ANTP-SmacN7融合肽辐射增敏作用机理的深入研究

Radiotherapy is an important strategy in achieving the local control of tumors, however, radioresistance limits the curable potential and contributes to local recurrences. Development of therapeutic agents which can sensitize tumor to radiation will be extremely important for improvement of radiotherapy. Increasing evidence suggest that radioresistance of tumor is associated with overexpression of inhibitor of apoptosis proteins (IAPs), a well-known family of anti-apoptotic proteins and plays an important role in radioresistance. Second mitochondria-derived activator of caspase (Smac) is a mitochondrial protein that is released to the cytoplasm in response to induction of apoptosis. Smac interacts with both BIR2 and BIR3 domains of multiple IAPs to release the binding of IAPs to both initiator (caspase 9) and effector (caspase 3/7) caspases. It has been known that Smac is low-expressed in radioresistant tumor cells, exogenous Smac is not allowed free to enter tumor cells. In our preliminary studies, we have synthesized a new peptide, ANTP-SmacN7 fusion peptide which has been demonstrated to sensitizes tumor cells to radiation-induced apoptosis in vitro and in vivo. Based on our previous study, in this study we will further the mechanism of ANTP-SmacN7 mediated radiosensitization in in vitro and in vivo models. Firstly, the expression of Smac, XIAP, cIAP1 and cIAP2 will be detected in tumor cells in which they exhibit different sensitivity to radiotherapy. The relationship between Smac and IAPs in ANTP-SmacN7 mediated radiosensitization will also be analyzed. Secondly, we will study the effect of ANTP-SmacN7 in combination with radiotherapy on XIAP, cIAP1, cIAP2 and other key proteins involved in apoptosis pathway. This is helpful for us to understand the roles of target proteins in ANTP-SmacN7 mediated radiosensitization. Thirdly, the phosphorylation of XIAP protein at ser87 in BIR1 domain is studied for a key factor for the stability of XIAP. Considering the inhibition and instability of XIAP caused by Smac, we will explore if ANTP-SmacN7 has a role for the phosphorylation of XIAP at Ser87. Finally, we will study if p53 takes part in the promotion on mitochondrial pathway of radiation-induced apoptosis mediated by ANTP-SmacN7. It is expected to provide further evidence for ANTP-SmacN7 acting as a new class of assistant anticancer drug, in effectively overcome of apoptosis resistance in different types of tumor cells with high levels of IAPs and sensitize tumor cells to radiotherapy. Indeed, the execution of this study will show great value in both of basis research and clinical application for tumor radiotherapy.

肿瘤的辐射耐受是目前肿瘤病人放疗的瓶颈问题。Smac作为促凋亡蛋白，可与凋亡抑制蛋白(IAPs) 特异结合，解除其凋亡抑制功能，从而提高肿瘤的辐射敏感性。但是辐射耐受的肿瘤细胞IAPs高表达，而Smac蛋白低表达，且外源的Smac不能进入细胞。我们前期研究以ANTP作为引导肽，合成了ANTP-SmacN7融合肽，成功把Smac的活性基团转入细胞并发挥了促凋亡作用。本项目将在前期研究基础上，采用肺癌体外细胞模型和体内动物模型深入研究该融合肽辐射增敏作用的机理，明确ANTP-SmacN7融合肽联合放疗对IAPs表达的改变以及对XIAP ser87磷酸化的影响，并探索P53在该融合肽通过线粒体通路促进细胞凋亡的作用。期望通过一系列临床前研究，为ANTP-SmacN7融合肽作为新的辐射增敏药物提供研究依据和必要的实验基础。因此，本研究无论在肿瘤放疗的基础研究还是将来临床应用都具有重要意义和应用价值

本项目在上一年度探讨了Tat-SmacN7在体内外对乳腺癌SK-BR-3细胞、食管癌Ec109细胞和非小细胞肺癌H460细胞两种不同肿瘤细胞株的辐射增敏作用，阐明了Tat-SmacN7融合肽的辐射增敏作用机制。.（1）细胞分为对照组、Tat-SmacN7组、照射组和联合组。通过细胞克隆形成实验和流式细胞术检测细胞凋亡，评价Tat-SmacN7对两种肿瘤细胞的辐射增敏作用。（2）PCR和Western blot实验观察细胞内caspase分子水平和XIAP蛋白水平的变化，ELISA方法检测caspase活性。利用caspase抑制剂阻断Tat-SmacN7的放射增敏作用，来探讨Tat-SmacN7的辐射增敏作用机制。.结果：（1）Tat-SmacN7单独使用对肿瘤细胞的促凋亡作用不明显，但是可以提高细胞对辐射的敏感性，从而提高肿瘤细胞的凋亡。使用微摩尔级的Tat-SmacN7处理细胞，在辐射相对耐受（H460）和相对敏感（Ec109）细胞株都发现放射增敏作用，对辐射耐受的H460细胞放射增敏作用更明显，SER分别为1.63和1.51。（2）PCR结果显示，联合组细胞的caspase-3、-8和-9的分子表达增高，ELASA证明caspase活性也被激活。使用caspase抑制剂（z-VAD-fmk）可阻断Tat-SmacN7的辐射增敏作用，H460和Ec109细胞的SER分别降到1.17和1.09。.结论：Tat-SmacN7是一种高效、潜在的新型放疗增敏药物。Tat-SmacN7对肿瘤细胞H460和Ec109的辐射增敏作用机制主要是通过提高caspase-3、-8和-9的分子表达，并且提高caspase的活性，体外实验发现有明显的辐射增敏效果。本项目将对发现新的辐射增敏药物、增强放疗效果具有重要的理论指导意义。