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

描述(申请人提供)：植物生物黄酮类，木犀草素(L或LUT)，以及两个合成配体(BMHPC和ZN-2)与高亲和力的II型雌二醇结合部位(Kd“5 nm)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。对组蛋白H4(和/或组蛋白H3/H4二聚体)上的II类位点作为配体结合域的鉴定表明，该位点的配体占有率调节翻译后修饰(乙酰化、甲基化、磷酸化等)。调节基因转录的组蛋白H3或H4。这代表了一种通过这些配体控制基因转录的新的表观遗传学机制。基因芯片和实时定量聚合酶链式反应(QPCR)研究表明，L、BMHPC或ZN-2除了调节与EGF信号通路有关的一些基因(EGFR、Grb2、SOS、RasGAP、MKK4、c-Fos、JNK1)外，还调节PC-3人前列腺癌PC-3细胞周期基因(CCNA2、CCNE2、CDKN1B、CDC25A、增殖细胞核抗原、PLK1)的表达。L对前列腺癌DU-145细胞中表皮生长因子信号转导和细胞周期通路基因的调控也得到证实。染色质免疫沉淀(ChIP)研究表明，L降低了与PLK1(而不是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，支持表观遗传控制假说。我们怀疑在L、BMHPC和ZN-2控制的表皮生长因子信号和细胞周期通路中的一些基因也是如此。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将确定L、BMHPC或ZN-2对表皮生长因子信号转导和细胞周期通路中基因表达(基因和蛋白质)以及细胞增殖的影响之间的时间关系。我们将研究表皮生长因子受体激动剂和拮抗剂吉非替尼对L、BMHPC和ZN-2调节表皮生长因子信号转导和细胞周期基因表达的影响(特异性靶点2)。具体目标3将包括对L、BMHPC或ZN-2处理的PC-3或DU-145细胞制剂的芯片研究。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)是否负责这些配体对这些细胞系中基因转录的调节。我们将(通过再芯片实验)确定招募到EGFR、c-Fos、PLK1和CDKN1B启动子上的特定协调控子(组蛋白乙酰转移酶；HAT‘s和组蛋白脱乙酰酶；HDAC’s)，并将研究L、BMHPC或ZN-2对这些协调控子与启动子结合的影响。最后，特异靶4将明确L、贝母HPC和ZN-2对正常前列腺组织和裸鼠PC-3或DU-145细胞移植瘤中表皮生长因子信号转导、细胞周期基因表达和细胞增殖的影响。这些研究将明确L、BMHPC和ZN-2在调控组蛋白功能、基因表达和前列腺癌细胞增殖中的作用机制，并可能开发基于新的II位点配体的药物，以控制与正常和恶性细胞增殖有关的EGF信号和细胞周期通路中的基因表达。公共健康相关性：本研究建议中描述的研究表明，饮食衍生的植物生物类黄酮影响表皮生长因子(EGF)信号转导和细胞周期途径中基因的活性。这些分子途径包含许多编码蛋白质的基因，这些蛋白质决定了正常和恶性前列腺细胞分裂的速度或速度。这是因为像木犀草素这样的生物黄酮类化合物模仿一种生物制剂(甲基对羟基苯基乳酸甲酯；MeHPLA)，这种生物制剂通常可以控制非恶性细胞的生长。这项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)在控制EGF信号通路基因的活性以及正常和恶性前列腺细胞分裂方面的作用，其作用类似于MeHPLA。这些研究可能导致开发用于癌症预防或治疗的新药和/或饮食成分。

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.