Serum Biomarkers of Hepatic Response to Cancer Chemopreventive Intervention

描述(由申请人提供)： 在肝癌的动物模型中，几乎任何化学预防药物的治疗都会导致肝脏蛋白质丰度的变化。在20世纪90年代的S，结构不同的化学保护剂被证明改变了许多实验致癌物的代谢活化和解毒之间的平衡。最近，转基因动物和寡核苷酸微阵列技术被用于探索化学预防药物发挥作用的转录调控途径。化学预防药物(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氧合酶发挥作用，现在发现通过促进癌细胞凋亡的额外机制发挥作用。今天，新的蛋白质组发现工具，如凝胶差异电泳(DGE)，能够检测蛋白质相对丰度的变化，而不需要事先识别蛋白质。虽然不是癌症终点本身，但其相对丰度受到调节的肝脏蛋白可以为化学预防药物的作用机制提供见解。这些肝脏蛋白可能被用来开发对化学预防治疗有反应的血清生物标志物。正常人血清中含有少量非分泌性蛋白质，主要分布在细胞核、线粒体和细胞质中。目前尚不清楚它们是如何进入血液的，但可能涉及到坏死、细胞凋亡和一种定义不清的现象，即“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的总丰度的稳定值。由于肝脏的大小，预计肝细胞将是正常人血清中非分泌性蛋白的主要贡献者。这项工作的长期目标是确定对化学预防干预反应的肝脏蛋白生物标志物，并开发其在血清中的质谱学分析方法。这些检测方法的成熟最终应该能够在临床试验中测量血清蛋白，以确定对肝癌化学预防干预的反应。最初对三萜类1-[2-cyano-3-12-dioxooleana-1，9(11)-dien-28-oyl]imidazole(CDDO-IM)的科学兴趣是因为它对一氧化氮产生的异常有效的抑制(本田2002)。除了抗炎特性外，CDDO同系物对健康细胞具有细胞保护作用，但对不同类型的癌细胞具有促凋亡和抗增殖作用。这些特性表明它们被用作癌症化学预防和治疗剂(Sporn和Liby 2005；Yore 2006)。三萜类化合物发挥生物学作用的机制仍未完全阐明。CDDO的同系物目前处于I期临床癌症试验中，CDDO-Im最近被证明是在大鼠肝脏肿瘤发生模型中测试过的最有效的化学预防药物(Yates 2006)。在这项建议中，CDDO-Im将应用于大鼠，以评估开发血清蛋白作为肝脏对化学保护干预反应的生物标志物的可行性。目的I：为了检测肝脏对治疗的反应性的候选生物标志物，将在用先前建立的CDDO-Im化学预防剂量方案治疗大鼠后进行DGE研究。通过凝胶内酶切、质谱分析和数据库搜索，将在细胞核、线粒体和细胞质亚细胞组分中鉴定差异表达的肝脏蛋白。目的II：将发展样品制备方法和定量质谱学分析方法，以测定AIM I中确定的漏出性肝蛋白的血清浓度。根据候选生物标志物蛋白的物理性质，将建立从血清中提取它们的技术。将开发同位素稀释质谱分析法来定量测量非分泌性蛋白的血清浓度，以用作肝脏对治疗反应的生物标志物。中试DGE数据显示，CDDO-Im可调节40多种肝细胞胞浆蛋白的相对浓度。它们的质谱学鉴定揭示了各种蛋白质类别，包括但不限于蛋白质小体复合体亚基、线粒体蛋白、脱氢酶、硒结合蛋白、醛还原酶和GSTs。在提取的血清中鉴定了几种GST，其中在概念验证演示中，GST@在CDDO-Im治疗的大鼠中显著升高。这项工作的长期目标是识别对癌症化学预防干预有反应的肝脏蛋白质生物标记物，并开发其在血清中的定量分析方法。这些检测方法的成熟最终应该能够在临床试验中测量血清蛋白，以确定肝脏对肝癌化学预防干预的反应。

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.