Serum Biomarkers of Hepatic Response to Cancer Chemopreventive Intervention

肝脏对癌症化学预防干预反应的血清生物标志物

• 批准号: 7545518
• 负责人: John D Groopman
• 金额: $ 17.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545518
• 关键词: Aldehyde Reductase; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Apoptotic; Biological; Biological Assay; Biological Markers; Blood; Carcinogens; Cell Nucleus; Cells; Chemopreventive Agent; Chemoprotective Agent; Clinical; Clinical Trials; Complex; Cytoplasm; Data; Databases; Detection; Digestion; Dose; Equilibrium; Extravasation; Gel; Hepatic; Hepatocyte; Human; Imidazole; Intervention; Isotopes; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Measurement; Measures; Metabolic Activation; Metabolism; Methods; Microarray Analysis; Mitochondria; Mitochondrial Proteins; Modeling; Necrosis; Nitric Oxide; Nuclear; Oligonucleotide Microarrays; Organ; Oxidoreductase; Phase; Preparation; Production; Property; Proteins; Proteomics; Rattus; Regulatory Pathway; Relative (related person); Sampling; Serum; Serum Proteins; Sulforaphane; Sulindac; Techniques; Testing; Therapeutic Agents; Tissues; Transgenic Animals; Treatment Protocols; Work; cancer cell; cancer therapy; cell type; detoxication; gel electrophoresis; insight; interest; multicatalytic endopeptidase complex; physical property; response; selenium-binding proteins; tool; tumorigenesis

DESCRIPTION (provided by applicant): In animal models of liver cancer, treatment with almost any chemopreventive agent produces changes in the abundance of hepatic proteins. In the 1990's, structurally dissimilar chemoprotective agents were shown to alter the balance between the metabolic activation and detoxication of many experimental carcinogens. More recently, transgenic animals and oligonucleotide microarray technologies were used to explore transcriptional regulatory pathways through which chemopreventive agents work. Chemopreventive agents (e.g. sulforaphane, sulindac) originally thought to primarily work by altering classically described phase I and II metabolism or directly inhibiting inducible cycloxygenase are being found to work through additional mechanisms that promote apoptosis in cancer cells. Today, new proteomic discovery tools, such as difference in gel electrophoresis (DIGE), enable the detection of changes in the relative abundance of proteins without the need to identify proteins in advance. Although not cancer endpoints themselves, hepatic proteins whose relative abundances are modulated can provide insights into mechanisms of chemopreventive agent action. These hepatic proteins may be exploited to develop serum biomarkers of responsiveness to chemopreventive treatment. Normal human serum contains small amounts of non-secreted proteins that are predominantly located in the nucleus, mitochondrion and cytoplasm. How they arrive in blood is unknown but probably involves necrosis, apoptosis and a poorly defined phenomenon called "tissue leakage". Conceptually, the serum concentration of a leaked protein could achieve a steady-state value reflecting its total abundance in different organs. Because of the liver's large size, hepatocytes are expected to be major contributors of non-secreted proteins found in normal human serum. The long-term objectives of this work are to identify hepatic protein biomarkers of responsiveness to chemopreventive intervention and to develop mass spectrometric methods for their analysis in serum. Maturation of these assays should ultimately enable the measurement of serum proteins to determine responsiveness to liver cancer chemopreventive interventions in clinical trials. Initial scientific interest in the triterpenoid 1-[2-cyano-3-12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO- Im) was generated by its unusually potent inhibition of nitric-oxide production (Honda 2002). In addition to their anti-inflammatory properties, CDDO congeners are cytoprotective of healthy cells but exert pro-apoptotic and anti-proliferative effects on different cancer cell types. These properties suggested their use as cancer chemopreventive and therapeutic agents (Sporn and Liby 2005; Yore 2006). The mechanisms through which triterpenoids exert their biological effects remain to be completely elucidated. CDDO congeners are currently in clinical phase I cancer trials and CDDO-Im was recently demonstrated to be the most potent chemopreventive agent ever tested in a rat model of hepatic tumorigenesis (Yates 2006). In this proposal, CDDO-Im will be administered to rats to evaluate the feasibility of developing serum proteins as biomarkers of hepatic responsiveness to chemoprotective intervention. Aim I: To detect candidate biomarkers of liver responsiveness to treatment, DIGE studies will be performed following the treatment of rats with a previously established chemopreventive dosing regimen of CDDO-Im. Differentially expressed hepatic proteins in nuclear, mitochondrial and cytoplasmic sub-cellular fractions will be identified via in-gel enzymatic digestion, mass spectrometric analysis and database searches. Aim II: To measure the serum concentration of leaked hepatic proteins identified in Aim I, sample preparation methods and quantitative mass spectrometric assays will be developed. Guided by the physical properties of the candidate biomarker proteins, techniques for their extraction from serum will be established. Isotope dilution mass spectrometric assays will be developed to quantitatively measure the serum concentration of non-secreted proteins for use as biomarkers of hepatic responsiveness to treatment. Pilot DIGE data demonstrate CDDO-Im modulates the relative concentration of over forty cytosolic liver proteins. Their mass spectrometric identification revealed a variety of protein classes that include but are not limited to proteosome complex subunits, mitochondrial proteins, dehydrogenases, Se-binding proteins, aldehyde reductases and GSTs. Several GSTs were mass spectrometrically identified in extracted serum where, in a proof-of-concept demonstration, GST @ was conspicuously elevated in CDDO-Im treated rats.Narrative The long-term objectives of this work are to identify hepatic protein biomarkers of responsiveness to cancer chemopreventive intervention and to develop mass spectrometric methods for their quantitative analysis in serum. Maturation of these assays should ultimately enable the measurement of serum proteins to determine hepatic responsiveness to liver cancer chemopreventive interventions in clinical trials.

描述（由申请人提供）： 在肝癌动物模型中，几乎所有化学预防剂的治疗都会导致肝蛋白丰度发生变化。在 20 世纪 90 年代，结构不同的化学保护剂被证明可以改变许多实验致癌物的代谢激活和解毒之间的平衡。最近，转基因动物和寡核苷酸微阵列技术被用来探索化学预防剂发挥作用的转录调控途径。化学预防剂（例如萝卜硫素、舒林酸）最初被认为主要通过改变经典描述的 I 期和 II 期代谢或直接抑制诱导型环加氧酶发挥作用，但现在发现其通过促进癌细胞凋亡的其他机制发挥作用。如今，新的蛋白质组学发现工具，例如差异凝胶电泳 (DIGE)，可以检测蛋白质相对丰度的变化，而无需提前识别蛋白质。虽然肝蛋白本身不是癌症终点，但其相对丰度受到调节的肝蛋白可以提供对化学预防剂作用机制的见解。这些肝蛋白可用于开发化学预防治疗反应性的血清生物标志物。正常人血清含有少量非分泌蛋白，主要分布在细胞核、线粒体和细胞质中。它们如何进入血液尚不清楚，但可能涉及坏死、细胞凋亡和一种定义不明确的现象，称为“组织渗漏”。从概念上讲，泄漏蛋白质的血清浓度可以达到反映其在不同器官中总丰度的稳态值。由于肝脏体积较大，预计肝细胞是正常人血清中非分泌蛋白的主要贡献者。这项工作的长期目标是确定对化学预防干预反应的肝蛋白生物标志物，并开发用于血清分析的质谱方法。这些测定的成熟最终应该能够测量血清蛋白，以确定临床试验中对肝癌化学预防干预措施的反应。三萜类化合物 1-[2-cyano-3-12-dioxooleana-1,9(11)-dien-28-oyl]imidazol (CDDO-Im) 最初的科学兴趣是由于其对一氧化氮产生的异常有效的抑制作用而产生的(Honda 2002)。除了抗炎特性外，CDDO 同源物还对健康细胞具有细胞保护作用，但对不同的癌细胞类型发挥促凋亡和抗增殖作用。这些特性表明它们可用作癌症化学预防和治疗剂（Sporn 和 Liby 2005；Yore 2006）。三萜类化合物发挥其生物效应的机制仍有待完全阐明。 CDDO同系物目前正处于临床I期癌症试验中，并且CDDO-Im最近被证明是在肝肿瘤发生大鼠模型中测试过的最有效的化学预防剂（Yates 2006）。在该提案中，将给大鼠施用 CDDO-Im，以评估开发血清蛋白作为肝脏对化学保护干预反应性生物标志物的可行性。目标 I：为了检测肝脏对治疗反应的候选生物标志物，将在用先前建立的 CDDO-Im 化学预防给药方案治疗大鼠后进行 DIGE 研究。将通过凝胶内酶消化、质谱分析和数据库搜索来鉴定核、线粒体和细胞质亚细胞部分中差异表达的肝蛋白。目标 II：为了测量目标 I 中鉴定的泄漏肝蛋白的血清浓度，将开发样品制备方法和定量质谱分析。在候选生物标志物蛋白质的物理特性的指导下，将建立从血清中提取它们的技术。将开发同位素稀释质谱测定法来定量测量非分泌蛋白的血清浓度，用作肝脏对治疗反应的生物标志物。 DIGE 试验数据表明 CDDO-Im 调节四十多种胞浆肝蛋白的相对浓度。他们的质谱鉴定揭示了多种蛋白质类别，包括但不限于蛋白酶体复合物亚基、线粒体蛋白、脱氢酶、硒结合蛋白、醛还原酶和 GST。在提取的血清中对几种 GST 进行了质谱鉴定，在概念验证演示中，CDDO-Im 治疗的大鼠中 GST @ 显着升高。 叙述 这项工作的长期目标是鉴定对癌症化学预防干预反应的肝蛋白生物标志物，并开发用于血清中定量分析的质谱方法。这些测定的成熟最终应该能够测量血清蛋白，以确定临床试验中肝脏对肝癌化学预防干预措施的反应。