新型Smac模拟物通过RIP1通路的辐射增敏机理研究

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. Increasing evidence suggested that radioresistance of tumor was associated with overexpression of inhibitor of apoptosis proteins (IAPs), a well-known family of anti-apoptotic proteins which played an important role in radioresistance. Smac is a mitochondrial protein that is released to the cytoplasm in response to induction of apoptosis. It has been well known that Smac is low-expressed in radioresistant tumor cells, exogenous Smac could not enter tumor cells freely. In our previous studies, we have synthesized a new peptide, ANTP-SmacN7, which has been demonstrated to sensitizes tumor cells to radiation-induced apoptosis in vitro and in vivo. We also discovered that it’s mechanism was that it can inhibited the activation of XIAP, a member of IAPs, by means of suppression on the phosphorylation of XIAP at Ser87. However, the mechanism of ANTP-SmacN7 interaction with the other two IAPs members, cIAP1 and cIAP2, is unknown till now. .Stimulation factors leads to the formation of an intracellular complex at the cytoplasmic membrane that includes RIP1, cIAP1 and cIAP2. RIP1 is involved in the activation of caspase8, to activate a caspase cascade to mediate apoptosis. However, cIAP1 and cIAP2 could combine with RIP1 and inhibited it’s dissociation. Thus, RIP1 could not activate caspase8. Furthermore, ubiquitination of RIP1 at K377 by cIAP1 leads to the activation of NF-κB pathway, and maintains tumor cell survival. So, deubiquitination of RIP1 is important in maintaining of apoptosis activation of RIP1..According to the facts above, we will explore the radiosensitization mechanisms of Smac mimetic through RIP1 pathway in this study. Firstly, the expression of Smac, cIAP1 and cIAP2 will be detected in tumor cells in which they exhibit different sensitivity to radiotherapy. Secondly, we will study the effect of ANTP-SmacN7 in combination with radiotherapy on the expression of cIAP1, cIAP2 and caspase in apoptosis pathway. Thirdly, ANTP-SmacN7 may promotes the degradation of cIAP1 and cIAP2, that in turn increases the dissociation of RIP1 and activation of caspase-8, then activates the caspase cascade of apoptosis. Finally, Ubiquitination of RIP1 at K377, IKK complex and the activation of NF-κB will be detected. We will study if ANTP-SmacN7 plays a key role in the deubiquitination of RIP1 and maintaining tumor cell apoptosis induced by radiotherapy. It is expected to provide further evidence for ANTP-SmacN7 acting as a new anticancer drug. Indeed, the execution of this study will present great value in both basis research and clinical application of ANTP-SmacN7 in tumor radiotherapy.

肿瘤的辐射耐受降低了放疗疗效，探索辐射增敏药物并明确其机制是放射医学急需解决的课题之一。Smac是目前辐射增敏研究的热点。我们在前期研究中研发并改良了新型Smac模拟物ANTP-SmacN7，可以抑制IAPs家族成员XIAP Ser87的磷酸化进而发挥辐射增敏作用。但该模拟物对其他成员cIAP1,cIAP2的作用尚不明确,它们与RIP1结合并抑制cas8，产生辐射耐受。据此推测ANTP-SmacN7通过RIP1通路来调控细胞凋亡。为验证此假设，我们拟在细胞和动物水平分析ANTP-SmacN7是否促进cIAP1、cIAP2与RIP1的解离，进而激活cas8，促进细胞凋亡，并进一步探索该模拟物能否促进RIP1的去泛素化，维持对cas8的激活。通过CHIP和RNAi等技术获得ANTP-SmacN7通过RIP1通路调控细胞凋亡的证据，明确其辐射增敏机制，为该模拟物作为新的辐射增敏药物提供科学依据。

肿瘤细胞的辐射耐受往往降低放疗疗效，明确肿瘤辐射耐受机制和寻求辐射增敏药物，是放射医学领域急需解决的课题之一。Smac模拟物目前是辐射增敏研究的热点。我们自主研发了新型Smac模拟物ANTP-SmacN7。本研究中，我们发现该模拟物对凋亡抑制蛋白家族其他成员cIAP1和cIAP2的抑制作用，而且可以激活RIP1，进一步激活caspase8，促进辐射诱导的肿瘤细胞凋亡，提高肿瘤的辐射敏感性。另外，我们与法国里昂大学合作，把辐射增敏药物做成纳米颗粒AGUIX，进一步探索了AGuIX纳米颗粒的辐射增敏作用及其机制；第三，项目组又开展了ANTP-SmacN7在高LET射线（重离子：铁粒子和碳粒子）照射的辐射增敏机制研究，发现我们研发的增敏化合物能明显抑制凋亡抑制蛋白的表达，激活重离子照射后肿瘤细胞的凋亡级联，发挥辐射增敏作用。本研究将为新的辐射增敏药物研究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意义和研究价值。