Effects of salinomycin and binding target proteins in pancreatic cancer

_________________________________________________________________________________________________________________________ 项目-2。盐霉素在胰腺癌中的作用及结合靶蛋白(PI：吴二喜博士) 项目摘要 胰腺癌(PC)是一种致命的疾病，其5年生存率约为6%。 诊断和治疗抵抗力。PC中肿瘤干细胞(CSCs)的存在被认为是主要的 PC治疗抵抗和PC患者治疗复发的原因。盐霉素，最广泛的 使用过的抗球虫药物，已被发现对CSCs具有深刻的疗效，并可克服多种药物 癌症中的抗药性。我们的初步数据显示盐霉素对PC具有很强的细胞毒性。 细胞。我们确定了盐霉素的两个潜在结合靶点：转录中介因子-1β(TIF1？) 和核仁素(NCL)在PC细胞中。然而，盐霉素在PC中的作用机制仍不清楚； 尤其是它的直接结合靶点。在本项目中，我们提出了盐霉素对人牙周炎的抑制作用。 癌症和CSCs可能是由于1)细胞增殖和存活的减少和/或2)诱导 细胞分化。这项拟议的研究的目标是确定盐霉素的结合靶蛋白和 它们在PC中的功能以及受盐霉素及其结合蛋白调控的信号通路。我们 假设盐霉素反应细胞中存在特定的靶蛋白，并且盐霉素启动 它的功能是通过其结合的靶蛋白实现的。三个具体的目标将被用来检验这一假设。目标1.目标 PC和PC-CSC中盐霉素结合靶蛋白的测定及其临床意义 使用病理标本。盐霉素与TIF1b和NCL的直接结合将用 免疫沉淀和免疫结合方法。盐霉素与其潜能的相互作用 目标将通过分析它们的关联和解离分布来进一步确认。与临床的相关性 TIF1b和NCL的表达水平将通过评估这两个基因的表达水平与 病人的结果。目的2.确定TIF1和NCL在盐霉素抗吉西他滨中的作用 耐药PC细胞。盐霉素在体外和体内的作用将在不同的条件下进行分析，包括 TIF1和/或NCL缺失或过表达。盐霉素及其联合用药的疗效观察 将使用转基因小鼠模型来确定PC上的吉西他滨。目标3.剖析信号 盐霉素、吉西他滨、它们的组合以及盐霉素结合蛋白调节的信号通路 PC细胞。盐霉素及其靶蛋白对小鼠骨髓瘤关键通路基因表达的影响 PC进展将使用定制的RT-PCR阵列来确定。已确定的目标将进一步 使用多种策略进行评估。本研究的提出将为进一步深入研究PC的发病机制提供新的思路 盐霉素或联合用药作为新型抗前列腺癌药物的耐药性及理论基础 养生法。 _________________________________________________________________________________________________________________________

_________________________________________________________________________________________________________________________ Project-2. Salinomycin's Effects and Binding Target Proteins in Pancreatic Cancer (PI: Dr. Erxi Wu) Project Summary Pancreatic Cancer (PC) is a deadly disease and its 5-year survival rate is approximately 6% due to late diagnoses and therapy resistance. The existence of cancer stem cells (CSCs) in PC is considered as a major cause for PC therapy resistance and PC patients' relapse from therapy. Salinomycin, one of the most widely used coccidiostats, has been found to possess profound efficacy towards CSCs and to overcome multiple drug resistance in cancers. Our preliminary data showed that salinomycin possesses strong cytotoxicity against PC cells. We identified two salinomycin's potential binding targets: transcription intermediary factor-1beta (TIF1¿) and nucleolin (NCL) in PC cells. However, the action mechanism of salinomycin in PC still remains unclear; especially its direct binding targets. In this project, we propose that the inhibitory effects of salinomycin on cancer and CSCs could be due to 1) the reduction of cell proliferation and survival and/or, 2) the induction of cell differentiation. The goal of this proposed study is to determine salinomycin's binding target proteins and their functions in PC as well as the signaling pathways regulated by salinomycin and its binding proteins. We hypothesize that specific target proteins exist in the salinomycin responsive cells and that salinomycin initiates its function via its binding target proteins. Three specific aims will be used to test the hypothesis. Aim 1. To determine the binding target proteins of salinomycin in PC and PC-CSC as well as their clinical relevance using pathological specimens. The direct binding of salinomycin to TIF1b and NCL will be determined using immunoprecipitation and immune-binding approaches. The interaction between salinomycin and its potential targets will be further confirmed by analyzing their association and dissociation profiles. The clinical relevance of TIF1b and NCL will be examined by assessing the correlation of the expression levels of both genes with patients' outcomes. Aim 2. To determine the roles of TIF1¿ and NCL for salinomycin against Gemcitabine resistant PC cells. The effects of salinomycin will be analyzed in vitro and in vivo at various conditions including lack or overexpression of TIF1¿ and/or NCL. The efficacies of salinomycin and the combination with Gemcitabine on PC will be determined using a transgenic mouse model. Aim 3. To dissect the signaling pathways regulated by salinomycin, gemcitabine, their combination, and the binding proteins of salinomycin in PC cells. The effects of salinomycin and its target proteins on the expression of genes in the key pathways in PC progression will be determined using customized RT-PCR array. The identified targets will be further evaluated using multiple strategies. This proposed study will provide new insight into the mechanism of PC therapy resistance and a theoretical basis for the use of salinomycin or combination with Gem as novel anti-PC regimens. _________________________________________________________________________________________________________________________