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

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）与II型[3H]雌二醇结合位点高亲和力（Kd ' 5nM）相互作用，在体内和体外抑制正常和恶性前列腺细胞的增殖。II型位点作为组蛋白H4（和/或组蛋白H3/H4二聚体）上的配体结合域的鉴定表明，配体占据该位点可调节组蛋白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一种新的表观遗传机制。微阵列和实时PCR （qPCR）研究表明，L、BMHPC或ZN-2在PC-3人前列腺癌细胞中调控细胞周期基因（CCNA2、CCNE2、CDKN1B、CDC25A、PCNA、PLK1）的表达，以及EGF信号通路相关基因（EGFR、GRB2、SOS、RasGAP、MKK4、c-Fos、JNK1）的表达。在DU-145前列腺癌细胞中也证实了EGF信号和细胞周期通路基因的L调节。染色质免疫沉淀（ChIP）研究显示，L降低了与PLK1（但不包括RB1）基因启动子相关的组蛋白H4（在5、8、12、16号赖氨酸）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。提出的研究的目标是确定这些II型位点配体的作用机制。特异性目标1将定义L、BMHPC或ZN-2对EGF信号通路和细胞周期通路中基因表达（mRNA和蛋白质）和细胞增殖的影响之间的时间关系。EGFR激动剂（EGF）和拮抗剂（吉非替尼）对L、BMHPC和ZN-2调控EGF信号和细胞周期基因表达（Specific Aim 2）的影响将被研究。特异性Aim 3将包括用L、BMHPC或ZN-2处理的PC-3或DU-145细胞制剂的ChIP研究。我们将确定与EGF信号（EGFR, c-Fos）或细胞周期（PLK1, CDKN1B）基因启动子相关的组蛋白H3和/或H4的翻译后修饰（乙酰化）是否负责这些配体在这些细胞系中的基因转录调控。将鉴定募集到EGFR、c-Fos、PLK1和CDKN1B启动子上的特异性共调节因子（组蛋白乙酰转移酶；HAT's和组蛋白去乙酰化酶；HDAC's）（通过re-ChIP测定），并研究L、BMHPC或ZN-2对这些共调节因子与启动子关联的影响。最后，Specific Aim 4将确定L、BMHPC和ZN-2对正常前列腺组织和裸鼠PC-3或DU-145细胞异种移植中EGF信号传导、细胞周期基因表达（mRNA、蛋白）和细胞增殖的影响。研究应明确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，并可开发基于II型配体的新型药物，用于控制正常细胞和恶性细胞增殖的EGF信号通路和细胞周期通路中的基因表达。

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.