GABA-B-R-mediated prevention of pancreatic cancer

项目摘要。 胰腺导管腺癌(PDAC)是导致癌症死亡的第四大原因，近100% 受害者在确诊后6个月内死亡。因此，预防PDAC的新战略是 急需之物。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 饮水加酒精诱导胰腺炎动物模型的建立 给它们注射尼古丁衍生的致癌亚硝胺4-(甲基亚硝基)-1-(3-吡啶)-1- 丁酮(NNK)。利用该动物模型和人PDAC异种移植模型进行小鼠体内移植和体外研究 与人PDAC细胞系和PDAC的假定起源细胞，胰腺管上皮细胞，我们的 已发表的和新的初步数据显示，腺酰环化酶依赖的细胞内信号下游 β-肾上腺素受体(b-ars)在刺激PDAC的同时，还触发了表皮生长的释放 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-Ars结合以及通过刺激a7nAChR介导的a7nAChR间接地 释放应激神经递质去甲肾上腺素和肾上腺素，这是b-ars的激动剂。全 这个刺激网络的组成部分在PDAC中上调，同时g-氨基丁酸也上调 通过阻断腺酰环化酶激活来抑制这一途径的GABA被抑制。治疗 GABA对体外培养的PDAC细胞或体内移植的PDAC细胞均有抑制作用。这些发现表明GABA 作为一种潜在的PDAC预防药物。为了检验这一假说，同时加深我们对 关于刺激的b-AR信号和抑制的GABA信号对PDAC的调节，我们提出了四种 明确的目标。 具体目标1：使用我们的仓鼠模型，我们将检验GABA或合成GABA 类似物巴洛芬可阻止PDAC的发展。 具体目标2：利用PCR微阵列、Western blotting和免疫组织化学方法，我们将研究 增殖、血管生成、转移、凋亡、细胞更新和cAMP信号转导标记物的调控 用GABA在仓鼠PDAC组织和正常胰腺组织中检测。 具体目标3：利用体外培养的人PDAC细胞系和胰管上皮细胞，我们将确定 GABA在b-AR和PGE2介导的信号通路中的抑制作用及可能的抑制作用 GABA对肿瘤坏死因子α(TNFa)和白介素1b(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 A4nAChR的表达和NNK诱导的应激性神经递质产生的刺激 A7nAChR参与刺激PDAC。 产生的数据将为GABA在预防PDAC中的使用提供临床前基础。

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.