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，几乎任何化学预防剂的治疗都会引起肝蛋白丰度的变化。在20世纪90年代，结构上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活化和解毒之间的平衡。最近，转基因动物和寡核苷酸芯片技术被用来探索转录调控途径，通过化学预防剂的工作。最初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起作用的化学预防剂（例如萝卜硫素、舒林酸）被发现通过促进癌细胞凋亡的额外机制起作用。今天，新的蛋白质组学发现工具，如凝胶电泳差异（DIGE），使得能够检测蛋白质相对丰度的变化，而不需要提前识别蛋白质。虽然不是癌症终点本身，肝蛋白的相对丰度的调制可以提供深入了解化学预防剂的作用机制。这些肝脏蛋白质可用于开发对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主要位于细胞核、胞浆和细胞质中的非分泌性蛋白。它们是如何进入血液的尚不清楚，但可能涉及坏死、凋亡和一种定义不清的现象，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的总丰度的稳态值。由于肝脏的大尺寸，预期肝细胞是正常人血清中发现的非分泌性蛋白质的主要贡献者。这项工作的长期目标是确定肝脏蛋白质生物标志物的反应，化学预防干预，并开发质谱方法，在血清中进行分析。这些检测方法的成熟最终将使血清蛋白的测量能够在临床试验中确定对肝癌化学预防干预的反应性。三萜类化合物1-[2-氰基-3-12-二氧代齐墩果烷-1，9（11）-二烯-28-酰基]咪唑（CDDO-Im）对一氧化氮产生的异常强效抑制作用引起了科学界的最初兴趣（本田，2002年）。除了它们的抗炎特性之外，CDDO同源物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。这些性质表明它们可用作癌症化学预防剂和治疗剂（Sporn和Liby 2005; Yore 2006）。三萜类化合物发挥其生物学效应的机制仍有待完全阐明。CDDO同源物目前处于临床I期癌症试验中，并且CDDO-Im最近被证明是在肝肿瘤发生的大鼠模型中测试的最有效的化学预防剂（Yates，2006年）。在该提案中，将向大鼠施用CDDO-Im以评估开发血清蛋白作为肝脏对化学保护干预的反应性的生物标志物的可行性。目标一：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的化学预防性给药方案治疗大鼠后进行DIGE研究。将通过凝胶内酶消化、质谱分析和数据库检索鉴定细胞核、线粒体和细胞质亚细胞组分中差异表达的肝蛋白。目标二：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测定法。在候选生物标志物蛋白的物理性质的指导下，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，以定量测量非分泌蛋白的血清浓度，用作肝脏对治疗反应性的生物标志物。中试DIGE数据表明CDDO-Im调节超过40种胞质肝蛋白的相对浓度。它们的质谱鉴定揭示了多种蛋白质类别，包括但不限于蛋白体复合物亚基、线粒体蛋白、丝氨酸蛋白酶、硒结合蛋白、醛还原酶和GST。几个GST的质谱鉴定提取的血清中，在一个概念验证示范，GST @显着升高CDDO-Im治疗rates.Narrative这项工作的长期目标是识别肝蛋白质生物标志物的反应癌症化学预防干预和开发质谱方法，其在血清中的定量分析。这些检测方法的成熟最终将使血清蛋白的测量能够在临床试验中确定肝脏对肝癌化学预防干预的反应性。

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.