肝細胞中檳榔對於AP-1及NF-κB基因之調控

鄭景暉臺灣大學:臨床牙醫學研究所楊婷婷Yang, Ting-TingTing-TingYang2010-05-262018-07-092010-05-262018-07-092008U0001-0107200818304900http://ntur.lib.ntu.edu.tw//handle/246246/184137嚼食檳榔在東南亞國家是一非常普遍之口腔嗜好，目前在全世界有六億之嚼食人口。嚼食檳榔已知為造成口腔白斑、口腔黏膜下纖維化、口腔癌及食道癌之主要危險因子之一。嚼食檳榔者由於可能會將唾液完全吞入上消化道，因此檳榔塊所產生的嚴重毒性作用也會在口腔以外的部位發生。許多的研究在動物實驗中發現檳榔子萃取物會對肝細胞造成基因毒性及細胞毒性。此外，轉錄因子AP-1及NF-κB與肝癌有很強烈的相關性，並在許多報告中皆指出肝炎病毒X 蛋白可活化AP-1 及NF-κB。近幾年來持續有針對於嚼食檳榔與肝癌相關性之流病報告，結果指出嚼食檳榔為一造成肝癌之獨立危險因子，並也對於慢性B 型肝炎病毒感染相關的肝癌有加成效果。由於台灣為B 肝病毒高帶原率的國家，因此本實驗欲針對檳榔成份合併B 型肝炎病毒兩大因素對於AP-1、NF-κB 轉錄因子分子調控機制與肝癌形成的關係進行探討。藉由報導基因分析我們發現檳榔子萃取物與檳榔素分別在800 μg/ml 及0.8 mM 的濃度下對於AP-1 及NF-κB 轉錄因子有活化之作用。在檳榔成份活化AP-1 的部分主要是經由Ras/Raf/ERK/MEK路徑，而在相同濃度的檳榔子萃取物作用之下，NF-κB 的部份則主要是經由PKC及MEK/ERK 但不經由Ras 路徑來達到活化。在B 肝病毒X 蛋白的存在下，對於檳榔子萃取物活化AP-1 及NF-κB 有協同活化效益，但對檳榔素則只有加成作用。我們利用RT-PCR 或西方墨點法研究檳榔子萃取物及檳榔素是否會誘導一些具有肝癌侵略性代表意義之相關指標分子的表現，結果發現檳榔子萃取物會增加tPA, PAI-1, IL-6 及IL-8 的基因表現，在HBx 存在下，IL-8 表現增加較為明顯; 檳榔素則會增加N-Cadherin, VCAM-1。總結來說，檳榔成份會對於轉錄因子AP-1及NF-κB 有活化作用，並在HBx 的存在下結果更加顯著，此外還會造成一些具有肝腫瘤侵略性之指標性分子之表現。因此我們提供了一個合併檳榔成份與肝炎病毒對於肝癌形成影響之可能分子機制，而對於臨床上之蘊含意義為在B 型肝炎病毒的帶原下，若加上嚼食檳榔之習慣可能會提高肝癌形成之危險性。Betel quid (BQ) chewing is a common oral habit in Southeast Asian contries. There are about six hundred million BQ chewers in the world. The evidence of BQ chewing as one of the major risk factors leading to leukoplakia, oral submucous fibrosis, oral cancer and esophagus cancers have been well published. The BQ chewers may swallow saliva completely and therefore the severe toxic effects of BQ may occurs at sites other than the oral cavity. The animal studies have demonstrated that the betel quid components causes hepatotoxicity and the genotoxic effects of hepatocytes. In addition, transcriptional factors AP-1 and NF-κB has strong correlation with liver carcinogenesis and can be activated by X protein of the HBV. Recently, the epidemiology reports have shown that the habitual BQ chewing is an independent risk factors of hepatocellular carcinoma (HCC) and may have an addiditve effect with HBV infection. The HBsAg carrier rate in general population of Taiwan is one of the highest in the world. In our study, we like to investigate the AP-1 and NF-κB molecular mechanisms combine with HBV factors for the association of BQ components with the development of HCC. By using the luciferase assay, we found that the AN extract (ANE) 800 μg/ml and Arecoline (ACO) 0.8 mM induced AP-1 binding site activation mainly through Ras/Raf/MEK/ERK pathways. At the same ANE concentrations, NF-κB binding sites were activated mainly through PKC and MEK/ ERK pathways, but not the Ras pathways. In the presence of HBx protein, ANE-induced AP-1 and NF-κB is synergistically activated and with MEK/ERK pathways involvement. Compare with ANE, ACO-induced AP-1 and NF-κB activation in the presence of HBx protein only reach to the additive effects. At the mRNA level, some HCC invasiveness markers were induced by ANE (tPA, PAI-1 and IL-6 ) or ACO (N-Cadherin, VCAM-1). At the protein level, ANE alone or combine with HBx both induced IL-6 expression. However, the protein expression of IL-8 was only found in ANE or ACO stimulation in the presence of HBx. In conclusion, BQ components activate AP-1 and NF-κB transcriptional factors and induced the HCC invasiveness markers expression at the mRNA and protein level. ANE combine with HBV synergistically induced AP-1 and NF-κB activation. We provide the possible molecular mechanism of betel nuts chewing and combine with viral factors in the development of HCC. Our clinical implication is betel but chewing in HBsAg carriers may increase the risk of HCC development.中文摘要------------------------------------------------i文摘要-----------------------------------------------iii 錄-------------------------------------------------v 次--------------------------------------------------x 錄---------------------------------------------------xiii 一章緒論.1 檳榔嚼食與口咽部腫瘤的關係---------------------------------------------1.2 檳榔塊之組成成分------------------------------------------------------------1.3 檳榔子萃取物的致癌性、突變性及基因毒性---------------------------2.4 檳榔鹼的致癌性、突變性及基因毒性------------------------------------3.5 活性氧所扮演的角色(ROS)-------------------------------------------------3.6 訊息傳導與肝癌之關連性---------------------------------------------------4.6.1 Ras/Raf/MEK/ERK訊息傳導對腫瘤形成之影響--------------4.6.2 AP-1及NF-κB之訊息傳導與肝癌之關係----------------------5.6.3 p53 腫瘤抑制基因---------------------------------------------------6.7 檳榔化學致癌性對肝臟之影響---------------------------------------------6.8 B型肝炎病毒與肝癌---------------------------------------------------------7.9 嚼食檳榔與肝癌關係之流行病學研究------------------------------------8.10 實驗動機與目的---------------------------------------------------------------9二章實驗材料方法.0 實驗材料與藥品來源--------------------------------------------------------10.1 檳榔子之水萃取--------------------------------------------------------------11.2 細胞株與細胞培養-----------------------------------------------------------11.3 細胞生長評估-----------------------------------------------------------------12.3.1 MTT dye配製--------------------------------------------------------12.3.2 MTT assay ------------------------------------------------------------13.4 報導基因分析-----------------------------------------------------------------13.4.1 報導基因相關質體--------------------------------------------------------13.4.2 HBV相關質體-------------------------------------------------------14.4.3 HBV相關質體製備-------------------------------------------------15.4.4 質體轉染(Transfection)---------------------------------------------15.4.4.1 報導基因分析(Reporter assay)-------------------------15.4.5 Dual-Luciferase Reporter assay------------------------------------16.4.6 Bio-Rad protein assay -----------------------------------------------16.5 西方點墨法(Western Blotting)----------------------------------------------17.5.1 蛋白質表現-----------------------------------------------------------17.5.2 蛋白質的萃取--------------------------------------------------------17.5.3 配置膠體與電泳分析-----------------------------------------------17.5.4 蛋白質轉漬(Transfer)-----------------------------------------------18.5.5 抗體使用--------------------------------------------------------------18.6 聚合酶鏈鎖反應 (polymerase chain reaction)---------------------------19.6.1 RNA分離法----------------------------------------------------------19.6.2 RNA定量-------------------------------------------------------------19.6.3 RNA反轉錄(Reverse Transcription)------------------------------20.6.4 聚合酶鏈鎖反應 (polymerase chain reaction, PCR)-----------20.7 HBsAg and HBeAg assay----------------------------------------------------21.8 統計分析-----------------------------------------------------------------------22三章實驗結果.1 檳榔子萃取物及檳榔素之肝細胞毒性.1.1 檳榔子萃取物之肝細胞毒性--------------------------------------23.1.2 檳榔素之肝細胞毒性-----------------------------------------------23.1.3 p53所扮演的角色---------------------------------------------------24.2 檳榔成份活化AP-1及NF-κB之機轉.2.1 檳榔萃取物與檳榔素活化AP-1----------------------------------25.2.2 檳榔子萃取物及檳榔素活化AP-1與PI3K之關係-----------25.2.2.1 檳榔子萃取物與檳榔素活化AP-1之作用無法由PI3K抑制劑LY294002所阻斷-----------------------------------------25.2.2.2 檳榔子萃取物與檳榔素活化AP-1之作用無法由PI3K抑劑劑Wortmannin所阻斷---------------------------------------26.2.3 檳榔子萃取物及檳榔素活化AP-1與PKC之關係-----------26.2.3.1 PKC抑制劑H7對檳榔子萃取物所活化之AP-1有抑制效果------------------------------------------------------------------ 27.2.3.2 PKC抑制劑H7對於檳榔素所活化之AP-1無抑制效果--------------------------------------------------------------------- 27.2.4 檳榔子萃取物及檳榔素活化AP-1與Ras-ERK訊息傳導之關係-----------------------------------------------------------------------27.2.4.1 檳榔子萃取物藉由Ras訊息傳導路徑活化AP-1---------28.2.4.2 檳榔素藉由Ras訊息傳導路徑活化AP-1------------------28.2.4.3 檳榔子萃取物藉由MEK/ERK訊息傳導路徑活化AP-1 --------------------------------------------------------------------- 29.2.4.4 檳榔素藉由ERK訊息傳導路徑活化AP-1 ----------------29.2.5 檳榔子萃取物與檳榔素皆可活化NF-κB-----------------------29.2.6 檳榔子萃取物活化NF-κB與PI3K之關係---------------------30.2.6.1 檳榔子萃取物活化NF-κB無法由PI3K抑制劑LY294002所阻斷------------------------------------------------------------ 30.2.6.2 檳榔子萃取物活化NF-κB無法由PI3K抑制劑Wortmanninn阻斷-----------------------------------------------30.2.7 檳榔子萃取物活化NF-κB與PKC之關係---------------------30.2.7.1 PKC抑制劑H7對於檳榔子萃取物所活化之NF-κB有抑制作用------------------------------------------------------------ 30.2.8 檳榔子萃取物活化NF-κB與Ras-ERK之關係----------------31.2.8.1 檳榔子萃取物活化NF-κB並無透過Ras之訊息傳導路徑--------------------------------------------------------------------- 31.2.9 檳榔子萃取物藉由MEK/ERK訊息傳導路徑活化NF-κB---31.3 活性氧及酵素對於檳榔子萃取物活化AP-1及NF-κB之影響.3.1 NAC (N-acetylcysteine)抑制檳榔子萃取物活化AP-1--------32.3.2 酯解酶(Esterase)抑制檳榔子萃取物活化AP-1----------------32.3.3 過氧化氫酶(Catalase)對檳榔子萃取物活化AP-1無抑制作用 --------------------------------------------------------------------------32.3.4 NAC、酯解酶及過氧化氫酶對於檳榔子萃取物活化NF-κB皆無抑制作用--------------------------------------------------------33.4 B型肝炎病毒X蛋白存在對檳榔成份活化AP-1/ NF-κB之協同(synergistic)及加成(additive)效應.4.1 基因型(genotype)A之B型肝炎病毒X蛋白(HBx)對檳榔子萃取物活化AP-1有協同效應(synergistic effects)----------------34.4.2 基因型(genotype)B之B型肝炎病毒X蛋白(HBx)對檳榔子萃取物活化AP-1有協同效應(synergistic effects)----------------34.4.3 基因型(genotype)A之B型肝炎病毒X蛋白(HBx)對檳榔素活化AP-1只有加成效應(additive effects)-------------------------34.4.4 基因型(genotype)B之B型肝炎病毒X蛋白(HBx)對檳榔素取物活化AP-1只有加成效應(synergistic effects)----------------35.4.5 基因型(genotype) A之B型肝炎病毒X蛋白(HBx)對檳榔子萃取物活化NF-κB有協同效應(synergistic effects)-----------35.4.6 基因型(genotype) B之B型肝炎病毒X蛋白(HBx)對檳榔子萃取物活化NF-κB有協同效應(synergistic effects)-----------35.4.7 HBx蛋白在HuH7細胞中的表現--------------------------------36.5 B型肝炎病毒X蛋白存在下對檳榔成份活化AP-1/ NF-κB訊息傳導路徑之影響.5.1 HBx蛋白對檳榔子萃取物活化AP-1之協同效應可由MEK/ERK抑劑U0126阻斷---------------------------------------36.5.2 HBx蛋白對檳榔子萃取物活化NF-κB之協同效應部份可由MEK/ERK抑制劑U0126阻斷------------------------------------36.5.3 HBx蛋白在HuH7細胞中的表現--------------------------------37.6 HuH7細胞mRNA 產物.6.1 檳榔子萃取物誘導tPA, PAI-1及IL-6 mRNA表現-----------37.6.2 檳榔素誘導N-Cadherin及VCAM-1 mRNA表現-------------37.7 IL-6及IL-8蛋白表現--------------------------------------------------------38.8 檳榔成份對HBsAg及HBeAg濃度的影響------------------------------38四章討論.1 檳榔子萃取物與檳榔素對於細胞生長的影響--------------------------40.2 檳榔成份活化AP-1 / NF-κB之機轉--------------------------------------41.3 活性氧對於檳榔子萃取物活化AP-1有較大的影響-------------------42.4 B型肝炎病毒X蛋白(HBx)對檳榔子萃取物活化AP-1/NF-κB有協同效應(synergistic effects) --------------------------------------------------43.5 不同基因型HBx蛋白之影響----------------------------------------------44.6 檳榔成份誘導HuH-7之mRNA及蛋白表現---------------------------45.7 檳榔成份對病毒複製的影響-----------------------------------------------46五章總結---------------------------------------------------------------------------------48考文獻---------------------------------------------------------------------------------------50次ig.1. Dose-dependent effects of ANE on cell proliferation in HuH7, HepG2 and primary hepatocyte.----------------------------------------------------------------60ig.2. Dose-dependent effects of Arecoline on cell proliferation in HuH7, HepG2 and primary hepatocyte -----------------------------------------------------------61ig.3. Effects of Pifithrin-α on ANE/Arecoline treated cell proliferation in HepG2. ----------------------------------------------------------------------------------------62ig.4. Activation of AP-1 binding site by (A) ANE (B) Arecoline in HuH7 cells.----------------------------------------------------------------------------------63ig.5. Fold activation of AP-1 binding site by ANE (A) and Arecoline (B) is resistant to inhibition by LY294002. --------------------------------------------64ig.6. Fold activation of AP-1 binding site by ANE (A) and Arecoline (B) is resistant to inhibition by Wortmannin -------------------------------------------65ig.7. PKC inhibitor H7 inhibits AP-1 binding site activation stimulated by ANE (A) but not Arecoline (B)----------------------------------------------------------66ig.8A. ANE induced AP-1 binding site activation is mediated via Ras pathway ----------------------------------------------------------------------------------------67ig.8B Arecoline induced AP-1 binding site activation is mediated via Ras pathway.-----------------------------------------------------------------------------68ig.9. ERK1/2 inhibitor U0126 inhibits AP-1 binding site activation stimulated by ANE (A) and Arecoline (B) ------------------------------------------------------69ig.10. Fold activation of NF-κB binding site by (A) ANE (B) Arecoline in HuH7 cells.----------------------------------------------------------------------------------70ig.11. Fold activation of NF-κB binding site by ANE is resistant to inhibition by LY294002----------------------------------------------------------------------------71ig.12. Fold activation of NF-κB binding site by ANE is resistant to inhibition by Wortmannin.-------------------------------------------------------------------------72ig.13. PKC inhibitor H7 partially inhibits NF-κB binding site activation stimulated by ANE.------------------------------------------------------------------------------73ig.14. Ras pathway does not involved in ANE induced NF-κB binding site activation.----------------------------------------------------------------------------74ig.15. ERK1/2 inhibitor U0126 inhibits NF-κB binding site activation stimulated by ANE.------------------------------------------------------------------------------75ig.16. ROS scavenger NAC (N-acetylcysteine) inhibits AP-1 binding site activation stimulated by ANE.----------------------------------------------------76ig.17. Esterase inhibits AP-1 binding site activation stimulated by ANE.----------77ig.18. AP-1 binding site activation stimulated by ANE is resistant to Catalase ----------------------------------------------------------------------------------------78ig.19. NF-κB binding site activation stimulated by ANE is resistant to NAC, Esterase and Catalase--------------------------------------------------------------79ig.20. Synergistic effects of ANE mediated AP-1 binding site activation by HBxA and HBxB ---------------------------------------------------------------------------80ig.21. Additive effects of Arecoline mediated AP-1 binding site activation by HBxA and HBxB. ------------------------------------------------------------------81ig.22. Synergistic effects of ANE mediated NF-κB binding site activation by HBxA and HBxB. ------------------------------------------------------------------82ig.23. Synergistic effects of ANE 800 ug/ml mediated AP-1 binding site activation by HBxA and HBxB was inhibited by MEK/ERK inhibitor (U0126) ------83ig.24. Synergistic effects of ANE 800 μg/ml mediated NF-κB binding site activation by 0.2 μg HBxA was partially inhibited by MEK/ERK inhibitor (U0126) ----------------------------------------------------------------------------- 84ig.25. Effects of ANE to mRNA expression of (A) Tissue plasminogen activator (B) PAI-1 and (C) IL-6.------------------------------------------------------------85ig.26. Effects of Arecoline to mRNA expression of (A) N-Cadherin (B) VCAM-1. ----------------------------------------------------------------------------------------86ig.27. Effects of ANE and Arecoline combine with HBx to IL-6 and IL-8 potein level expression.--------------------------------------------------------------------87ig.28. Inhibition of HBsAg and HBeAg expression in HuH-7 by (A)ANE and (B)Arecoline.------------------------------------------------------------------------88ig.29. (A)HBsAg and (B)HBeAg expression in HepG2.2.15 (HBV-producing cell line) stimulated by ANE-----------------------------------------------------------89ig.30. (A)HBsAg and (B)HBeAg expression in HepG2.2.15 stimulated by Arecoline.----------------------------------------------------------------------------90ig.31. (A)HBsAg and (B)HBeAg expression in high confluence of HepG2.2.15 cells stimulated by ANE-----------------------------------------------------------91ig.32. (A)HBsAg and (B)HBeAg expression in high confluence of HepG2.2.15 cells stimulated by Arecoline -----------------------------------------------------92ig.33. Possible signal transduction pathway of AP-1 activated by areca nut extract (ANE) and Arecoline (ACO).-----------------------------------------------------93ig.34. Possible signal transduction pathway of NF-κB activated by areca nut extract (ANE)-----------------------------------------------------------------------94ig.35. ANE-induced AP-1 and NF-application/pdf2415897 bytesapplication/pdfen-US檳榔子萃取物AP-1NF-κBB型肝炎病毒X 蛋白肝癌Areca nut extractHBxHCC[SDGs]SDG3肝細胞中檳榔對於AP-1及NF-κB基因之調控Mechanism of AP-1 and NF-κB activation and signaling y betel quid components in liver cellshttp://ntur.lib.ntu.edu.tw/bitstream/246246/184137/1/ntu-97-R94422013-1.pdf