Mechanisms of Chemoprevention by Isothiocyanates

描述(申请人提供)：诱导细胞凋亡被认为是异硫氰酸酯(ITCs)抑制癌变的一个重要机制。许多信号通路已被证明与这一过程有关；然而，ITCs的上游靶标尚未得到充分研究。我们的研究表明，作为亲电体，ITCs一旦进入细胞内就很容易与谷胱甘肽和蛋白质结合，细胞内的蛋白质最终成为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它们诱导细胞凋亡的活性密切相关。我们发现微管蛋白是ITCs的蛋白质靶标之一，它与ITCs的结合可以引发构象改变、微管网络的破坏、选择性降解，最终导致细胞周期停滞和细胞凋亡。最近，我们做了一个有趣的观察：ITC治疗可以耗尽人类癌细胞中突变型P53，但不能耗尽野生型P53，并且伴随着野生型P53活性的恢复。由于获得了功能，突变的p53细胞具有更强的增殖能力和抵抗细胞死亡的能力。因此，突变型P53的去除和野生型活性的恢复可能是ITC诱导的细胞凋亡消除肿瘤细胞的重要机制。由于超过50%的人类癌症含有P53突变，在这项建议中，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蛋白的结合可以触发构象变化，导致突变型P53蛋白枯竭，恢复野生型活性，从而导致细胞发生凋亡。我们提出了五个目标来验证这一假设：目标1将研究ITCs选择性耗尽突变型p53并恢复野生型活性的机制；目标2将确定突变型p53耗尽及其野生型功能恢复的功能作用；目标3将研究ITCs的结构-活性关系(SARS)，以寻找更有效的化合物来耗尽突变型p53并恢复野生型活性；目标4将在转基因突变型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研究饮用富含PEITC的豆瓣汁对重度吸烟者口腔细胞突变型P53和细胞凋亡的影响。 公共卫生相关性：人们认为，诱导细胞凋亡是异硫氰酸酯(ITCs)作为各种癌症的化学预防药物的一个重要机制。我们观察到ITC处理可以耗尽突变型P53，但不能耗尽野生型P53，并且这种耗竭伴随着野生型样活性的恢复。这些发现提示了一种机制，即ITCs可以使带有突变型P53的细胞对凋亡诱导敏感。由于超过50%的人类癌症存在P53突变，在本项目中，我们将研究ITCs耗尽突变型P53并恢复其野生型功能的机制，并了解其功能后果。此外，我们还将在突变型p53转基因动物模型中测试ITCs的作用，并使用从重度吸烟者饮用富含苯乙基ITCs的西洋菜汁前后收集的口腔细胞来测试ITCs的效果。我们还建议开展ITCs的结构-活性关系(SAR)研究，以发现新的先导化合物。这项研究将允许设计更有效的与ITCs相关的小分子化合物，这些化合物针对突变的p53，用于癌症预防和治疗。

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.