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標題: | 以定量磷酸化蛋白體分析找尋肝癌細胞在蕾莎瓦處理下所產生之替代訊息傳導路徑 Quantitative Phosphoproteome Analysis to Reveal Alternative Signaling Pathways in Hepatocellular Carcinoma Cell under Sorafenib Treatment |
作者: | Chun-Yao Wang 王俊堯 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 肝細胞癌,PI3K/AKT 訊息傳導路徑,替代訊息傳導路徑,蕾莎瓦,Y-box binding protein, Hepatocellular carcinoma,PI3K/AKT signaling pathway,Alternative signaling pathway,Sorafenib,Y-box binding protein, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 肝細胞癌(hepatocellular carcinoma, HCC)在全球癌症致死率排名第三,有九成都屬於原發性肝癌(Primary liver cancer),其中B型及C型肝炎病毒感染或是肝硬化的發展是造成HCC的主要元兇。由於末期肝細胞癌之腫瘤較具有侵略性且病人預後較差,因此治療末期肝癌的過程通常較為複雜,主要的治療方法為肝臟手術性之切除,但由於肝細胞癌末期病人常常有腫瘤細胞轉移以及肝功能不全等現象,因此大多數病人並不適用肝臟切除手術。近年來,多重激酶抑制劑蕾莎瓦(multi-kinase inhibitor)作為標靶藥物用來治療病人成為末期肝細胞癌症病人的另一治療選擇,此藥物對於末期肝細胞癌病人的存活率已有所提升。
在本研究中,我們利用質譜定量之技術結合SILAC(Stable Isotope Labeling of Amino acids in Cell culture)技術來分析比較處理蕾莎瓦前後之HuH7細胞,一共鑑定到了562個磷酸化蛋白,其中包含了2180條磷酸化胜肽(在蕾莎瓦處理下發現了72個表現量上升、116個表現量下降之磷酸化位點),接著使用motif-xR、STRING及IPAR分析軟體分析後,發現有些蛋白在蕾莎瓦處理下改變量有明顯增減者多屬於MAPK signaling pathway,其中一個表現量下降之蛋白為DNA結合蛋白Y-box binding protein 1(YB-1),由文獻資料得知這是一個同時會被MAPK signaling pthway 以及 PI3K/AKT signaling pathway所調控之下游轉錄因子,在眾多癌症中均有大量表現並與癌症發展以及抗藥性有關。在本研究中我們發現YB-1在HuH7細胞中被MAPK-ERK signaling pathway所調控,在U0126(MEK抑制劑)治療下其磷酸化表現量下降;意外的是我們發現YB-1磷酸化表現量在對sorafenib具有抗藥性之HuH7R細胞中高度上升,亦有文獻指出YB-1是AKT的受質,而由AKT磷酸化YB-1所影響之細胞功能尚不明朗。 根據本研究,YB-1在HuH7R細胞中是被PI3K/AKT signaling pathway所調控,細胞之侵襲能力(invasion)與爬行能力(migration)接隨著YB-1被抑制(knock down)而下降,藉由訊息傳導路徑以及功能分析,我們認為YB-1藉由調控PI3K/AKT/GSK3β signaling pathway來影響細胞之侵襲以及爬行能力,同時我們也找到了一些被YB-1所調控之下游分子如PI3K, P53和VEGFA可能對於細胞存活能力有所影響。 最後,我們確立了YB-1在HuH7及HuH7R細胞中截然不同的功能,其中YB-1在HuH7細胞中是被MAPK signaling pathway所調控,在HuH7R細胞中則是被另一條替代訊息傳導路徑PI3K/AKT signaling pathway所調控。藉由了解同一個分子在不同的條件下所扮演的角色,對於嶄新抗癌藥物的發展或是替代療法的選擇上將有所助益。 Hepatocellular carcinoma(HCC) is now the third leading cause of cancer deaths worldwide, accounting for 90% of primary liver cancers. Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis). Advanced HCC is often a complex condition with highly aggressive tumor and poor prognosis to common therapies. The mainstay of therapy is surgical resection, but the majority of patients are not eligible because of tumor extent or underlying liver dysfunction. Recently, a therapeutic option for advanced HCC is the use of sorafenib, a muti-kinase inhibitor, which was showed to be able to increase survival in HCC patients. In this study, we applied quantitative proteomics approach using nanoflow LC MS/MS in combination with SILAC technology to compare HuH7 and sorafenib treated HuH7 cells. Through phosphopeptide enrichment by titanium dioxide affinity column, a total of 2180 phosphopeptides from 562 phosphorylated proteins were identified. The SILAC based quantification described 72 up-regulated and 116 down-regulated phosphorylation sites upon sorafenib treatment. By using Motif-x, STRING and IPA analyses, we found that most proteins showing significant fold change under sorafenib treatment are involved in MAPK-ERK signaling pathway. One of the down-regulated proteins we found is the Y-Box binding protein 1(YB-1), which was reported as a transcription factor regulated by MAPK-ERK and PI3K-AKT signaling pathway. YB-1 is overexpressed in many cancers and associated with tumor progression and drug resistance. In this study we found that YB-1 is a downstream target of MAPK-ERK signaling cascade, its phosphorylation is decreased under U0126(MEK inhibitor) treatment. Surprisingly, we found that phosphorylation of YB-1 is increased in sorafenib- resistant cell line HuH7R. Recent studies indicated YB-1 as a direct substrate of AKT. The functional significance of AKT-mediated phosphorylation remains largely unknown. Based on our findings, YB-1 is regulated by PI3K/AKT signaling pathway instead of MAPK signaling pathway in HuH7R cell. Invasion and migration ability are decreased in YB-1-knock-down HuH7R cell. After signaling pathway and functional analysis, results indicated that YB-1 regulates cell invasion and migration ability through activation of PI3K/AKT/GSK3βsignaling pathway. We further identified some YB-1-regulated downstream genes like PI3K, P53 and VEGFA that may contribute to cell viability. In conclusion, we defined different roles of YB-1 in HuH7 and HuH7R cell. YB-1 is regulated by MAPK signaling pathway in HuH7 cell but it is regulated by an alternative signaling pathway, PI3K/AKT signaling pathway, in HuH7R cell. Understanding different regulation mechanisms of the same signal molecule at different circumstances may contribute to further development of novel anti-cancer drug and alternative therapies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55656 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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