iTRAQ技术在消化道肿瘤研究中的应用进展

近年来,同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantification,iTRAQ)技术已成为蛋白质组学定量研究中一种强有力的工具,尤其在肿瘤相关领域备受关注,其突出优势为测定蛋白范围广泛、分析结果可靠、精确度高、重复性好等。大量文献也表明,iTRAQ技术在消化道肿瘤,如食管癌、胃癌、肝癌等肿瘤标志物筛查研究、肿瘤发生发展机制研究以及肿瘤治疗预后探索等方面得到广泛应用。在大规模定量生物学时代,iTRAQ技术在分子水平上深入了解消化道肿瘤的相关机制研究中越来越不可或缺。因此本文对iTRAQ技术在消化道肿瘤中的研究进展进行简要综述。     

蛋白质组学在肿瘤生物标记筛选中的应用

蛋白质组学的快速发展为肿瘤生物标记的研究提供了科学的理论依据,在乳腺癌、结直肠癌、鼻咽癌及肝细胞癌等癌症的早期诊断方面发挥了重要作用,一些新的肿瘤生物标记相继被发现,如细胞质泛素、钙结合蛋白、血浆铜蓝蛋白和胆绿素还原酶等.了解这些标记物的表达机制,对癌症的诊断和治疗有重要意义.     

蛋白质组学在肿瘤中的应用

蛋白质组学是从细胞水平及整体水平研究蛋白质构成及其变化规律的新兴学科.近年来,蛋白质组学研究已在多种肿瘤中展开,并在阐明肿瘤发生发展机制、发现新的特异性标志物、药物治疗靶标以及肿瘤生物学行为预测等方面取得了一定的进展.     

肿瘤蛋白质组学研究进展

蛋白质组学研究是后基因组时代肿瘤学研究的重点。激光捕获微切割、蛋白质芯片、同位素包被亲和标记等新技术促进了蛋白质组学的发展及其在各种癌症研究中的应用。蛋白质组技术给肿瘤标志物筛选和个体化治疗等领域带来了新的途径。     

蛋白质组学在肿瘤研究中的应用

1994年,Wlkins 和Williams在第一届国际双向电泳会议上首先提出了蛋白质组（proteome）的概念,1995年,Wasinger VC.等在《Electrophoresis》杂志上首次发表了蛋白质组的研究文章.蛋白质组被定义为：1个基因组所编码的全部蛋白质（proteome refers to the total protein complement of a genome）.     

蛋白质组学在肿瘤防治研究中的应用

随着人类基因组测序计划(human genome sequence project)的完成．产生了蛋白质组学(proteomics)——研究细胞内全部蛋白质的组成及动态变化的一门新兴学科。恶性肿瘤是一种多种基因和蛋白质参与的复杂疾病。综述了在肿瘤防治中蛋白质组学的主要策略和技术．及其在肿瘤标志物的筛选和鉴定、肿瘤分类、预后、治疗效果的评价及肿瘤发生机制等方面的研究。     

蛋白质组学在肿瘤研究中的应用

蛋白质组学是研究细胞、组织和器官内所有蛋白质的组成及其动态变化的科学，是在蛋白质水平上定量、动态、整体地研究生物体，并建立完整的蛋白质文库。应用蛋白质组学技术分离鉴定肿瘤细胞与常起源细胞之间的差异表达蛋白，可为寻找肿瘤的特异标志物，揭示肿瘤的发病机制，研究肿瘤转移、耐药机制及肿瘤治疗等提供更多的研究思路和理论依据。     

蛋白质组学在寻找肿瘤生物标记物中的技术与应用

蛋白质组学(proteomics)的主要任务是识别，鉴定细胞、组织或机体的全部蛋白质，并分析蛋白质的功能和结构。它集中体现了当代基因组学、计算机学、分析仪器学和生化等学科的最新发展水平。蛋白质组学摒弃了传统上以小项目形式分割地研究蛋白质的弊端，采用立体的、全面的和快速的分析方法，以透视全体基因在特定细胞或组织的表达为基本目标。它的产生无论从研究策略上还是其负载的技术，     

分泌蛋白质组学在肿瘤标志物研究中的现状及进展

＂分泌蛋白＂是指由活细胞分泌的一系列分子,另外也包括活细胞表面流出分子。分泌蛋白在细胞信号传导、通信和迁移中扮演重要的角色。随着肿瘤生物标记研究的进展,肿瘤细胞分泌蛋白的研究逐渐成为热点,可能成为肿瘤诊断和预后评估的工具,也可能成为药物治疗的靶点。近年来,随着蛋白组学技术的发展,极大促进了分泌蛋白组学的研究和发展。然而,分泌蛋白组学研究仍旧面临诸多困难。本文系统回顾了所发现的常见肿瘤细胞分泌蛋白及其分析方法。     

分泌蛋白质组学在肿瘤标志物研究中的现状及进展

分泌蛋白是指由活细胞分泌的一系列分子,另外也包括活细胞表面流出分子。分泌蛋白在细胞信号传导、通信和迁移中扮演重要的角色。随着肿瘤生物标记研究的进展,肿瘤细胞分泌蛋白的研究逐渐成为热点,可能成为肿瘤诊断和预后评估的工具,也可能成为药物治疗的靶点。近年来,随着蛋白组学技术的发展,极大促进了分泌蛋白组学的研究和发展。然而,分泌蛋白组学研究仍旧面临诸多困难。本文系统回顾了所发现的常见肿瘤细胞分泌蛋白及其分析方法。     

Isobaric tags for relative and absolute quantitation-based proteomics analysis of the effect of ginger oil on bisphenol A-induced breast cancer cell proliferation

Several chemicals in the environment, particularly those with estrogenic activity and small amounts (micromolar or lower) of environmental estrogen can cause changes in cell function and interfere with endocrine functions of animals and humans. These compounds enter the human body and increase the load of estrogen in the body, leading to an increasing incidence of estrogen-related tumors in breast cancer, ovarian cancer and endometrial cancer. Previous studies have demonstrated that ginger can inhibit the expression of estrogen receptors, while the bioactive ingredients of ginger sig-nificantly inhibit proliferation and promote the apoptosis of breast cancer cells. In the present study, a quantitative proteomics technique based on relative and absolute quanti-tative isobaric labeling was used to determine the effect of ginger essential oil (GEO) and BPA combined treatment on the proteomic characteristics of MCF-7 cells. In total, 5,084 proteins were detected. Proteins that were upregulated >1.2-fold and downregu-lated by >0.8-fold were differentially expressed. Overall, 528 differentially expressed proteins were identified. Compared with the control group, MCF-7 cells treated with GEO, BPA and GEO-BPA resulted in 45 (14 up- and 31 downregulated), 481 (141 up- and 340 downregulated) and 34 (13 up- and 21 downregulated) differentially ex-pressed proteins, respectively. Compared with the BPA group, MCF- 7 cells treated with GEO-BPA resulted in 210 (117 up- and 93 downregulated) differentially expressed proteins, among the 210 differentially expressed proteins in the GEO-BPA group, 10 proteins were associated with oxidative phosphorylation pathways, while succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB), succinate dehydrogenase cytochrome b560 subunit, mitochondrial (SDHC), cytochrome c oxidase subunit 2 and superoxide dismutase (Mn), mitochondrial (SOD2) expression was decreased with GEO-BPA combined treatment. Through the analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, the cellular localization, functional annotation and biological pathways of differentially expressed proteins were ex-amined. The results indicated that GEO-BPA may act through the oxidative phosphory-lation pathway, decreased the expression of SDHB and SDHC, affected the tricarbox-ylic acid cycle and decreased the expression of SOD2. This may have led to oxidative stress and the death of breast cancer cells, and the SDH signaling pathway may be an important mediator of the inhibitory effects of GEO in MCF-7 breast cancer cells. GEO can inhibit the proliferation of breast cancer MCF-7 cells induced by BPA, and the underlying mechanism may be associated with oxidative phosphorylation. These results may aid the development of future treatment strategies for breast cancer caused by environmental estrogen exposure.     

The spectra count label-free quantitation in cancer proteomics

Mass spectrometry is used routinely for large-scale protein identification from complex biological mixtures. Recently, relative quantitation approach on the basis of spectra count has been applied in several cancer proteomic studies. In this review, we examine the mechanism of this technique and highlight several important parameters associated with its application.     

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.     

蛋白质组学及其在常见肿瘤研究中的应用进展

蛋白质组学是运用各种技术手段分析细胞内动态变化的蛋白质组成成分、表达水平和修饰状态,了解蛋白质之间的相互作用与联系,揭示蛋白质功能与细胞生命活动规律.从蛋白质组学的角度研究肿瘤,可以更深层次地了解肿瘤组织的起源、演进过程及特点,从而掌握肿瘤的发生发展规律.     

New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis

Despite the revolutionary effects of imatinib on advanced gastrointestinal stromal tumors (GISTs), most patients eventually develop disease progression following primary resistance or acquired resistance driven by secondary‐resistant mutations. Even in radiographically vanishing lesions, pathology has revealed persistent viable cells during imatinib therapy, which could lead to the emergence of drug‐resistant clones. To uncover the mechanisms underlying these clinical issues, here we examined imatinib‐induced phosphoproteomic alterations in GIST‐T1 cells, using our quantitative tyrosine phosphoproteomic analysis method, which combined immunoaffinity enrichment of phosphotyrosine‐containing peptides with isobaric tags for relative and absolute quantitation (iTRAQ) technology. Using this approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins, with 11 proteins exhibiting greater than 1.5‐fold increases in tyrosine phosphorylation. Among them, we evaluated FYN and focal adhesion kinase (FAK), both of which are reportedly involved in proliferation and malignant alteration of tumors. We confirmed increased tyrosine phosphorylation of both kinases by western blotting. Inhibition of FYN and FAK phosphorylation each increased tumor cell sensitivity to imatinib. Furthermore, a FAK‐selective inhibitor (TAG372) induced apoptosis of imatinib‐resistant GIST‐T1 cells and decreased the imatinib IC₅₀. These results indicate that FYN or FAK might be potential therapeutic targets to overcome resistance to imatinib in GISTs. Additionally, we showed that the iTRAQ‐based quantitative phosphotyrosine‐focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance.     

Plasma membrane proteomics identifies bone marrow stromal antigen 2 as a potential therapeutic target in endometrial cancer

This report utilizes a novel proteomic method for discovering potential therapeutic targets in endometrial cancer. We used a biotinylation‐based approach for cell‐surface protein enrichment combined with isobaric tags for relative and absolute quantitation (iTRAQ) technology using nano liquid chromatography–tandem mass spectrometry analysis to identify specifically overexpressed proteins in endometrial cancer cells compared with normal endometrial cells. We identified a total of 272 proteins, including 11 plasma membrane proteins, whose expression increased more than twofold in at least four of seven endometrial cancer cell lines compared with a normal endometrial cell line. Overexpression of bone marrow stromal antigen 2 (BST2) was detected and the observation was supported by immunohistochemical analysis using clinical samples. The expression of BST2 was more characteristic of 118 endometrial cancer tissues compared with 59 normal endometrial tissues (p < 0.0001). The therapeutic effect of an anti‐BST2 antibody was studied both in vitro and in vivo. An anti‐BST2 monoclonal antibody showed in vitro cytotoxicity in BST2‐positive endometrial cancer cells via antibody‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity. In an in vivo xenograft model, anti‐BST2 antibody treatment significantly inhibited tumor growth of BST2‐positive endometrial cancer cells in an NK cell‐dependent manner. The anti‐BST2 antibody had a potent antitumor effect against endometrial cancer both in vitro and in vivo, indicating a strong potential for clinical use of anti‐BST2 antibody for endometrial cancer treatment. The combination of biotinylation‐based enrichment of cell‐surface proteins and iTRAQ analysis should be a useful screening method for future discovery of potential therapeutic targets.     

蛋白质组学在消化系统恶性肿瘤中的研究进展

蛋白质组学作为肿瘤研究的新平台,通过从整体水平研究蛋白质间的网络调控,对胰腺癌、食管癌、胃癌、大肠癌、肝癌等消化系统恶性肿瘤的早期诊断、预防和治疗有着积极的意义,尤其在筛选消化系统恶性肿瘤的早期特异性标记物中有独特的优越性。