线粒体肽NDDP4抑制卵巢癌恶性增殖机制及靶向阻遏研究

Mitochondria are involved in energy metabolism, respiration and apoptosis, and the concept of mitochondrial dysfunction has been set forth as a new hot topics and hard work in cancer research. While there is current agreement that measures of mitochondrial activity and fidelity are abnormal in individuals with cancer, the true role of mitochondrial dysfunction in cancer remains controversial. In my previous work as a postdoctor in UCLA, we propose that a previously unrecognized class of molecules, which we have named the mitochondrial derived peptides (MDPs), constitute key biological molecules that are a central function of mitochondria. In the preliminary unpublished data, we hypothesize that understanding the basic biology and physiological roles of MDPs will shed valuable new light on the nature of mitochondrial function and will delineate currently unexplored new directions in drug design and cancer therapy. If our hypotheses are true, the resultant knowledge will represent a seminal contribution to biology and medicine with paradigm-shifting implications to the field of mitochondrial biology, and may also provide potential novel diagnostic and therapeutic targets for patients with cancer. Our initial characterization of the biological activity of NDDP4 indicates that it is a potent bioactive molecules acting to induce cell apoptosis through the cell-extrinsic pathway and further initiating the intrinsic apoptosis pathway. These observations suggest that the mitochondria is not simply an organelle that integrates signals from other sites within the cell to facilitate respiration and apoptosis; but that it is a critical source of bioactive peptides or named mitokine that serve as signals and mediators of cell survival and apoptosis and whole organism metabolic function. Ingeniously, when the results mentioned above is incorporated with the novel oncolysis adenovirus which combined the characteristics of oncolysis with targeted gene amplication and constructed by ourself previously, tumor-selective replication of the E1A mutant adenovirus would activate the native adenovirus E3 promoters to express the NDDP4 cDNA preferentially in tumor cells and amplify the NDDP4 protein. Thus, the aim of this project is beginning from the unknown small peptide derived from the mitochondria, and from the novel angle of the relationship between the mitochondrial dysfunction and cancer, to further explore the mechanism of the apoptosis induced by NDDP4 and the signal pathway which NDDP4 will be take part in by set up the new technique of mitochondrial activity and fidelity and oncolysis adenovirus construction technique. We believe this targeting NDDP4 gene immunotherapy of ovarian cancer residue/metastasis will shed the new light on cancer recurrence and metastasis, span the key technique barrier in adenovirus gene therapy and ultimately provide a new strategy to prevent the recurrence and metastasis of cancer.

21世纪肿瘤代谢成为肿瘤研究的新热点，而执行着细胞代谢、呼吸和凋亡三大功能的细胞器- - 线粒体与肿瘤的研究成为新的热点及难点。申请人赴美国加州大学UCLA深入研究了线粒体与肿瘤的关系，提出了线粒体肽的概念，成功地筛选出了线粒体凋亡肽，进而提出细胞内可能存在线粒体因子的科学假说，并巧妙地与前期工作中自主创新构建的选择性复制溶瘤腺病毒基因载体的研究成果相结合，从核外遗传物质线粒体DNA编码的未知小分子肽出发，从线粒体功能异常与肿瘤的独特视角，以线粒体凋亡肽NDDP4为切入点，以困扰临床治疗实际的卵巢癌残留/转移灶为突破口，深入探讨NDDP4抑制卵巢癌恶性增殖的机制，并引入线粒体功能分析技术和选择性复制溶瘤病毒构建技术，从应用角度出发探索一条有效清除卵巢癌残留/转移病灶的靶向基因免疫治疗新途径，从而达到探索肿瘤转移治疗新方法、解决腺病毒基因治疗关键技术障碍，最终实现靶向遏制肿瘤复发和转移的目的。

线粒体作为执行细胞代谢、呼吸和凋亡三大功能的细胞器，其与肿瘤的研究是当代国际肿瘤研究领域新的热点及难点。肿瘤患者线粒体的活性及保守性均发生改变，但线粒体异常与肿瘤究竟有无关联？线粒体肽是否就是线粒体因子？能否利用线粒体肽的特点及其凋亡诱导作用，从而指导临床对卵巢癌更加高效、特异的抗肿瘤治疗？. 基于前期研究提出的线粒体肽的概念，本项目利用美国seahorse海马细胞能量代谢实时测定仪等，在对ND-1所编码的5个线粒体肽进行功能分析后，成功地筛选出了线粒体凋亡肽NDDP4。它能引起Caspase-3和9活化，剪切底物产生cleaved PARP蛋白，从而引发以线粒体为核心的内源性细胞凋亡。基因芯片分析提示NDDP4可能是TNF超家族的新成员。在联合生物抗体公司制备针对NDDP4抗原的高纯度特异性抗体后，用131I标记His/NDDP4多肽，使用Pulldown技术结合质谱等方法，我们证实NDDP4通过与细胞表面DR3结合进而连接接头蛋白的死亡结构域，通过激活Caspase-8诱导外源性细胞凋亡。另一方面，我们建立并优化针对NDDP4的in-house-Elisa检测体系，检测它在正常卵巢和卵巢癌患者血浆和体液中的表达及不同种属组织器官的分布，证明了NDDP4是线粒体因子可能性。针对卵巢癌的治疗，我们结合自主创新构建的选择性复制溶瘤腺病毒基因载体，构建以NDDP4为靶点的Adv5/dE1A-NDDP4（M9），通过caspase凋亡通路，骨髓间充质干细胞运载复制型M9选择性的杀伤卵巢癌细胞，而对正常细胞无杀伤作用，从而抑制卵巢癌恶性增殖。. 本项目从应用角度出发探索一条有效清除卵巢癌残留/转移病灶的靶向基因免疫治疗新途径，从而达到探索肿瘤转移治疗新方法、解决腺病毒基因治疗关键技术障碍，为最终实现靶向遏制肿瘤复发和转移的目的提供理论支持。在项目执行期内，经费使用合理，除完成既定目标外还充实了研究内容，为所在实验室开创了选择性复制溶瘤腺病毒靶向基因载体构建的新的技术平台。研究结果已发表SCI论文8篇。