SMAC及IAP家族蛋白对直肠癌新辅助同步放化疗疗效的影响及其机理研究

Neoadjuvant chemoradiotherapy is recommended for stage II/III rectal cancer. The concurrent drugs usually used included 5-fluorouracil, capecitabine, oxaliplatin and/or irinotecan. With neoadjuvant chemoradiotherapy, the pathological response rate could reach 60% to 70%, that is, two thirds patients had residual tumor. It is important to improve the chemoradiotherapy sensitization for these patients to improve long-term survival..Apoptosis is essential for tumor development and plays an important role in the responsiveness to chemotherapy and radiotherapy. The intrinsic apoptotic pathway is mediated by mitochondria and activated in response to intracellular stresses including oncogene activation and DNA damage. Second mitochondria-derived activator of caspase [Smac; also called DIABLO, direct inhibitor of apoptosis protein (IAP) binding protein with low pI], a 23-kDa protein produced as a dimmer within the intermembrane space of the mitochondria; SMAC is released from mitochondria in response to anticancer drug or radiation stimuli and promotes apoptosis by antagonizing IAPs. Recent work suggests that SMAC mediated apoptosis is important for the apoptotic responses induced by several anticancer agents and radiation. Overexpression of SMAC and chemically synthesized SMAC mimetics can enhance apoptosis induced by chemotherapy and radiotherapy in several types of tumor cells including rectal cancer cell lines. However, little is known about the radiosensitivity and prognosis prediction of SMAC and IAP family proteins for rectal cancer patients who received neoadjuvant chemoradiotherapy. Therefore, it is essential to initiate systemic and comprehensive study on tissue immunohistochemistry expression of SMAC and IAP family proteins for these patients to explore the prognostic effects. Meanwhile, the effects of Smac knockdown and SMAC mimetics on the radiosensitivity of rectal cancer cells and the molecular mechanisms by which SMAC, IAPs and SMAC mimetics modulate radiosensitivity need be determined. Xenograft models will be used to compare the therapeutic responses of rectal cancer cells with different SMAC status.

新辅助同步放化疗是直肠癌治疗的重要组成部分，但病理完全缓解率偏低。而SMAC模拟物可提高放疗或化疗引起的多种细胞系的凋亡，有放射增敏的作用。但SMAC与直肠癌同步放化疗之间的关系尚缺乏系统研究。课题组前期研究从细胞-裸鼠到临床水平证实了SMAC模拟物对食管癌的化疗增敏作用，据此提出SMAC模拟物对直肠癌的放化疗具有增敏作用的假设。本课题组拟通过对临床标本检测同步放化疗前后SMAC及IAPs蛋白的表达，分析其与临床特征及疗效的相关性；建立SMAC基因敲降的稳定细胞株及XIAP高表达细胞株，并加入SMAC小分子模拟物，研究SMAC敲降、XIAP高表达及模拟物对细胞凋亡的影响；并将细胞系注射裸鼠皮下，建立动物模型，体内验证SMAC对放化疗的增敏效果；检测多种蛋白的表达，分析SMAC增敏机制，了解SMAC及IAPs蛋白在直肠癌发病、预后及疗效中的作用及SMAC模拟物对SMAC低表达直肠癌的增敏效应

尽管经过多学科综合治疗，局部晚期直肠癌的疗效得到很大提高，但这部分患者存在很大的异质性，约2/3的患者经过术前同步放化疗仍有肿瘤残存，甚至约10%的患者为放化疗无效，表现为对治疗抗拒。而我院研究显示同步放化疗后降期明显（术后分期yp0-II期）是总生存率及无病生存率的独立预后因素，表明同步放化疗的近期疗效可以转化为长期生存的获益。如何将放疗抗拒的患者转化为放疗敏感的患者，是临床亟需解决的问题。. 细胞实验表明两种结肠癌细胞SW480和HCT116均为SMAC高表达，另外SW480为cIAP1高表达，HCT116为cIAP1低表达，结合克隆形成实验显示相同剂量下HCT116细胞对放疗比SW480敏感，推测SW480细胞对放疗抗拒可能是凋亡通路相关的。但由于SMAC抗体停产，故结合本课题组成员前期研究，将新的研究策略调整为：YM155对结直肠癌细胞放化疗增敏的可能。虽然SW480细胞表现为对放疗抗拒，但YM155作用后细胞死亡明显增加，有早期死亡和晚期死亡，且S期和G2/M期细胞比例增多，但未观察到凋亡相关或PARP相关蛋白表达增加。转录分析和RT-PCR验证进一步表明YM155对SW480细胞损伤可能与通过抑制DNA修复有关。而放疗对肿瘤细胞杀伤的机制包含DNA单链和双链断裂，放疗诱导的DNA单链断裂为可逆损伤，若结合抑制DNA损伤修复药物，则可将这部分损伤转化为不可逆，从而起到增加放疗疗效的作用。因此，YM155有望成为放疗增敏的药物。