肿瘤耐药相关新蛋白Bex1促凋亡机制及其逆转耐药功能研究

Our previous study found that a small molecule protein Bex1 was silenced significantly in Imatinib/Vincristine and other multi-drugs secondary resistance tumor cells, re-expression of Bex1 in drug-resistant tumor cells can restore neoplasm drug sensitivity by inducing apoptosis. Further studies showed that Bex1 interact with Bcl-2, a multi-functional molecule in anti-apoptosis, and the apoptosis induced by Bex1 is probably associated with the Bex1 translocation from cytosol to mitochondria, while the exact molecular mechanism of apoptosis induced by Bex1 and the relationship with Bcl-2 should be further study. To investigate the complex biological functions of Bex1, We will use multiple technical methods such as Molecular Docking and Site-Mutation in the first step to clarify the crucial amino acids/peptide chain in binding with Bcl-2 domain. Then we will use the changed Bex1 which has mutation or deletion in binding domain as control, to dissect the wild-type Bex1 pro-apoptotic molecular mechanism from exogenous and endogenous as well as endoplasmic reticulum stress signaling pathway, and investigate the biologic effect of interaction between Bex1 and Bcl-2, by using the systemic technologic methods which integrated flow cytometry, fluorescence resonance energy transfer and multiple molecular biology technology. we also will explore the possibility of bex1 binding domain peptide as drug sensitizer and reversal agents by Bcl-2 blocking. In this study, the elucidation of Bex1 pro-apoptotic molecular mechanism provide a basis to further clarify the mechanism of secondary drug resistance of tumor cells and new breakthrough point for the reversal of tumor resistance.

我们前期研究发现Imatinib、Vincristine等多种药物继发性耐药过程中小分子蛋白Bex1显著沉默，重新表达Bex1能促进细胞凋亡恢复药物敏感性。进一步研究发现Bex1能与Bcl-2相互作用，Bex1诱导凋亡与其移位至线粒体有关，但Bex1促进细胞凋亡的确切分子机制及与Bcl-2结合的生物效应尚需进一步深入研究。为此，我们将在本研究中运用分子对接和定点突变技术明确结合关键肽链/氨基酸，继而以缺失/突变结合域的Bex1作为对照，综合流式细胞术、荧光共振能量转移等多种分子生物学技术，从外源性、内源性以及内质网应激凋亡通路研究野生型Bex1的促凋亡机制及Bex1与Bcl-2相互作用的生物学效应，同时我们也将探讨Bex1的Bcl-2结合域作为Bcl-2的封闭多肽开发肿瘤药物增敏剂和耐药逆转剂的可能性。本研究对阐明和完善肿瘤耐药分子机制有重要理论价值，并为逆转肿瘤耐药提供新的突破方案。

本项目前期研究发现Imatinib继发性耐药过程中小分子蛋白BEX1显著沉默，重新表达BEX1能促进细胞凋亡恢复药物敏感性，进一步研究发现BEX1能与BCL-2相互作用，但BEX1促进细胞凋亡的确切分子机制及与BCL-2结合的生物效应尚不明确。本课题的研究内容是解析BEX1与BCL-2结合关键肽链，阐明BEX1调控的细胞凋亡分子机制，明确BEX1与BCL-2的结合对细胞凋亡的影响，并进一步探索BEX1结合结构域作为小分子多肽药物提高抗肿瘤药物疗效、逆转肿瘤耐药性的可行性及适用范围。通过分子克隆技术和免疫共沉淀技术，发现BEX1与BCL-2结合的关键肽链在于第33-64氨基酸，蛋白质细胞内定位分析也显示野生型BEX1蛋白可定位至BCL-2表达的主要细胞器线粒体，而缺失33-64肽链的突变体不能定位至线粒体；BEX1诱导细胞凋亡的机制主要通过与BCL-2分子结合后，竞争性抑制BH3-only蛋白BAX与BCL-2二聚体形成，导致游离BAX水平增加进而启动细胞凋亡；体内试验初步发现BD-BEX1多肽段可部分逆转耐药细胞对Imatinb并呈剂量依赖，且在裸鼠体内具有良好生物安全性，但是BD-BEX1多肽并无逆转肿瘤细胞5-FU耐药的能力。此外，通过研究BEX1在结直肠癌中的功能，发现BEX1表达与肿瘤分期正相关，BEX1沉默后通过JNK/c-Jun途径抑制结直肠癌细胞生长。本课题深入揭示BEX1促进细胞凋亡的确切分子机制，对阐明和完善肿瘤耐药机制有重要理论价值，为逆转肿瘤细胞imtinib耐药现象提供新的解决方案；初步探索BEX1在结直肠肿瘤中的功能和作用机制，有助于进一步深入理解结直肠肿瘤分子生物学机制。本课题目前已接收及发表研究论著9篇，申请专利1项，参加AACR举办的国际性肿瘤研究大会一次，壁报展示BEX1研究成果；出国交流学习2人，与国际顶尖肿瘤专家建立了良好的合作伙伴关系；培养博士研究生4名，硕士研究生3名。