in the reversal of chemo-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：由本课题合作单位大连理工大学开发的BH3-only蛋白模拟物S1可通过抑制抗凋亡蛋白Bcl-2/Mcl-1来诱导卵巢癌顺铂耐药细胞凋亡，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：内质网应激信号通路可能在BH3-only蛋白模拟物诱导的肿瘤细胞凋亡过程中发挥重要作用，Bcl-2可能是耐药细胞应答反应的核心调控因子。本课题拟以BH3-only蛋白模拟物ABT-737为对照，比较S1、ABT-737分别作用于卵巢癌顺铂耐药细胞诱导的内质网应激、自噬及凋亡之间的差异，探讨内质网应激和自噬在S1抗肿瘤过程中的作用；并使用分子生物学与生物信息学等实验方法，进一步明确抗凋亡蛋白Bcl-2调控内质网应激信号网络的机制，为S1逆转肿瘤耐药机制提供理论依据。

肿瘤细胞为了适应特殊的环境、保证自身生长与增殖，需要自噬、内质网应激等适应性反应维持其特有的内稳态。如何打破这个特有内稳态是抑制肿瘤细胞增殖、克服肿瘤耐药的新的途径。为了达到这些目的，我们首先从细胞凋亡和自噬等角度探讨卵巢癌顺铂耐药机制，首次证明作为自噬底物的p62 还可通过调控Keap1-Nrf2-ARE 信号途径，激活细胞内抗氧化基因的表达，降低了细胞ROS 水平，导致细胞耐药的形成。发现化疗药物在诱导肿瘤细胞发生凋亡的同时，伴有自噬的发生，自噬通过清除泛素化的错误折叠蛋白，可以减弱内质网应激水平，减少凋亡的发生是导致肿瘤耐药发生的重要机制之一。其次，从线粒体、内质网、溶酶体等亚细胞器角度，探讨Ca2+信号在细胞器之间直接与间接交叉对话，进一步阐明细胞自噬与凋亡之间的交互调控机制，发现顺铂可以诱导Ca2+从内质网进入线粒体，引起线粒体的钙超载导致顺铂敏感的肿瘤细胞发生凋亡，而肿瘤耐药细胞对内质网应激耐受可以抑制内质网应激诱导的细胞凋亡，阻断内质网和线粒体Ca2+信号的失衡，维持细胞的存活。最后，利用抗凋亡蛋白Bcl-2抑制剂S1、ABT737等BH3-only蛋白小分子模拟化合物，发现Bcl-2参与了内质网应激-自噬途径的调控，既增强氧化应激的细胞毒作用，造成自噬-内质网失衡，又可能促进线粒体分裂、诱导过度线粒体自噬，还可上调Sirt3的表达，并促进其从细胞核转位至线粒体，抑制肿瘤细胞的糖代谢，明显增强抗肿瘤药物敏感性，说明Bcl-2家族可以通过多途径调控肿瘤的耐药，Bcl-2是耐药肿瘤细胞复杂信号网络中的关键基因，从分子水平阐明Bcl-2调控内质网应激相关蛋白诱导自噬的机制，对于未来抗肿瘤药物开发具有重要参考价值。