Smac类似物ANTP-SmacN7通过IAPs通路的辐射增敏机制研究

Radiotherapy is an important strategy in achieving the efficient control of tumor. However, radio-resistance of tumor cells limits the curable potential and contributes to local recurrences. Discovery for mechanisms of tumor radiation tolerance and development of therapeutic agents which can sensitize tumor to radiation will be extremely important for improvement of radiotherapy. It has been well known that Smac is low expressed in radioresistant tumor cells, exogenous Smac could not enter tumor cells freely. This project to perform the study on tumorous radiation sensitization using the two endpoints of Smac and IAPs. It is one of the advancing front of radiation biology and radiation oncology. The high level expression of IAPs in tumor cell can suppress the apoptosis caspase, then suppress the apoptosis which induced by radiation and depress the tumor sensitivity to radiotherapy. Smac can disengage the suppress function to apoptosis of IAPs by binding it directly. But the express of Smac in the radiation resistant cell is low level. Smac can enhance the apoptosis which induced by radiation when import it to tumor cells. This study will link SmacN7, the active group of Smac, with ANTP, a leader peptide, then the SmacN7 can be imported to tumor cells by ANTP. Therefore we can study the influence to XIAP, cIAP1, cIAP2, caspase-9, caspase-3 and caspase-8 by the fusion protein, ANTP-SmacN7. 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, thephosphorylation 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.Then elucidate the mechanism of ANTP-SmacN7 enhance cell apoptosis induced by radiation. The great value and practical importance of this study is to explore the new radiation sensitization drugs and increase curative effect of radiotherapy.

众所周知，肿瘤的辐射耐受机制和寻求辐射增敏靶点，是放射生物学领域的前沿热点问题。肿瘤细胞内Smac低表达是产生辐射抗性的分子基础，且外源的Smac不能进入细胞。我们在前期工作中以ANTP作为引导肽，合成了Smac类似物ANTP-SmacN7，把外源的Smac活性基团导入乳腺癌细胞内，发现其具有辐射增敏作用，但其具体机制尚待明确。因此，本项目拟在前期工作基础上，采用乳腺癌细胞模型和动物模型深入研究该融合肽的辐射增敏机制，明确其对细胞凋亡关键因子IAPs家族成员XIAP、cIAP1和cIAP2等的影响，阐明ANTP-SmacN7是否通过抑制XIAPser87的磷酸化进而抑制其功能。本研究将为该模拟物作为新的辐射增敏药物提供研究依据和必要的实验基础，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的研究价值和实用意义。

电离辐射为很多患者提供了有效的治疗手段，然而，细胞往往具有不同程度的辐射耐受，因此，明确细胞产生耐辐射的机制和寻求辐射增敏方法和药物是需要解决的课题。Smac是促凋亡蛋白，可降低凋亡抑制蛋白XIAP的活性，提高细胞的辐射敏感性，但Smac与XIAP作用机理尚不明确。辐射耐受的细胞XIAP高表达，而Smac低表达，且外源的Smac不能进入细胞。我们前期研究合成了ANTP-SmacN7融合肽，成功把Smac转入细胞并发挥辐射增敏作用，发现该多肽可诱导PKC失活。本项目将在前期基础上，采用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的作用机理，探索ANTP-SmacN7通过诱导PKC失活，降低XIAP ser87的磷酸化水平，促使其失活，发挥辐射增敏作用，并探索P53是否参与了该融合肽在线粒体通路促进细胞凋亡的作用。本研究首先明确了 Smac 和 cIAP1 和 cIAP2 蛋白在不同肿瘤细胞中的表达水平与放射敏感性的关系；其次，明确了 ANTP-SmacN7 联合照射对 cIAP1 和 cIAP2 蛋白水平的影响；第三，明确了ANTP-SmacN7融合肽联合辐射对细胞周期、凋亡和DNA损伤的影响。第四，明确了ANTP-SmacN7融合肽与XIAP特异结合后，XIAP结构的变化与其Ser87位点磷酸化的影响。明确了Ser87位点磷酸化在ANTP-SmacN7融合肽放射增敏的作用。本研究结果为ANTP-SmacN7作为新的辐射增敏药物提供基础研究依据，这将在临床应用中具有重要意义。