GABA-B-R-mediated prevention of pancreatic cancer

项目摘要。 胰腺导管腺癌（PDAC）是癌症死亡率的第四大原因， 受害者在确诊后六个月内死亡因此，预防PDAC的新策略 迫切需要。使用PDAC的两个危险因素（胰腺炎和吸烟），我们建立了一个仓鼠 通过饮用水中的乙醇诱导动物胰腺炎，同时另外 注射尼古丁衍生的致癌物质亚硝胺4-（甲基亚硝胺）-1-（3-吡啶基）-1- 丁酮（NNK）。在小鼠和体外研究中使用该动物模型以及来自人PDAC的异种移植物 用人PDAC细胞系和PDAC的假定起源细胞，胰腺导管上皮细胞， 已发表的和新的初步数据表明，腺苷酸环化酶依赖的细胞内信号下游 β-肾上腺素受体（b-AR）刺激PDAC，同时额外触发表皮生长的释放 因子、血管内皮生长因子和花生四烯酸。NNK直接激活这一信号通路 通过与b-AR结合并间接通过刺激α 7烟碱乙酰胆碱受体（α 7 nAChR）介导的 释放应激神经递质去甲肾上腺素和肾上腺素，它们是b-AR的激动剂。所有 这种刺激网络的组分在PDAC中上调，而同时g-氨基丁酸 通过阻断腺苷酸环化酶的活化来抑制该途径的GABA被抑制。治疗 GABA对体外PDAC细胞或体内PDAC移植瘤均有抑制作用。这些发现表明GABA 作为潜在的PDAC预防剂。为了验证这一假设，同时进一步了解 在通过刺激性b-AR信号和抑制性GABA信号调节PDAC方面，我们提出了四个 具体目标。 具体目标1：使用我们的仓鼠模型，我们将测试GABA或合成GABA 类似物巴氯芬可阻止PDAC的发展。 具体目标2：利用PCR微阵列、蛋白质印迹和免疫组织化学，我们将研究 调节增殖、血管生成、转移、凋亡、细胞更新和cAMP信号传导的标志物 GABA在仓鼠PDAC和正常胰腺组织中的作用。 具体目标3：使用体外培养的人PDAC细胞系和胰腺导管上皮细胞，我们将确定 GABA在b-AR和PGE 2介导的信号通路中的抑制作用，并探索潜在的减少 GABA对TNF α和IL-1b的影响。 具体目标4：我们将测试由于NNK诱导的脱敏而抑制GABA产生的假设 和刺激应激神经递质的生产由于NNK诱导的上调， a7 nAChR参与PDAC的兴奋。 所产生的数据将为使用GABA预防PDAC提供临床前基础。

Project Summary. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality with near 100% of the victims succumbing within 6 month of diagnosis. New strategies for PDAC prevention are therefore urgently needed. Using two risk factors for PDAC (pancreatitis and smoking), we have established a hamster model of PDAC by inducing pancreatitis in the animals via ethanol in the drinking water while additionally injecting them with the nicotine-derived carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). Using this animal model as well as xenografts from human PDAC in mice and in vitro studies with human PDAC cell lines and the putative cell of origin of PDAC, pancreatic duct epithelial cells, our published and new preliminary data show that adenylyl cyclase-dependent intracellular signaling downstream of beta-adrenoreceptors (b-ARs) stimulates PDAC while additionally triggering the release of epidermal growth factor, vascular endothelial growth factor and arachidonic acid. NNK activates this signaling pathway directly by binding to b-ARs and indirectly by stimulating the a7nicotinic acetylcholine receptor (a7nAChR)-mediated release of the stress neurotransmitters noradrenaline and adrenaline, which are agonists for b-ARs. All components of this stimulatory network are upregulated in PDAC while at the same time g-aminobutyric acid (GABA) which inhibits this pathway by blocking the activation of adenylyl cyclase is suppressed. Treatment of PDAC cells in vitro or PDAC xenografts in vivo with GABA had inhibiting effects. These findings suggest GABA as a potential PDAC preventive agent. To test this hypothesis and at the same time further our understanding on the regulation of PDAC by stimulatory b-AR signaling and inhibitory GABA signaling we propose four specific aims. Specific Aim 1: Using our hamster model, we will test the hypothesis that GABA or the synthetic GABA analogue baclophen prevent the development of PDAC. Specific Aim 2: Using PCR microarrays, Western blotting and immunohistochemistry, we will investigate the modulation of markers for proliferation, angiogenesis, metastasis, apoptosis, cell renewal, and cAMP signaling by GABA in tissues from hamster PDAC and normal pancreatic tissue. Specific Aim 3: Using human PDAC cell lines and pancreatic duct epithelial cells in vitro, we will determine the inhibitory actions of GABA in b-AR and PGE2-mediated signaling pathways and explore a potential reduction in TNFa and IL-1b by GABA. Specific Aim 4: We will test the hypothesis that inhibited GABA production due to NNK-induced desensitization of the a4nAChR and stimulation of stress neurotransmitter production due to NNK-induced upregulation of the a7nAChR contribute to the stimulation of PDAC. Data generated will provide a preclinical basis for the use of GABA in the prevention of PDAC.