c-MET对人食管鳞癌Eca109细胞放射敏感性影响的相关性实验研究

发布时间：2017-12-28 17:38

本文关键词：c-MET对人食管鳞癌Eca109细胞放射敏感性影响的相关性实验研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景与目的:食管癌是最常见的消化道恶性肿瘤之一,全世界每年新发病例在增加,其中,半数以上食管癌发生在中国。据《2015中国癌症统计》报道,我国食管癌发病率已跃居全国恶性肿瘤发病的第3位,死亡居第4位。手术、放疗以及化疗等相结合的多学科综合治疗是食管癌的主要治疗模式。虽然近年来各种治疗手段都有了很大程度的发展,但食管癌总体预后仍较差,5年生存率仅15%～25%。另外,患者预后个体差异也较大,这很大程度上取决于食管癌的生物学行为,尤其是不同基因特征对肿瘤增殖、侵袭、转移及治疗疗效产生至关重要的影响。目前食管癌的特异性分子标志物并不明确。MET(Mesenchymal-epithelial Transition Factor)是肝细胞生长因子(Hepatocyte Growth Factor,HGF)的细胞表面受体,也被叫做c-Met或HGFR(Hepatocyte Growth Factor Receptor),属于酪氨酸激酶受体(Receptor Tyrosine Kinase,RTK)家族成员,由c-Met基因编码。异常的RTK信号途径在肿瘤的发生、发展、侵袭、转移以及耐药的过程中具有重要作用,而c-Met信号通路正属于RTK信号通路。研究发现c-Met信号通路的激活与多种肿瘤的发生、发展、转移及放化疗耐受密切相关,如结肠癌、胃癌、卵巢癌、头颈部鳞癌、肺癌、乳头状肾癌、三阴性乳腺癌、前列腺癌等。在食管腺癌中,c-Met的表达率为340%～540%,且与预后明显相关。而在亚裔食管鳞癌中,其同样存在着较高的表达率,约34%～69%。因此,c-Met可能成为食管鳞癌治疗新的靶点。另一方面,放射治疗是食管癌的重要治疗手段,虽然近年来放疗设备和放疗技术有了很大的改进,但放疗抵抗依然是影响食管鳞癌放疗疗效的一个重要原因。放疗抵抗的产生是一个多基因、多因子、多机制共同作用的复杂过程,与细胞周期阻滞、相关基因改变、肿瘤微环境变化、自噬性调节及肿瘤干细胞的存在等多种因素相关。一些临床研究显示c-Met的表达水平与放疗疗效存在相关性。基础研究表明肿瘤细胞和/或间质细胞受到电离辐射后,能够激活c-Met传导通路,进而引起肿瘤细胞的放射抵抗。因此,抑制c-Met信号通路有可能减少放疗抵抗,从而提高放疗疗效。综上所述,研究c-Met与食管鳞癌放射敏感性的相关性具有理论基础和积极的临床意义。我们的前期研究已提示c-Met蛋白在食管鳞癌中的表达存在差异,且与患者的预后显著相关(p=0.001)。亚组分析还提示,在接受术后辅助放疗的患者中,c-Met高表达者预后显著差于低表达者(p=0.002)。在随后的研究中,我们对其作用机制进行研究,期望为临床食管鳞癌新的靶向治疗药物的应用及精准的多学科综合治疗的开展提供新思路。材料与方法:在前期研究中,选取在本院行食管癌手术的180例食管鳞癌患者术后肿瘤组织石蜡块进行免疫组化检测,分析c-Met蛋白表达与患者肿瘤病理特点及预后的相关性,并分析其对术后辅助放疗疗效的影响。在随后的研究中,首先选用c-Met抑制剂BPI-9016M进行干预,探讨HGF/c-Met信号通路抑制对人食管鳞癌Eca109细胞株生物学行为及放射敏感性的影响;然后,将Eca109细胞分对照组、BPI-9016M组、辐射组和辐射+BPI-9016M组共4组,采用流式细胞仪技术检测c-Met抑制剂BPI-9016M对Eca109细胞细胞周期和细胞凋亡的影响,并用蛋白免疫印迹法(Western Blot,WB)检测细胞凋亡相关蛋白及DNA损伤修复相关蛋白含量的改变,进一步探讨其具体的分子作用机制;最后,建立人食管鳞癌裸鼠移植瘤模型,将荷瘤裸鼠分对照组、BPI-9016M组、辐射组和辐射+BPI-9016M组共4组,通过肿瘤体积计算肿瘤生长延缓情况、利用增长系数(Enhancement Factor,EF)评价c-Met抑制剂BPI-9016M的放射增敏效果。结果:1.在前期针对临床病例的免疫组化分析中,我们发现c-Met蛋白在食管鳞癌中的表达存在差异,且与患者预后显著相关,高表达者预后差(p=0.001)。亚组分析提示,接受术后辅助放疗的患者,c-Met高表达者预后显著差于低/阴性表达者(p=0.002)。2.体外细胞学实验显示,c-Met抑制剂BPI-9016M明显抑制Eca109人食管鳞癌细胞株的体外增殖生长,存在量-效关系;Eca109细胞的生长受放射线的影响,放射剂量越大,Eca109细胞生长越受限制,并且c-Met抑制剂BPI-9016M对Eca109细胞有放射增敏作用,用多靶单击模型计算的放射增敏比为1.40。流式细胞仪检测结果显示,c-Met抑制剂BPI-9016M联合辐射对Eca109细胞的细胞周期影响不大,但显著增加其细胞凋亡率(p0.05);蛋白免疫印迹法检测结果显示,与单纯辐射组相比较,Eca109细胞经BPI-9016M处理后再联合辐射,使凋亡相关的蛋白P53和Bcl-2下调、活化的Caspase 3和Caspase 9上调。进一步的DNA损伤修复相关蛋白检测结果还提示:Eca109细胞经放射线照射后,引起了 ATM、ATR、Chk1、Chk2和H2AX的显著磷酸化,然而各蛋白的磷酸化水平在经BPI-9016M预处理后均有所下降,以γ H2AX下降得最为明显。3.体内动物模型研究显示,Eca109细胞皮下接种于裸鼠成瘤后,分别经对照组、BPI-9016M组、辐射组以及辐射+BPI-9016M组的不同处理,从肿瘤生长曲线来看,相较于空白对照组,BPI-9016M组、辐射组以及辐射+BPI-9016M组肿瘤的生长均受到不同程度的抑制,其中辐射+BPI-9016M组较其他治疗组均更明显(p0.05),联合组c-Met抑制剂BPI-9016M的放射增敏系数(EF)为1.51。讨论:已有文献报道,食管癌c-Met蛋白的过表达与肿瘤恶性生物学行为相关。在西方国家的研究中发现食管腺癌c-Met蛋白的过表达与患者的预后生存相关,而在食管鳞癌患者中,其与预后的相关性尚不清楚。我们的前期研究显示,食管鳞癌患者c-Met蛋白的高表达与其预后差显著相关,与国内一些针对食管鳞癌的免疫组化研究结果报道相一致,提示c-Met基因有可能是食管鳞癌药物治疗的潜在靶点。我们前期的免疫组化亚组分析还提示,接受术后辅助放疗的患者,c-Met高表达者预后显著差于低表达者,分析原因,与c-Met可能参与多种放疗抵抗的机制相关。我们的体外细胞学实验证实,放射线抑制人食管鳞癌Eca109细胞的生长和增殖,而c-Met抑制剂BPI-9016M增加了 Eca109细胞对放射线的敏感性。流式细胞仪检测显示,c-Met抑制剂BPI-9016M联合辐射对Eca109细胞的细胞周期影响不大,但显著增加其细胞凋亡率。蛋白免疫印迹法实验提示c-Met抑制剂BPI-9016M预处理Eca109细胞后可增加放射后促细胞凋亡相关蛋白的含量。在我们的研究中,BPI-9016M联合辐射组活化的Caspase 3和Caspase 9蛋白较其他组别显著增加,促进了细胞凋亡的发生。Caspase家族成员大多数是细胞凋亡的启动子或效应子,Caspase 9为凋亡启动子,Caspase 3为凋亡效应子,在细胞凋亡过程中均发挥重要作用,尤其是Caspase 3在细胞凋亡过程中起关键作用,一旦被激活,即发生下游的级联反应,使凋亡不可避免。在我们的研究中,BPI-9016M组Eca109细胞中的P53蛋白稍有减少,辐射组其表达量与对照组接近,而在辐射+BPI-9016M组P53显著减少,认为BPI-9016M联合放射线可通过减少P53的表达来调控细胞凋亡;在BPI-9016M联合辐射组,功能与P53相似的抑制细胞凋亡的Bcl-2也明显减少。进一步的研究显示,Eca109细胞经辐射处理后,与DNA损伤修复相关的蛋白ATM、ATR、Chk1、Chk2和H2AX的磷酸化水平显著增加,而细胞预先经c-Met抑制剂BPI-9016M处理,再予以辐射,这些DNA损伤相关的关键蛋白的磷酸化被不同程度抑制。从机制上解读,辐射可通过诱导DNA双链断裂从而导致细胞死亡,放射抵抗的主要原因是由于肿瘤细胞内源性或者获得性的辐射抵抗,主要表现在DNA损伤修复能力的增强。DNA损伤发生后,细胞可激活相应的修复通路对其进行修复,其反应通路主要由感受因子、传导通路及效应因子组成。DNA损伤发生后,感受因子RAD9-HUS1-RAD1复合体可通过一个包含有RAD17的蛋白复合体募集至DNA损伤位点,以促进ATR-介导的效应蛋白激酶Chk1的磷酸化及激活,从而调节细胞周期S期的进展及G2/M期的阻滞。另一个感受因子MRE11-RAD50-NBS1(MRN)复合体,可通过检测双链断裂位点,在DNA双链断裂区域周围募集ATM并促进ATM介导的组蛋白H2AX磷酸化,随后一系列的修复相关因子及蛋白招募至DNA损伤区域,形成损伤点,参与DNA的损伤修复进程。从我们的研究可以推测c-Met抑制剂增加食管鳞癌放射敏感性的机制可能部分是通过抑制ATR-Chk1、ATM-Chk2传导通路的活化从而抑制细胞DNA损伤修复促进细胞凋亡实现的。我们的研究表明c-Met可能是食管鳞癌放疗增敏的重要靶点之一,靶向抑制c-Met联合放疗在提高临床食管鳞癌放疗疗效中具有重要的研究意义。结论:我们的研究探讨了 c-Met抑制剂BPI-9016M联合放射线对食管鳞状细胞癌细胞系Eca109细胞生物学特性的影响,可能能部分解释c-Met抑制剂对放疗的增敏作用机制,对临床治疗方案的选择亦能提供一定的理论基础,至于其详细机制,还有待于进一步研究。
[Abstract]:Background and purpose: esophageal cancer is one of the most common malignant tumors of digestive tract. The number of new cases is increasing every year, and more than half of esophageal cancers occur in China. According to the 2015 China cancer statistics, the incidence of esophageal cancer in China has leaped into the third place of the country's malignant tumor, and the death ranks fourth. Multidisciplinary integrated treatment, combined with surgery, radiotherapy and chemotherapy, is the main mode of treatment for esophageal cancer. Although a variety of treatments have been developed in recent years, the overall prognosis of esophageal cancer is still poor, and the survival rate of 5 years is only 15% to 25%. In addition, the individual differences in prognosis of patients are also large, which largely depends on the biological behavior of esophageal cancer, especially the different genetic characteristics, which have a crucial impact on tumor proliferation, invasion, metastasis and therapeutic effect. The specific molecular markers of esophageal cancer are not clear at present. MET (Mesenchymal-epithelial Transition Factor) is the hepatocyte growth factor (Hepatocyte Growth, Factor, HGF) of the cell surface receptor, also known as c-Met or HGFR (Hepatocyte Growth Factor Receptor), which belongs to the tyrosine kinase receptor (Receptor Tyrosine, Kinase, RTK) encoding by c-Met gene family members. Abnormal RTK signaling pathway plays an important role in tumor occurrence, development, invasion, metastasis and drug resistance. C-Met signaling pathway is a RTK signaling pathway. It is found that activation of c-Met signaling pathway is closely related to the occurrence, development, metastasis and chemoradiation tolerance of multiple tumors, such as colon cancer, gastric cancer, ovarian cancer, head and neck squamous cell carcinoma, lung cancer, papillary renal cell carcinoma, three negative breast cancer, prostate cancer and so on. In the adenocarcinoma of the esophagus, the expression rate of c-Met is 340% ~ 540%, and it is closely related to the prognosis. In Asian esophageal squamous cell carcinoma, it also has a high rate of expression, about 34% to 69%. Therefore, c-Met may be a new target for the treatment of squamous cell carcinoma of the esophagus. On the other hand, radiotherapy is an important treatment for esophageal cancer. Although radiotherapy equipment and radiotherapy technology have been greatly improved in recent years, radiotherapy resistance is still an important reason that affects the radiotherapy efficacy of esophageal squamous cell carcinoma. The emergence of radiation resistance is a complex process of multiple genes, multiple factors and multiple mechanisms, which is related to cell cycle arrest, related gene alterations, tumor microenvironment changes, autophagy regulation and the existence of cancer stem cells. Some clinical studies have shown that there is a correlation between the expression level of c-Met and the therapeutic effect of radiotherapy. Basic studies have shown that tumor cells and / or interstitial cells can activate the c-Met pathway after ionizing radiation and then cause radiation resistance of tumor cells. Therefore, inhibition of c-Met signaling pathway may reduce radiation resistance and improve the therapeutic effect of radiotherapy. To sum up, the study of the correlation between c-Met and radiosensitivity of squamous cell carcinoma of the esophagus has a theoretical basis and positive clinical significance. Our previous studies have suggested that the expression of c-Met protein in esophageal squamous cell carcinoma is different and has a significant correlation with the prognosis of the patients (p=0.001). Subgroup analysis also suggested that in patients with postoperative adjuvant radiotherapy, the prognosis of c-Met high expression was significantly lower than that of low expression (p=0.002). In subsequent studies, we studied the mechanism of action, hoping to provide new ideas for the application of new targeted therapy drugs and precise multidisciplinary comprehensive treatment for esophageal squamous cell carcinoma. Materials and methods: in previous studies, selected in our hospital surgery of esophageal cancer in 180 cases of patients with esophageal squamous cell carcinoma after resection of tumor tissue paraffin block immunohistochemical analysis of c-Met protein expression correlated with the pathological characteristics and prognosis of patients with cancer, and to analyze the effect of postoperative adjuvant radiotherapy curative effect. In a subsequent study, first select c-Met inhibitor BPI-9016M intervention, to investigate the inhibitory effects of HGF/c-Met signaling pathway on the biological behavior of human esophageal squamous cell carcinoma cell line Eca109 and Radiosensitivity; then, Eca109 cells were divided into control group, BPI-9016M group, radiation group and radiation group +BPI-9016M 4 group, the effect of c-Met inhibitor BPI-9016M flow detection cytometry on Eca109 cell cycle and apoptosis, and by Western blotting (Western Blot, WB) detection of apoptosis related protein and DNA repair related protein content changes, further explore its molecular mechanism; finally, to establish the xenograft model of human esophageal squamous cell carcinoma in nude mice bearing tumor the nude mice were divided into control group, BPI-9016M group, radiation group and radiation group +BPI-9016M 4 group, through the calculation of tumor volume tumor growth delay, using growth coefficient (E Nhancement Factor, EF) was used to evaluate the radiosensitization effect of c-Met inhibitor BPI-9016M. Results: 1.. In the early stage of immunohistochemical analysis of clinical cases, we found that the expression of c-Met protein in esophageal squamous cell carcinoma was different, and was significantly correlated with the prognosis of patients. The prognosis of patients with high expression was poor (p=0.001). Subgroup analysis showed that the prognosis of c-Met high expression patients was significantly lower than that of low / negative expression (p=0.002) patients who received postoperative adjuvant radiotherapy. 2. in vitro experiments, c-Met inhibitor BPI-9016M significantly inhibited the proliferation of Eca109 human esophageal squamous carcinoma cell line in vitro, the dose effect relationship; influence the growth of Eca109 cells by radiation, the radiation dose, Eca109 cell growth is restricted, and the c-Met inhibitor BPI-9016M has radiosensitizing effect on Eca109 cells, calculation multi target single hit model radiosensitization ratio is 1.40. Flow cytometry showed that c-Met inhibitor BPI-9016M combined with radiation on the cell cycle of Eca109 cells is not affected, but significantly increased the apoptosis rate (P0.05); the detection results of Western blotting showed that compared with radiation group, Eca109 cells treated by BPI-9016M combined with radiation, the apoptosis related protein P53 and the down-regulation of Bcl-2 and activation of Caspase 3 and C
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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