Bioflavonoid Effects on EGF Signaling and Cell Cycle Pathways in Prostate Cancer

生物类黄酮对前列腺癌 EGF 信号传导和细胞周期途径的影响

• 批准号: 8006439
• 负责人: BARRY Matthew MARKAVERICH
• 金额: $ 30.9万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006439
• 关键词: Acetylation; Affinity; Agonist; Animal Model; Applications Grants; Binding Sites; Bioflavonoid; Biological; Biological Assay; Biological Products; Breast; CCNA2 gene; CCND1 gene; CCNE2 gene; CDC25A gene; CDKN1B gene; Cell Count; Cell Cycle; Cell Cycle Progression; Cell Line; Cell Proliferation; Cell division; Cells; Coupled; Cyclohexanones; Data; Development; Diet; Dietary Component; Drug Controls; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epigenetic Process; Estradiol; FOS gene; Figs - dietary; GRB2 gene; Gefitinib; Gene Expression; Gene Expression Regulation; Gene Proteins; Genes; Genetic Transcription; Genistein; Goals; Growth; Health; Histone Deacetylase; Histone H2A; Histone H3; Histone H4; Histones; Human; In Vitro; LNCaP; Lead; Ligand Binding Domain; Ligands; Luteolin; Lysine; MAPK8 gene; MCF7 cell; Malignant - descriptor; Malignant neoplasm of prostate; Mediating; Messenger RNA; Methylation; Modification; Molecular; Mus; Non-Malignant; Normal Cell; Nucleosomes; Nude Mice; Oligonucleotide Microarrays; PC3 cell line; PLK1 gene; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Plants; Post-Translational Protein Processing; Preparation; Progress Reports; Prostate; Proteins; Quercetin; RB1 gene; Recruitment Activity; Regulation; Research Proposals; Signal Pathway; Signal Transduction; Signaling Pathway Gene; Site; Son of Sevenless Proteins; Time; Tissues; Transferase; Western Blotting; Xenograft procedure; base; c-myc Genes; cancer cell; cancer prevention; cancer therapy; carbene; cdc Genes; chromatin immunoprecipitation; cyqualon; daidzein; design; dimer; histone acetyltransferase; in vivo; mRNA Expression; methyl 4-hydroxyphenyllactate; novel; promoter; research study; response

DESCRIPTION (provided by applicant): The plant bioflavonoid, luteolin (L or LUT), and two synthetic ligands (BMHPC and ZN-2) interact with type II [3H]estradiol binding sites with high affinity (Kd" 5nM) and inhibit normal and malignant prostate cell proliferation in vivo and in vitro. The identification of type II sites as a ligand-binding domain on histone H4 (and/or histone H3/H4 dimers) suggests that ligand occupancy of this site modulates post-translational modifications (acetylation, methylation, phosphorylation, etc.) of histones H3 or H4 that regulate gene transcription. This represents a novel epigenetic mechanism for controlling gene transcription by these ligands. Microarray and real time PCR (qPCR) studies demonstrate that L, BMHPC or ZN-2 regulate cell cycle gene (CCNA2, CCNE2, CDKN1B, CDC25A, PCNA, PLK1) expression in PC-3 human prostate cancer cells in addition to a number of genes involved in the EGF Signaling Pathway (EGFR, GRB2, SOS, RasGAP, MKK4, c-Fos, JNK1). L modulation of EGF Signaling and Cell Cycle Pathway genes was also confirmed in DU-145 prostate cancer cells. Chromatin immunoprecipitation (ChIP) studies revealed that L reduced the acetylation state of histone H4 (at Lys 5, 8, 12, 16) associated with the PLK1 (but not RB1) gene promoter, supporting the epigenetic control hypothesis. We suspect this will be the case for a number of genes in the EGF Signaling and Cell Cycle Pathways controlled by L, BMHPC and ZN-2. The goal of the proposed studies is to define the mechanism of action of these type II site ligands. Specific Aim 1 will define temporal relationships between L, BMHPC or ZN-2 effects on gene expression (mRNA and protein) in the EGF Signaling and Cell Cycle Pathways and cell proliferation. The effects of EGFR agonists (EGF) and antagonists (Gefitinib) on L, BMHPC and ZN-2 regulation of EGF Signaling and cell cycle gene expression (Specific Aim 2) will be studied. Specific Aim 3 will consist of ChIP studies on preparations from PC-3 or DU-145 cells treated with L, BMHPC or ZN-2. We will determine if post-translational modifications (acetylation) of histones H3 and/or H4 associated with EGF Signaling (EGFR, c-Fos) or cell cycle (PLK1, CDKN1B) gene promoters are responsible for the regulation of gene transcription by these ligands in these cell lines. Specific coregulators (histone acetyl transferases; HAT's and histone deacetylases; HDAC's) recruited to EGFR, c-Fos, PLK1 and CDKN1B promoters will be identified (via re-ChIP assays) and L, BMHPC or ZN-2 effects on the association of these coregulators with the promoters will be studied. Lastly, Specific Aim 4 will define the effects of L, BMHPC and ZN-2 on EGF Signaling and cell cycle gene expression (mRNA, protein) and cell proliferation in normal prostate tissue and in PC-3 or DU-145 cell xenografts in nude mice. The studies should define the mechanism of action of L, BMHPC and ZN-2 in the regulation of histone function, gene expression and prostate cancer cell proliferation and could lead to the development of new type II site ligand based drugs for the control of gene expression in EGF Signaling and Cell Cycle Pathways responsible for the proliferation of normal and malignant cells. PUBLIC HEALTH RELEVANCE: The studies described in this research proposal indicate that diet-derived plant bioflavonids influence the activity of genes in the epidermal growth factor (EGF) Signaling and cell cycle pathways. These molecular pathways contains a number of genes encoding proteins that determine how slow or fast normal and malignant prostate cells divide. This is because bioflavonoids like luteolin mimic a biological agent (methyl p- hydroxyphenyllactate; MeHPLA) that normally controls the growth of non-malignant cells. The experiments proposed in this grant application will define the mechanism of action of luteolin, and two drugs (BMHPC and ZN-2) designed to act like MeHPLA, in the control of the activity of the EGF Signaling Pathway genes and normal and malignant prostate cell division. These studies may lead to the development of new drugs and/or dietary components for use in cancer prevention or treatment.

描述（由申请人提供）：植物双黄酮、毛地黄黄酮（L或LUT）和两种合成配体（BMHPC和ZN-2）以高亲和力（Kd-5 nM）与II型[3 H]雌二醇结合位点相互作用，并在体内和体外抑制正常和恶性前列腺细胞增殖。将II型位点鉴定为组蛋白H4（和/或组蛋白H3/H4二聚体）上的配体结合结构域表明，该位点的配体占据调节翻译后修饰（乙酰化、甲基化、磷酸化等）。调节基因转录的组蛋白H3或H4。这代表了通过这些配体控制基因转录的一种新的表观遗传机制。微阵列和真实的时间PCR（qPCR）研究表明，L、BMHPC或ZN-2调节PC-3人前列腺癌细胞中细胞周期基因（CCNA 2、CCNE 2、CDKN 1B、CDC 25 A、PCNA、PLK 1）的表达，以及参与EGF信号通路的许多基因（EGFR、GRB 2、SOS、RasGAP、MKK 4、c-Fos、JNK 1）。在DU-145前列腺癌细胞中也证实了EGF信号传导和细胞周期途径基因的L调节。染色质免疫沉淀（ChIP）研究表明，L减少组蛋白H4（在赖氨酸5，8，12，16）与PLK 1（但不是RB 1）基因启动子的乙酰化状态，支持表观遗传控制假说。我们怀疑这将是由L，BMHPC和ZN-2控制的EGF信号传导和细胞周期途径中的许多基因的情况。拟议研究的目标是确定这些II型位点配体的作用机制。具体目标1将定义L、BMHPC或ZN-2对EGF信号传导和细胞周期途径中的基因表达（mRNA和蛋白质）和细胞增殖的影响之间的时间关系。将研究EGFR激动剂（EGF）和拮抗剂（吉非替尼）对L、BMHPC和ZN-2调节EGF信号传导和细胞周期基因表达（特异性目的2）的影响。具体目标3将包括对用L、BMHPC或ZN-2处理的PC-3或DU-145细胞制备物的ChIP研究。我们将确定是否与EGF信号（EGFR，c-Fos）或细胞周期（PLK 1，CDKN 1B）基因启动子相关的组蛋白H3和/或H4的翻译后修饰（乙酰化）负责这些配体在这些细胞系中调控基因转录。将鉴定募集至EGFR、c-Fos、PLK 1和CDKN 1B启动子的特异性辅调节剂（组蛋白乙酰转移酶; HAT和组蛋白脱乙酰酶; HDAC）（通过re-ChIP测定），并研究L、BMHPC或ZN-2对这些辅调节剂与启动子的关联的影响。最后，具体目标4将确定L、BMHPC和ZN-2对正常前列腺组织和裸鼠中PC-3或DU-145细胞异种移植物中EGF信号传导和细胞周期基因表达（mRNA、蛋白质）和细胞增殖的影响。这些研究应该确定L、BMHPC和ZN-2在调节组蛋白功能、基因表达和前列腺癌细胞增殖中的作用机制，并可能导致开发新的基于II型位点配体的药物，用于控制负责正常和恶性细胞增殖的EGF信号传导和细胞周期途径中的基因表达。公共卫生关系：这项研究计划中描述的研究表明，饮食来源的植物biopropionids影响表皮生长因子（EGF）信号传导和细胞周期途径中基因的活性。这些分子通路包含许多编码蛋白质的基因，这些蛋白质决定正常和恶性前列腺细胞分裂的快慢。这是因为类毛地黄黄酮类化合物模拟了一种通常控制非恶性细胞生长的生物制剂（对羟基苯乳酸甲酯; MeHPLA）。本资助申请中提出的实验将定义毛地黄黄酮和两种药物（BMHPC和ZN-2）的作用机制，这两种药物旨在像MeHPLA一样控制EGF信号通路基因的活性以及正常和恶性前列腺细胞分裂。这些研究可能导致开发用于癌症预防或治疗的新药和/或膳食成分。