Mechanisms of Chemoprevention by Isothiocyanates

描述（由申请人提供）：诱导细胞凋亡被认为是异硫氰酸酯（ITCs）抑制癌变的重要机制。许多信号通路已被证明与这一过程有关；然而，国际贸易中心的上游目标尚未得到充分调查。我们的研究表明，ITCs作为亲电试剂，一旦进入细胞，就很容易与谷胱甘肽和蛋白质结合，并且细胞内蛋白质最终成为ITCs的主要结合靶点。我们发现苯乙基ITC （PEITC）和萝卜硫素（SFN）的总蛋白结合亲和力与其诱导细胞凋亡的活性密切相关。我们发现微管蛋白是ITCs的蛋白靶点之一，其与ITCs的结合可以引发构象变化、微管网络破坏、选择性降解，最终导致细胞周期阻滞和凋亡。最近，我们做了一个有趣的观察，ITC治疗可以消耗人类癌细胞中的突变p53，但不能消耗野生型p53，并且这种消耗伴随着野生型活性的恢复。由于功能的获得，突变的p53细胞具有更强的增殖能力和抗细胞死亡能力。因此，去除p53突变体并恢复其野生型活性可能是通过tc诱导的凋亡消除致癌细胞的重要机制。由于超过50%的人类癌症含有p53突变，在本提案中，我们将重点研究ITCs对p53蛋白的影响。我们的主要假设是ITCs与突变型p53蛋白结合可引发构象变化，导致突变型p53耗损并恢复野生型活性，从而导致细胞凋亡。我们提出了五个目标来检验这一假设：目标1将研究ITCs选择性地消耗突变型p53并恢复野生型活性的机制；目的2将确定突变型p53缺失及其野生型功能恢复的功能作用；目的3将检查ITCs的结构-活性关系（SARs），以发现更有效的化合物来消耗突变型p53并恢复野生型活性；目的4在转基因突变型p53 a /J小鼠模型中研究ITCs对突变型p53蛋白水平和苯并[a]芘诱导的肺肿瘤形成的影响；目的5将研究饮用豆瓣菜汁（富含PEITC）对重度吸烟者口腔细胞中p53突变体和细胞凋亡的影响。

DESCRIPTION (provided by applicant): Induction of apoptosis is believed to be an important mechanism by which isothiocyanates (ITCs) inhibit carcinogenesis. A number of signaling pathways have been shown to link to this process; however, the upstream targets of ITCs have not been fully investigated. Our studies have shown that, as electrophiles, ITCs readily bind to glutathione and proteins once inside the cells and that intracellular proteins eventually serve as the predominant binding target of ITCs. We found that the total protein binding affinities of phenethyl ITC (PEITC) and sulforaphane (SFN) correlate well with their activities toward apoptosis induction. We identified tubulin as one of the protein targets of ITCs and its binding by ITCs can trigger conformational changes, disruption of microtubule network, selective degradation and, finally, cell cycle arrest and apoptosis. Recently, we made an intriguing observation that ITC treatment can deplete mutant p53 in human cancer cells, but not wild-type p53, and that depletion is accompanied by restoration of the wild-type-like activities. Due to gain of function, the mutant p53 cells are more proliferative and resistant to cell death. Therefore, the removal of mutant p53 and the restoration of its wild-type activity could be an important mechanism by which oncogenic cells can be eliminated by ITC-induced apoptosis. Because greater than 50% of human cancers contain p53 mutation, in this proposal we will focus our studies on the effects of ITCs on p53 protein. Our main hypothesis is that binding to the mutant p53 protein by ITCs can trigger conformational changes, leading to the mutant p53 depletion and restoration of the wild-type activities and consequently, causing cells to undergo apoptosis. We propose five aims to examine this hypothesis: Aim 1 will investigate the mechanisms by which ITCs selectively deplete mutant p53 and restore the wild-type activity; Aim 2 will determine the functional roles of mutant p53 depletion and its wild- type function restoration; Aim 3 will examine Structure-Activity Relationships (SARs) of ITCs to discover more efficacious compounds for depleting mutant p53 and restoring the wild-type activities; Aim 4 will study effects of ITCs on mutant p53 protein levels and lung tumor formation induced by benzo[a]pyrene in a transgenic mutant p53 A/J mouse model; and Aim 5 will investigate the effects of drinking watercress juice (rich in PEITC) on mutant p53 and apoptosis in oral cells collected from heavy smokers. PUBLIC HEALTH RELEVANCE: It is believed that apoptosis induction is an important mechanism by which isothiocyanates (ITCs) act as chemopreventive agents for various cancers. We observed that ITC treatment can deplete mutant p53, but not wild type p53, in cells and the depletion is accompanied by restoration of the wild-type-like activities. These findings suggest a mechanism by which ITCs can sensitize cells with mutant p53 to apoptosis induction. Because more than 50% human cancers have p53 mutation, in this program we will investigate the mechanisms by which ITCs deplete mutant p53 and restore its wild-type functions and understand the functional consequences. In addition, we will test the effects of ITCs in a mutant p53 transgenic animal model and by using oral cells collected from heavy smokers before and after drinking watercress juice rich in phenethyl ITCs. We also propose to carry out Structural-Activity Relationship (SAR) studies of ITCs for the discovery of new lead compounds. This research will allow design more effective small compounds related to ITCs that target to mutant p53 for cancer prevention and therapy.