Genetic Identification of Sulforaphane's Mechanism of Action in Yeast Cell Death

描述（由申请人提供）：萝卜硫素（SFN）是一种异硫氰酸酯，在西兰花和西兰花芽中大量存在，已证明在细胞培养和致癌物诱导的遗传动物癌症模型中是一种有效的化学保护剂。最近，几个研究小组已经表明，SFN能够诱导哺乳动物细胞中两种形式的程序性细胞死亡（PCD），称为凋亡（PCD I）和自噬（PCD II）。有趣的是，一个实验室已经确定哺乳动物细胞可以诱导自噬以保护自己免受SFN诱导的细胞凋亡。然而，SFN如何诱导自噬仍不清楚。 为了填补这一知识空白，我们正在努力了解SFN在诱导程序性细胞死亡，特别是自噬中的作用机制，使用芽殖酵母，酿酒酵母，作为真核系统的模型。这项工作是创新的，因为这是第一次在遗传上易于处理的模型系统中研究萝卜硫素的作用机制。我们想表征一系列的萝卜硫素抗性酵母突变体，我们已经在我们的实验室分离。这些突变体应该有助于我们确定参与介导SFN在芽殖酵母细胞死亡中的作用机制的遗传途径，也可能参与SFN诱导哺乳动物细胞死亡，特别是自噬的遗传途径。 我们希望我们的研究计划能够产生积极的影响，因为所确定的遗传途径应该为药理学干预提供新的靶点，从而增强SFN作为临床药物的化学保护和化疗作用。拟议的研究还可能导致识别可能在未来临床试验中有用的基于机制的生物标志物。 最后，除了扩大我们的知识基础，在癌症化学预防更普遍和萝卜硫素的作用机制更具体，资助研究中描述的这一地区R15赠款提案也将允许我的实验室和我们的部门在这里普罗维登斯学院继续培训和准备有才华的本科生在生物医学科学的成功职业生涯。) 公共卫生相关性：萝卜硫素是一种在西兰花和西兰花芽中大量发现的化学成分，已被证明是一种有效的抗癌药物，无论是在细胞培养还是在癌症动物模型中。目前尚不清楚这种药物是如何杀死癌细胞的，尽管它被认为参与触发细胞自杀或程序性细胞死亡的过程。 我们想用酵母细胞来发现萝卜硫素是如何起作用的。通过鉴定耐药的酵母突变体，我们希望能鉴定出参与细胞对萝卜硫素反应的基因。我们希望这项研究不仅能帮助我们更好地了解细胞自杀的过程，还能提高药物在治疗癌症患者的临床试验中的疗效。

DESCRIPTION (provided by applicant): Sulforaphane (SFN) is an isothiocyanate found in abundance in broccoli and broccoli sprouts that has proven to be an effective chemoprotective agent both in cell culture and in carcinogen-induced and genetic animal cancer models. Recently, several research groups have shown that SFN is able to induce two forms of programmed cell death (PCD) in mammalian cells known as apoptosis (PCD I) and autophagy (PCD II). Paradoxically, one laboratory has determined that mammalian cells can induce autophagy to protect themselves against SFN-induced apoptosis. However, how SFN induces autophagy remains unclear. To fill this knowledge gap, we are working to understand the mechanism of action of SFN in the induction of programmed cell death, especially of autophagy, using the budding yeast, Saccharomyces cerevisiae, as a model eukaryotic system. The proposed work is innovative, because it is the first time that sulforaphane's mechanism of action has been investigated in a genetically tractable model system. We would like to characterize a series of sulforaphane-resistant yeast mutants that we have isolated in our laboratory. These mutants should help us to identify the genetic pathway(s) involved in mediating SFN's mechanism of action in budding yeast cell death, genetic pathways that may also be involved in SFN's induction of cell death, especially of autophagy, in mammalian cells. We expect that our research program to have a positive impact because the identified genetic pathway(s) should provide new targets for pharmacological interventions that could enhance SFN's chemoprotective and chemotherapeutic effects as a clinical drug. The proposed research could also lead to the identification of mechanism-based biomarkers potentially useful in future clinical trials. Finally, in addition to expanding our knowledge base in cancer chemoprevention more generally and in sulforaphane's mechanism of action more specifically, funding the research described in this AREA R15 grant proposal will also allow my laboratory and our department here at Providence College to continue to train and to prepare talented undergraduate students for successful careers in the biomedical sciences. ) PUBLIC HEALTH RELEVANCE: Sulforaphane is a chemical ingredient found in abundance in broccoli and broccoli sprouts that has proven to be an effective anti-cancer drug both in cell culture and in animal models of cancer. It is not clear how this drug kills cancer cells though it is thought to be involved in triggering the process of cell suicide or programmed cell death. We would like to use yeast cells to discover how sulforaphane works. By identifying yeast mutants that are resistant to the drug, we hope to identify the genes involved in a cell's response to sulforaphane. We expect this research not only to help us better understand the process of cell suicide but also to enhance the drug's efficacy in clinical trials to treat cancer patients.