Molecular Mechanisms of Prostate Cancer Chemoprevention by Apigenin

描述(由申请人提供)：本次竞争性续签申请是为了继续研究芹菜素对前列腺癌的抗癌作用的分子机制。在之前的资助期间，我们研究了芹菜素在细胞培养、肿瘤异种移植和前列腺癌转基因小鼠模型中对各种蛋白激酶、IGF-I生长轴、β-连环蛋白和核因子-B信号通路的影响。我们发现芹菜素可以抑制多种信号转导途径，因此，芹菜素作用的潜在靶点包括磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B/Akt、经典的MAPK/ERK1/2和细胞周期蛋白依赖性激酶。有效抑制这些靶点的芹菜素剂量范围与抑制信号和前列腺癌发生的剂量范围相似，在人体内是生理上可以达到的。在这一新的应用中，我们假设I(B激酶：IKA/？)在介导芹菜素在多个信号转导通路上的抗癌作用中起着中心枢纽的作用。我们的假说是基于我们之前发表的发现和我们的初步数据，这些数据表明：1)IKK活性在人类前列腺癌及其可能的前体中上调；2)芹菜素抑制IKK活性及其磷酸化；3)芹菜素抑制多条信号转导通路，特别是IGF-I轴、PI3K-Akt、NF-(B和β-catenin)通路；以及iv)IKK复合体对所有这些不同的信号通路都具有调节作用。为了验证这一假设，我们将使用最先进的技术，包括核磁共振氢谱、X射线结晶学和先进的分子生物学程序，我们的研究将在敲入和敲除细胞培养物和转基因(TRAMP)小鼠上进行。我们提出的具体目标包括：1)Ikka/β在细胞转化中的作用和芹菜素的抑制活性；2)Ikka在maspin抑制中的作用及其被芹菜素的调节；3)ikk？对叉头盒O3a的活性和细胞分布的作用及其被芹菜素的调节；以及4)Ikka/？作为芹菜素体内抗癌作用的分子靶点。我们预计，了解芹菜素抗癌作用的分子机制可能有助于根据这些关键分子机制更有效地开发有效的化学预防/治疗策略，并减少相关副作用。 与公共健康相关：大量研究表明，芹菜素(4‘，5，7-三羟基黄酮)是一种天然存在的植物黄酮，广泛存在于常见水果和蔬菜中，具有良好的癌症预防和治疗特性。它毒性低，无致突变性，在不影响正常细胞的情况下，显示出抑制细胞生长和诱导癌细胞凋亡的选择性作用。其分子靶点和分子机制还没有得到充分的研究。我们先前已经证明芹菜素抑制多种信号转导通路，特别是IGF-I轴、PI3K-Akt、NF-(B)和β-catenin通路。在这个新的应用中，我们假设芹菜素结合并抑制Ikka/？，不依赖于NF-(B)调节，从而抑制前列腺癌的发生和发展。了解芹菜素的分子机制可能会揭示开发更有效、副作用更少的药物的关键分子靶点，以便将来用于化学预防/治疗方案。

DESCRIPTION (provided by applicant): This competitive renewal application is for continued funding to investigate the molecular mechanisms underlying the anticancer effects of apigenin on prostate cancer. During the previous funding period, we investigated the effects of apigenin on various protein kinases, the IGF-I growth axis, ¿-Catenin and NF-(B signaling pathways in cell cultures, tumor xenografts and a genetically-modified mouse model of prostate cancer. We found that apigenin inhibits multiple signal transduction pathways; consequently, potential targets of apigenin action include phosphatidylinositol 3-kinase (PI3K), protein kinase B/Akt, classical MAPK/ERK1/2, and cyclin-dependent kinases. The dose range of apigenin that effectively inhibits these targets is similar to that which suppresses signaling and prostate tumorigenesis and is physiologically attainable in humans. In this renewal application, we hypothesize that I(B kinase: IKKa/¿ acts as a central hub in mediating the anticancer effects of apigenin on multiple signal transduction pathways. Our hypothesis is based upon our previously published findings and our preliminary data showing that i) IKK activity is upregulated in human prostate cancer and its putative precursor ii) apigenin inhibits IKK activity and its phosphorylation, iii) apigenin inhibits multiple signal transduction pathways, specifically, the IGF-I axis, PI3K-Akt, NF-(B and ¿-Catenin pathways, and iv) the IKK complex exhibits regulatory effects on all of these distinct signaling pathways. To test this hypothesis, we will use state-of-the-art techniques, including 1H NMR, X-ray crystallography, and advanced molecular biology procedures, and our studies will be performed on knock-in and knockout cell cultures and on transgenic (TRAMP) mice. Our proposed specific aims include investigation of i) the role of IKKa/¿ in cell transformation and the inhibitory activity of apigenin, ii) the role of IKKa on maspin suppression and its modulation by apigenin, iii) the role of IKK¿ on activity and cellular distribution of forkhead box O3a and its modulation by apigenin, and iv) IKKa/¿ as a molecular target for the anticancer effects of apigenin in vivo on TRAMP mice. We expect that understanding the molecular mechanisms underlying the anti-cancerous effects of apigenin may be helpful in developing effective chemopreventive/therapeutic strategies based upon targeting these key molecular mechanisms more effectively and with fewer associated side-effects. PUBLIC HEALTH RELEVANCE: Numerous studies have shown that apigenin (4', 5, 7-trihydroxyflavone), a naturally occurring plant flavone abundantly present in common fruits and vegetables, possess promising cancer preventive and therapeutic properties. It has low toxicity, is non-mutagenic, and has shown selective effects in inhibiting cell growth and inducing apoptosis in cancer cells without affecting normal cells. The molecular targets and molecular mechanisms are not fully investigated. We have previously demonstrated that apigenin inhibits multiple signal transduction pathways, specifically the IGF-I axis, PI3K-Akt, NF-(B and ¿-Catenin pathways. In this renewal application, we hypothesize that apigenin binds to and inhibit IKKa/¿, independent of NF-(B regulation thereby suppressing prostate cancer development and progression. Understanding the molecular mechanisms of apigenin may reveal key molecular targets for the development of more effective agents with fewer side-effects for future use in chemopreventive/therapeutic regimens.