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

描述(申请人提供)：萝卜硫醚(SFN)是一种异硫氰酸酯，在西兰花和西兰花芽中大量存在，已被证明在细胞培养和致癌物诱导和遗传动物癌症模型中都是有效的化学保护剂。最近，几个研究小组发现SFN能够在哺乳动物细胞中诱导两种形式的程序性细胞死亡(PCD)，即凋亡(PCD I)和自噬(PCD II)。矛盾的是，一家实验室已经确定哺乳动物细胞可以诱导自噬来保护自己免受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在诱导细胞程序性死亡，特别是自噬中的作用机制，使用发芽酵母作为真核系统的模型。这项拟议的工作是创新的，因为这是第一次在遗传易处理的模型系统中研究萝卜硫素的作用机制。我们想要对我们在实验室中分离到的一系列耐萝卜硫素的酵母突变株进行鉴定。这些突变体将帮助我们确定参与介导SFN在萌芽酵母细胞死亡中的作用机制的遗传途径(S)，这些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。我们预计我们的研究计划将产生积极影响，因为已确定的遗传途径(S)将为药理干预提供新的靶点，从而增强三七总皂苷作为临床药物的化学保护和化疗效果。这项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记物。最后，除了更广泛地扩展我们在癌症化学预防和萝卜硫素作用机制方面的知识库外，资助这一领域R15拨款提案中描述的研究还将使我的实验室和我们普罗维登斯学院的系继续培训有才华的本科生，为他们在生物医学科学领域的成功职业生涯做好准备。) 与公众健康相关：萝卜硫素是一种化学成分，在西兰花和西兰花芽中大量存在，在细胞培养和癌症动物模型中都被证明是一种有效的抗癌药物。目前尚不清楚这种药物是如何杀死癌细胞的，尽管它被认为参与了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工作的。通过识别对这种药物有抗药性的酵母突变株，我们希望能识别与细胞对萝卜硫素反应有关的基因。我们期待这项研究不仅能帮助我们更好地了解细胞自杀的过程，还能提高该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.