利用蛋白質體學鑑定受幽門螺旋桿菌調控之宿主細胞因子並分析其功能特性

Abstract

胃癌是全世界人口的一大健康威脅，它在全球的常見癌症中排行第二，在台灣地區也分居男性十大腫瘤的第四名與女性十大腫瘤的第六位。許多流行病學與動物模式的研究中早已確立胃癌的發生與幽門螺旋桿菌的感染是息息相關的，而國際癌症研究署(IARC)也於西元1994年將幽門螺旋桿菌定義為第一類的致癌因子。目前發現世界上超過半數的人口會受到幽門螺旋桿菌的感染，受感染的個體中約有百分之十會發展成嚴重的胃部疾病，例如:胃潰瘍與胃部發炎萎縮，只有百分之ㄧ會造成胃癌的發生。幽門螺旋桿菌的感染會造成如此歧異的致病結果，除了本身病毒因子的差異與環境因素的影響外，宿主本身的細胞因子也扮演了決定性的角色。 為了說明幽門螺旋桿菌與宿主細胞因子之間的交互作用以了解腫瘤的發生過程並找出有哪些宿主的細胞因子參與其中的調控，我們試著探討有那些胃部細胞的蛋白質在受到幽門螺旋桿菌感染後，該蛋白質的表現情形會有差異。首先，我們建立了一個體外的細胞模式系統並找出可以模擬幽門螺旋桿菌感染胃上皮細胞情況的條件。我們採取蛋白質體學的方式，包括利用螢光二次元差異性電泳(2D-DIGE) 合併液相層析偶合串聯式質譜儀(nanoLC-MS/MS) 來分析胃上皮細胞的蛋白質在受幽門螺旋桿菌感染前後有何差異。經過三重複的實驗後，我們發現其中有28個蛋白質在受幽門螺旋桿菌感染後會改變其表現情形，這些蛋白質依功能可分為:1.蛋白質合成與蛋白質摺疊之相關因子; 2.細胞骨架蛋白質; 3.細胞代謝酵素; 4.轉錄與轉譯相關因子; 5.血管新生與腫瘤轉移相關因子; 6.細胞訊息傳遞相關因子; 7.氧化調控蛋白; 8.致癌蛋白。參考文獻報導後，我們選出8個癌症相關蛋白質做後續的研究，包括: lamininγ-1 chain precursor、valosin-containing protein (VCP)、 heat shock 70-kDa protein (Hsp70)、 mitochondrial matrix protein P1 (MMP-P1)、 FK506-binding protein 4 (FKBP4)、T-complex protein 1 (TCP1)、enolase α與 14-3-3 β。我們收集了10對臨床胃癌病人的檢體，利用西方墨點法與免疫組織染色分析來驗證上述8個蛋白質在胃部的腫瘤組織與週邊的正常組織中是否的確會有差異性的表現情形。根據實驗的結果，我們發現VCP, MMP-P1, TCP1, enolase α, 與14-3-3 β胃癌組織中的確會有過度表現的情形, 推測這些蛋白質可能透過降低細胞凋亡與細胞增生的方式來參與幽門螺旋桿菌所誘發的胃癌致癌過程。 幽門螺旋桿菌在胃部生長會造成胃部黏膜的發炎，而持續性的慢性發炎會促進胃部細胞凋亡並且造成剩餘組織的細胞增生。細胞增生與細胞凋亡之間的平衡關係對於維持胃部黏膜的正常生長代謝是必須的。一旦細胞凋亡減少或者細胞增生情形增加時，就會促成胃部的癌化發生。在上述可能參與幽門螺旋桿菌所誘發的胃癌相關因子中，VCP被報導在人類的骨癌細胞株中具有降低TNFα所引發的細胞凋亡的功能。本篇論文進ㄧ步分析VCP在幽門螺旋桿菌感染所引發的細胞凋亡中是否也同樣具有降低細胞凋亡的效應，並探討在幽門螺旋桿菌的致病機轉中VCP媒介了哪些細胞的生理反應。我們建立了一株會過度表現VCP的胃上皮細胞株，並發現無論是在養份缺乏或是幽門螺旋桿菌感染情形下，VCP的過度表現均會壓制胃上皮細胞的凋亡。藉由VCP的siRNA以降低VCP的表現量後，此種抑制細胞凋亡的效應也會隨之消失。進ㄧ步地，我們整合了基因微陣列技術(DNA microarray)與蛋白質體學的方式來研究VCP所調控的細胞因子，並利用專一性的抗體加以驗證。我們發現VCP透過活化NFκB的訊息傳遞及下游的細胞因子而促使細胞凋亡的減少、細胞轉移與增生。本篇論文發現在幽門螺旋桿菌的感染下，持續發炎的胃部細胞可能透過VCP的過度表現，進而媒介細胞凋亡的減少與細胞增生等生理反應而促成胃癌的發展。 蛋白質體學的技術提供了一個系統性的平台以研究幽門螺旋桿菌的治病機轉，並且解釋胃部細胞可能透過哪些細胞因子的調控而促使發炎的細胞走向腫瘤生成的過程。更重要的，透過研究幽門螺旋桿菌所誘發胃癌的治病機轉將有助於了解其他更複雜癌症的生成，並針對發炎所誘發腫瘤的研究提供了一個範本。

Gastric cancer remains a major health problem being the second commonest malignancy in the world. In Taiwan, gastric cancer also stays the fourth-common cause of cancer death in male, and the sixth in female. Epidemiological and animal studies demonstrated a link between gastric cancer and chronic infection with Helicobacter pylori(H. pylori), and H. pylori infection has been classified in 1994 by the International Agency for Research against Cancer (IARC) as a carcinogen type 1 for gastric cancer. More than 50% world-wide population was infected by H. pylori, though, approximately 10% of the infected subjects will develop more severe gastric pathologies like peptic ulcer disease and atrophic gastritis, and only 1% of infected individuals will development gastric cancer. In addition to H. pylori virulence factors and environmental influences, host factors determine such divergent disease outcome. In order to elucidate the H. pylori-host cell interactions that underline the neoplastic process and identify which host cell factors are involved, we tried to characterize proteins differentially expressed in H. pylori-infected gastric epithelial AGS cells. An in vitro model was established using a multiplicity of infection of 100 and evaluating the effectiveness of H. pylori infection by functional analyses. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The expression of many proteins was found to be altered, and 28 of these were identified and classified as protein synthesis- and folding-related proteins, cytoskeleton proteins, metabolic enzymes, transcription- and translation-related proteins, angiogenesis/metastasis-related proteins, cell communication/signal transduction-related proteins, or others (oxygen-regulated protein and oncoprotein). The expression profiles of eight of these proteins, lamininγ-1 chain precursor, valosin-containing protein (VCP), heat shock 70-kDa protein (Hsp70), mitochondrial matrix protein P1 (MMP-P1), FK506-binding protein 4 (FKBP4), T-complex protein 1 (TCP1), enolase α, and 14-3-3 β were further examined in cancerous and paired surrounding normal tissues by immunoblot assay and immunohistochemical staining to identify molecular targets that may be involved in the pathogenesis of H. pylori-induced gastric diseases. On the basis of our results, VCP, MMP-P1, TCP1, enolaseα, and 14-3-3βmay play a crucial role in H. pylori-induced gastric carcinogenesis by mediating antiapoptotic and proliferative responses. It is already known that H. pylori colonization would cause gastric mucosal inflammation, and such chronic inflammation promotes apoptosis, which leads to a compensatory proliferative response by the remaining tissues. The dynamic balance between cell proliferation and apoptosis is essential for maintaining mucosal homeostasis. Decreased apoptosis and increased proliferation may favor the carcinogenesis process. Among these identified cancer-associated proteins which are involved in H. pylori pathogenesis, VCP was reported to decrease TNFα-induced apoptosis in osteosarcoma cell line. To analyze the anti-apotosis effect of VCP and realize the cellular responses mediated by VCP in the H. pylori pathological mechanism. We established a stable VCP-overexpressing AGS cell line and found that VCP overexpression suppressed the apoptosis of these cells induced by serum starvation, or H. pylori infection. Moreover, this resistance to apoptosis was attenuated by introduction of VCP siRNA. Integrated transcriptomic and proteomic approaches were used and many novel VCP-regulated gene/proteins were identified. Based on a bioinformatic analysis and a literature search, the use of antibodies against VCP-regulated factors allowed us to conclude that VCP activates NFκB signaling and their subsequent downstream effectors may promote gastric epithelial cells toward anti-apoptosis, cell invasion or cell cycle progression. These VCP-mediated cellular responses may constitute the missing link of chronic inflammation to gastric cancer induced by H. pylori. Proteomic approach provides a systematic platform to consider insight into the pathogenesis of H. pylori, and the prospect of cellular responses that promoted gastric epithelial cells from inflammation toward carcinogenic process. More importantly, the study of H. pylori-induced gastric carcinogensis offers a paradigm for understanding more complex human cancers, especially those inflammation-associated tumors.