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: 7385183
• 负责人: John D Groopman
• 金额: $ 21.4万
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7385183
• 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; Class; Clinical; Clinical Trials; Complex; Cytoplasm; Data; Databases; Detection; Digestion; Dose; End Point; Equilibrium; Extravasation; Gel; Glutathione S-Transferase; 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; Thinking; Tissues; Today; Transgenic Animals; Treatment Protocols; Work; cancer cell; cancer therapy; cell type; concept; detoxication; gel electrophoresis; insight; interest; multicatalytic endopeptidase complex; physical property; response; selenium-binding proteins; size; 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丰富的肝蛋白质变化。在1990年代，结构不同的化学保护剂被证明可以改变代谢激活与许多实验性致癌物解毒之间的平衡。最近，使用转基因动物和寡核苷酸微阵列技术来探索化学预防剂可通过的转录调节途径。化学预防剂（例如硫烷，苏莱达克）最初被认为主要是通过改变经典描述的I期代谢或直接抑制可诱导的环氧酶的作用，可以通过促进癌细胞中凋亡的其他机制起作用。如今，新的蛋白质组学发现工具，例如凝胶电泳（DIGE）的差异，可以检测蛋白质相对丰度的变化，而无需事先识别蛋白质。尽管不是癌症终点本身，但调节相对丰度的肝蛋白可以提供对化学预防剂作用机制的见解。这些肝蛋白可能被利用以发展对化学预防治疗的反应性的血清生物标志物。正常的人血清包含少量的非分泌蛋白，这些蛋白主要位于细胞核，线粒体和细胞质中。它们如何到达血液是未知的，但可能涉及坏死，细胞凋亡和称为“组织泄漏”的现象不当。从概念上讲，泄漏蛋白质的血清浓度可以实现稳态值，反映其在不同器官中的总丰度。由于肝脏的大尺寸，肝细胞有望是正常人血清中发现的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性的肝蛋白生物标志物，并开发出质谱法以在血清中分析。这些测定法的成熟最终应使血清蛋白的测量能够确定临床试验中对肝癌化学预防干预措施的反应。三萜1- [2-Cyano-3-12-dioxooleana-1,9（11）-dien-28-28-oyl]咪唑（CDDO- IM）的初始科学兴趣是由于其对一氮氧化物产生的异常有效抑制而产生的（Honda 2002）。除了其抗炎特性外，CDDO同源物还具有健康细胞的细胞保护作用，但对不同的癌细胞类型产生促凋亡和抗增殖作用。这些特性表明它们用作癌症化学预防和治疗剂（Sporn and Liby 2005； Yore 2006）。三萜类化的生物学作用的机制尚未完全阐明。 CDDO同源物目前正在临床I期癌症试验中，最近证明CDDO-IM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）。在此提案中，CDDO-IM将被施用给大鼠，以评估将血清蛋白作为对化学保护干预的肝反应性的生物标志物的可行性。目的I：为了检测肝脏对治疗的候选生物标志物，在使用先前确定的CDDO-IM的化学预防剂量方案治疗大鼠后，将进行DIGE研究。将通过凝胶酶消化，质谱分析和数据库搜索来鉴定在核，线粒体和细胞质亚细胞级分中差异表达的肝蛋白。 AIM II：将开发出在AIM I中鉴定出的泄漏的肝蛋白的血清浓度，将开发样品制备方法和定量质谱测定法。在候选生物标志物蛋白的物理特性的指导下，将建立从血清中提取的技术。将开发同位素稀释质谱分析，以定量测量非分泌蛋白的血清浓度，以用作对治疗的肝反应性的生物标志物。 Pilot Dige数据显示CDDO-IM调节了40多种胞质肝蛋白的相对浓度。它们的质谱鉴定显示了各种蛋白质类别，包括但不限于蛋白体复合亚基，线粒体蛋白，脱氢酶，SE结合蛋白，醛还原酶和GST。在提取的血清中识别了几种GST质谱，其中，在概念验证的证明中，GST @在CDDO-IM处理的大鼠中显着升高。该工作的长期目标是确定肝蛋白生物标记物具有对癌症化学介入和质量谱分析的质量分析的肝蛋白生物标志物，以进行大量分析。这些测定法的成熟最终应使血清蛋白的测量能够确定肝癌化学预防干预措施的肝癌反应性。