【作者】 沈杰；

【导师】 刘昭前；

【作者基本信息】 中南大学 ， 临床药理学， 2014， 博士

【摘要】 研究背景与目标：肺癌是我国及世界范围最常见的恶性肿瘤,其发病率和死亡率均居首位。化学治疗是最重要的肿瘤治疗手段,寻找肺癌化学治疗的影响因子及作用靶点,是当前极为关注的研究课题。肿瘤中异常表达的基因可能参与肿瘤发生发展过程中的关键环节,并影响其它肿瘤相关因子和通路的状态。这些基因表达状态的异常对肿瘤的化疗及预后可能具有重要的预测意义,其本身可能成为抗肿瘤治疗的潜在靶点。eIF3a是真核翻译起始因子3的最大亚基,其在多种肿瘤呈现高表达现象。已知eIF3a参与调控翻译和分化,并与细胞周期密切相关。然而对其在肺癌等肿瘤表达的机制和意义并不清楚。本课题组前期对eIF3a在肺癌的表达及分子机制进行了初步探索,发现eIF3a的高表达可增加肺癌患者对铂类化疗的敏感性,并证实eIF3a从翻译水平调控影响DNA修复的NER途径。我们由此推测eIF3a的表达可能对于肺癌的预后及化疗效果具有预测意义。同时,近来的相关研究中,发现了一些与eIF3a密切相关的重要因子和通路,如p27/Kipl和ERK通路。这些因子和通路的状态,本身也具有重要的临床预测意义。并且,多数细胞水平研究显示eIF3a的过表达促进肿瘤的恶性表型和耐药性。因此,目前对于eIF3a对肿瘤发生和预后的意义存在着不同的观点。本研究拟采用临床研究的方法,从组织学角度对eIF3a及与其相关的通路及因子进行证实研究。非小细胞肺癌(NSCLC)占肺癌的85%,是肺癌的主要类型。化疗可以有效改善NSCLC患者术后生存。本研究计划在肿瘤组织水平研究与eIF3a相关的因子和信号通路的表达情况,并对NSCLC手术完全切除患者进行回顾性研究,调查eIF3a表达水平及相关因子对肺癌预后及术后化疗的预测意义。含羞草酸为一类含有α-氨基酸结构的吡啶酮类天然化合物,抑制eIF3a的表达,阻滞细胞周期。我们推测,eIF3a抑制剂可能产生特异的抗肿瘤增殖活性。并且,在研究eIF3a功能与肿瘤相关关系的时候,eIF3a抑制剂具有重要的先导化合物价值。因此本研究准备从抑制eIF3a及抗肿瘤增殖两方面,筛选一系列吡啶酮类衍生化合物生物活性。研究方法：基于以上研究背景,本课题拟采用下述研究方法以达到研究目的：(1)采用分子克隆及免疫方法制备eIF3a的原核蛋白及多克隆抗体；(2)采用免疫组织化学方法在NSCLC组织中调查eIF3a及p27/Kip1和ERK通路等相关因子和信号通路的表达水平,采用Spearman等级相关系数法评价各生物标志物表达水平的相关程度,采用卡方检验生物标志物之间及其与人口统计学资料及病理特征等临床参数之间的相关程度；(3)采用Kaplan-Meier法分析疾病相关生存期(DSS)及无病生存期(DFS),采用log-rank检验(单因素分析),建立Cox比例风险模型进行多因素分析,调查eIF3a表达水平与相关因子对肺癌术后及化疗预后的预测意义；(4)筛选具有抑制肿瘤细胞生长及调控eIF3a表达的合成吡啶酮类衍生化合物。研究结果：研究中我们获得下列结果：(1)获得了针对eIF3aC端的原核表达载体及高效价的eIF3a多克隆抗体；(2)eIF3a, p27/Kip1和ERK在NSCLC组织中的表达普遍,且存在正相关关系。特别是eIF3a和p27/Kipl在亚细胞部位(胞浆及胞核)的表达呈较强的正相关关系：Spearman等级相关系数r=0.653(胞浆),r=0.716(胞核)。而p-ERK在NSCLC组织中显著低表达,阳性表达呈局部点灶状。尚不能评价p-ERK/ERK与eIF3a在组织表达水平的相关关系。同时eIF3a在分化较好,鳞癌组织中表达较高；p27/Kipl在分化较好的NSCLC中表达较高；(3)对537例NSCLC术后患者进行探索研究,发现胞核表达的eIF3a和p27/Kip1对NSCLC术后生存有积极意义,两者均高表达为独立的预后预测指标(死亡风险比值比HR=0.360,95%CI=0.109-0.782,P=0.028)。化疗预后分析显示胞浆高表达的eIF3a对Ⅱ期NSCLC患者具有化疗预测价值。对扩大样本的Ⅱ期NSCLC患者进行验证研究显示,胞浆高表达eIF3a(P--0.036),胞核低表达p27/Kip1(P=0.031)的NSCLC患者能从术后化疗获益；(4)以吡非尼酮为先导化合物,合成其不同侧链修饰的衍生化合物。通过MTT法考察衍生化合物对高表达eIF3a的肺癌细胞A549及低表达eIF3a的肺纤维细胞NIH3T3的抑制作用,进行结构-选择性分析和构效分析,确定活性化合物,并从蛋白和核酸表达水平进行验证。研究结论：通过本研究,我们得出下列结论：(1)eIF3a与p27/Kipl在NSCLC组织中的表达状态存在显著相关；(2)eIF3a和p27/Kipl在NSCLC组织的不同表达状态及相关关系,有助于预测NSCLC完全切除患者的中期预后,并可一定程度预测术后化疗的可能获益,可能成为NSCLC预后的候选生物标志物；(3)具有“Y”型结构的1-苯基-5-苯氧甲基-2吡啶酮衍生物呈现对eIF3a和肿瘤细胞的显著抑制活性.其中间二苯胺类化合物抑制eIF3a表达作用较强。这些新化合物的活性,理化性质均优于含羞草酸,可开发为新型抗肿瘤先导化合物。创新点：自主获得eIF3a的C端重组蛋白及相应抗体；从病理组织学角度证实了eIF3a与p27/kipl的相互关系,首次探讨eIF3a作为肺癌预后和化疗预测标志物的临床意义；筛选了能够调节eIF3a表达而抑制肿瘤细胞生长的新型衍生化合物,为抗肺癌治疗提供新的研究思路。更多还原

【Abstract】 Background and AimLung cancer is the most common malignancy in our country and the world wide. The incidence and mortality of lung cancer lead the first of all tumors. Chemotherapy is the most important strategy for tumor management. Both impactive factor and targets proteins of cancer chemotherapy are long of great concern. Gene abnormal expressing in tumors may be involved with the key points in oncogenesis and development of tumor, and have impacts on status of other factors and pathways associated with tumor. The abnormality of such gene may have important prognostic significance for tumor management and chemotherapy, and itself could become potential target for chemotherapy as well.eIF3a, the biggest subunit of eukaryotic translation initiation factor3, was found over-expressing in a variety kinds of tumors. It is known for us that eIF3a was involved in the regulation of translation and differentiation, and was closely related to the cell cycle. However, the mechanism and significance of its over-expression in lung cancer and other tumors are unclear. Our group had conducted preliminary exploration on the expression status of eIF3a in lung cancer and its molecular mechanisms as well, we found over-expression of eIF3a improved chemo-responses of lung cancer patients to platinum-based chemotherapy, and we confirmed that eIF3a inhibited the NER pathway in translational level to regulate DNA repairs in vitro. So expression of eIF3a might be indicative for a sensitive phenotype of NSCLC. Herein, the underlying hypotheses of these studies were the expression of eIF3a might predict clinical benefits due to increase of cisplatin sensitivity in NSCLC. On the other hands, there were some reports on the factor and signaling pathways associated with eIF3a, for example:p27/Kipl and ERK pathway. What is more, most studies in vitro suggested the over-expression of eIF3a promote malignant phenotype as well as resistance to chemotherapy. Therefore, the prognostic value of eIF3a is still not clear, as it seems participate in both the protection from and induction of oncogenesis.Non-small cell lung cancer (NSCLC), the main types of lung cancer, accounts for85%of all lung cancers. Chemotherapy improves survival in patients with NSCLC. We wish to confirm the correlation of eIF3a with its related factors and pathways in NSCLC tissues. We also design retrospective study on radically resected NSCLC patients, to investigate the clinical significance of eIF3a, as well as its associated factors, in predicting the prognosis of NSCLC and chemotherapy. Mimosine, a natural pyridone containing α-amino acid structure, can inhibite eIF3a expression and protein synthesis, and is commonly used in cell synchronization, reversibly arresting cells in late G1phase. We postulated eIF3a inhibitors may produce specific anti-tumor proliferative activity. What is more, eIF3a inhibitor might be lead compounds in the study on functions of eIF3a and its correlation with tumors. In our study, we plan to screeen derivative compounds of pyridones with the activities to inhibite eIF3a and anti-proliferation of tumor cells. MethodsBased on the above background, This research intends to use the following methods to achieve the purposes as followed:(1) preparate prokaryotic protein of eIF3a and its polyclonal antibodies;(2) investigate the expression level of eIF3a and related signaling pathways in NSCLC tissues by immunohistochemical methods, analysize the degree of correlation betwwen eIF3a and related factors with Spearman rank correlation coefficient method, analysize the correlation between the biomarkers and clinical parameters such as demographic data and pathologic characteristics using the chi-square test;(3) collect clinical information of complete surgical resection of NSCLC and Follow up of survival, analysize prognostic significance of eIF3a and its related factors in signaling pathways in radically resected NSCLC and postoperative chemotherapy with Kaplan-Meier method and Cox regression model;(4) modify the side chains of lead compound (pirfenidone) and synthesis new derivatives. Investigate the anti-proliferative activities of new derivatives on lung cancer cell A549, which.express high level eIF3a, and lung fibroblast cell NIH3T3, which express low level eIF3a. Validate the screening results in protein and RNA level. ResultsWe got following results in this research:(1) we obtained prokaryotic expression vector for eIF3a C-terminal and its polyclonal antibodies with high titer;(2) the subcellular expression of eIF3a was strongly correlated with status of p27/Kip1(Spearman rank coefficient correlation for cytoplasmic eIF3a and p27/Kipl=0.653, for nuclear staining=0.716). Expression of p-ERK was significantly lower in NSCLC tissues:the positive expression was localized spotty; relationship of p-ERK/ERK with eIF3a expression levels in the tissues was not yet evaluated. Expression of eIF3a was higher in well differentiated squamous cell carcinoma tissues; p27/Kip1highly expressed in well differentiated in NSCLC;(3) In537radically resected NSCLC patients, eIF3a and p27/Kipl expressed in the nucleus has a positive meaning on NSCLC survival. Survival analysis revealed favorable prognostic impact of nuclear eIF3a, p27, and the combination high nuclear staining on NSCLC (Hazards Ratio=0.360,95%CI=0.109-0.782, P=0.028). Chemotherapy prognostic analysis showed high expression of cytoplasmic eIF3a has predictive value for chemotherapy in stage II NSCLC patients. Moreover, In addition, interaction research between biomarkers and chemotherapy status disclosed cisplatin-based regimen trend to prolong DSS of stage II NSCLC patients with high eIF3a-C (P=0.036) and low p27-N (P=0.031);(4)Some1-substituted phenyl-5-phenoxymethyl-2-pyridones with similar Y-type structure synthesized had been improved to inhibite eIF3a and proliferation of lung cancer cell. The two aniline series exhibited significant activities on inhibition of eIF3a and anti-proliferation on A549but not NIH3T3. ConclussionWe arrived following conclusions in the study:(1) the expression status eIF3a and p27/Kipl were significantly related in NSCLC tissues;(2) eIF3a and p27/K.ipl expression in different states NSCLC tissues and their relationships, could help predict mid-term prognosis in patients with completely resected NSCLC, as well as predict the possible benefit of postoperative chemotherapy;(3)The derivative pyridones with similar structure of mimosine could inhibite the express of eIF3a and the proliferation of lung cancer cell significantly, while it does not restrain the growth of lung fibroblast cells. Such derivatives are potential anti-tumor lead compounds and merits intensive development in future. Innovation pointsWe prepared recombinant protein of C terminal in eIF3a and the corresponding antibody; we histopathology confirmed the close relationship eIF3a with p27/kipl and provide the latest clinical evidence for prognostic and predictive significance of eIF3a for lung cancer, we screened derivatives inhibiting eIF3a and tumor growth, provided novel ideas for therapy of lung cancer.更多还原