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: 7569613
• 负责人: BARRY Matthew MARKAVERICH
• 金额: $ 31.85万
• 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/7569613
• 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; 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; 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; bcl-1 Genes; c-myc Genes; cancer cell; cancer prevention; carbene; cdc Genes; chromatin immunoprecipitation; cyqualon; daidzein; design; dimer; histone acetyltransferase; in vivo; mRNA Expression; methyl 4-hydroxyphenyllactate; novel; promoter; public health relevance; 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）：植物生物类黄酮、木犀草素（L 或 LUT）和两种合成配体（BMHPC 和 ZN-2）以高亲和力 (Kd" 5nM) 与 II 型 [3H] 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。II 型位点作为配体结合的鉴定 组蛋白 H4（和/或组蛋白 H3/H4 二聚体）上的结构域表明该位点的配体占据调节组蛋白 H3 或 H4 的翻译后修饰（乙酰化、甲基化、磷酸化等），从而调节基因转录。这代表了通过这些配体控制基因转录的新表观遗传机制。微阵列和实时 PCR (qPCR) 研究 证明 L、BMHPC 或 ZN-2 调节 PC-3 人前列腺癌细胞中的细胞周期基因（CCNA2、CCNE2、CDKN1B、CDC25A、PCNA、PLK1）表达，以及许多参与 EGF 信号通路的基因（EGFR、GRB2、SOS、RasGAP、MKK4、c-Fos、JNK1）。 L 调节 EGF 信号和细胞周期通路基因也在 DU-145 前列腺癌细胞中得到证实。染色质免疫沉淀 (ChIP) 研究表明，L 降低了与 PLK1（但不是 RB1）基因启动子相关的组蛋白 H4（位于 Lys 5、8、12、16）的乙酰化状态，支持了表观遗传控制假说。我们怀疑 EGF 信号传导中的许多基因都会出现这种情况 以及由 L、BMHPC 和 ZN-2 控制的细胞周期途径。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基因表达（mRNA 和蛋白质）的影响与细胞增殖之间的时间关系。 EGFR 激动剂 (EGF) 和拮抗剂的作用 (吉非替尼)对L、BMHPC和ZN-2对EGF信号传导和细胞周期基因表达的调节(具体目标2)进行了研究。具体目标 3 将包括对经 L、BMHPC 或 ZN-2 处理的 PC-3 或 DU-145 细胞制备物进行 ChIP 研究。我们将确定组蛋白 H3 和/或 H4 的翻译后修饰（乙酰化）是否与 EGF 信号传导相关 （EGFR、c-Fos）或细胞周期（PLK1、CDKN1B）基因启动子负责在这些细胞系中通过这些配体调节基因转录。将鉴定招募到 EGFR、c-Fos、PLK1 和 CDKN1B 启动子的特定辅助调节因子（组蛋白乙酰转移酶；HAT 和组蛋白脱乙酰酶；HDAC）（通过 re-ChIP 测定）和 L， 将研究 BMHPC 或 ZN-2 对这些辅助调节子与启动子的结合的影响。最后，具体目标 4 将定义 L、BMHPC 和 ZN-2 对正常前列腺组织和裸鼠 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。研究应确定 L 的作用机制， BMHPC 和 ZN-2 调节组蛋白功能、基因表达和前列腺癌细胞增殖，并可能导致开发基于新型 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。公共健康相关性：本研究提案中描述的研究表明，饮食来源的植物生物黄酮素影响基因的活性 表皮生长因子 (EGF) 信号传导和细胞周期途径。这些分子途径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速度。这是因为木犀草素等生物类黄酮模仿了通常控制非恶性细胞生长的生物制剂（对羟苯基乳酸甲酯；MeHPLA）。的 本次拨款申请中提出的实验将确定木犀草素的作用机制，以及两种药物（BMHPC 和 ZN-2）的作用类似于 MeHPLA，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。这些研究可能会导致用于癌症预防或治疗的新药物和/或饮食成分的开发。