(3.235.139.152) 您好!臺灣時間:2021/05/11 12:44
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:張志強
研究生(外文):Chih-Chiang Chang
論文名稱:探討ENO1(αenolase)在乳癌細胞中所扮演之角色與Tamoxifn活化ENO1(αenolase)蛋白表現之機制
論文名稱(外文):Studies on the role of α-enolase(ENO1) in Breast Cancer andits Molecular Mechanism of Tamoxifen Induces α-enolase Protein Expression
指導教授:楊沂淵楊沂淵引用關係
指導教授(外文):Yi-Yuan Yang
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:97
中文關鍵詞:乳癌
外文關鍵詞:ENO1α enolasebreast cancer
相關次數:
  • 被引用被引用:0
  • 點閱點閱:382
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
ENO1是一個存在於細胞質的糖解作用酵素,主要功能在於催化phosphoenolpyruvate 形成。但在近幾年的研究中指出ENO1是一個多功能性蛋白,在哺乳動物中,目前發現三種不同的isoform,分別為enolase-α(ENO1)、enolase-β(ENO3)、enolase-γ(ENO2)。ENO1存在於大部份的組織中,ENO2、ENO3主要存在於神經細胞或肌肉細胞中。這一些isoform是具有組織特異性的,它們一般以homodimers 或heterodimers 形式作用,使2-phosphoglycerate 變成 phosphoenolpyruvate。ENO1跟c-myc binding protein 1(MBP-1) 來自於相同的一個基因,經由不同的轉譯修飾過程所產生。在本實驗中我們證實Tamoxifen 在MCF-7細胞中,可以活化ENO1蛋白之表現,實驗中我們也有發現Tamoxifen 活化NFκB、ER α、ER α p118等蛋白之表現。在尋找轉錄因子結合位的Database(TFSEARCH: Searching Transcription Factor Binding Sites)上,我們發現到ENO1 轉錄調控區有轉錄因子NFκB的結合位。核質分離技術證實加入Tamoxifen可以使NFκB被活化,而進到細胞核裡面。接著使用NFκB的活化抑制劑PDTC,發現到當NFκB被抑制之後, ENO1的表現量下降了。在此我們證實Tamoxifen可以經由轉錄因子NFκB,結合到ENO1的轉錄調控區,因而活化ENO1蛋白之表現。利用即時性PCR(Real-time PCR)定量mRNA 的表現量,發現腫瘤組織中ENO1的表現量有明顯高於正常組織中的ENO1。在乳癌病人的病理切片中,我們有發現到在乳癌病人的乳腺管癌化的細胞中表現量比較多。因此我們認為ENO1 乳癌細胞中大量表現,一定具有臨床上的意義。
Enolase α(ENO1) is a cytoplasmic glycolytic enzyme involved in the formation of phosphoenolpyruvate. More recent evidence, however ,shows that Enolase is a multifunction protein.In mammalian cells,three isoforms of enolase have been found. There are designated as enolase-α(ENO1)、enolase-β(ENO3)、enolase-γ(ENO2).ENO1 is widely distributed in a variety of tissues, whereas ENO2 and ENO3 are exclusively found in neuron/neuronendocrine and muscles tissues, respectively. The expression of the those isoforms is developmentally regulated in a tissue-specific manner.Enolases from heterodimers or homodimers to convert 2-phosphoglycerate into phosphoenolpyruvate in glycolysis. Enolase αand c-myc binding protein originate from a single gene through alternative use of translational starting site. We previously demonstrated that Tamoxifen induces ENO1 expression in MCF-7 cell line. In this report, we found Tamoxifen induces ENO1、ERα、ERα p118、NFκB protein expression. In the database (TFSEARCH: Searching Transcription Factor Binding Sites),we found NFκB binding site in ENO1 promoter. Nuclear extraction suggested that Tamoxifen inducing NFκB translocation into nuclear. We use PDTC, a NFκB inhibitor, blocks NFκB tronslocation into nucleus. PDTC inhibitor was down-regulated ENO1 protein expression. We investigated NFκB regulation ENO1 expression by binding on ENO1 promoter.Quantitative assays of the mRNA levels of the ENO1 was measured by Real-time PCR analysis technique and revealed that expression of the ENO1 was higher in tumor tissue when compared to normal tissue which dissected form the tumor margin. Tissue section from patients with breast caner were examined Immunohistochemically. We found ENO1 overexpression in breast ductal carcinoma.
目錄(Contents)
頁數
中文摘要 (Abstract in Chinese) I
英文摘要 (Abstract in Chinese) III
目錄 (Contents) V
圖目錄 (Figures and Tables lists) X
縮寫表 (Abbreviations) XII

第一章 緒論 (Introduction)
前言 2
一、 Enolase 在細胞中之角色 3
二、 Estrogen receptor(ER)的介紹 5
三、 NFκB的介紹 6

第二章 實驗材料與方法 (Materials and Methods)
一、 藥品試劑 10
二、 常用溶液 15
三、 常用儀器 19
四、 實驗方法 21
1. 細胞培養 Cell Culture 21
2. 反轉錄-聚合酶連鎖反應 RT-PCR 21
3. 西方墨點法Western Blotting Assay 24
4. 免疫沉澱法Immunoprecipitation (IP) 26
5. 核質分離 Nucleus Extraction 27
6. 染色質免疫沉澱法 Chromatin Immunoprecipitation; CHIP assay 28
7. MTT cell viability assay 30
8. Transfection 30
9. Promoter region construct 31
10. 免疫組織染色(Immuo-histochemistry Staining) 33
11. 免疫螢光染色(Immuno-fluorescence staining) 34
12. Real time PCR 35

第三章 實驗結果 (Results) 36
一、乳癌病人之normal 和tumor tissue,以PCR證實,ENO1表現量在 tumor tissue > normal tissue 37
二、乳癌病人之normal 和tumor tissue,以Real-time PCR證實,ENO1表現量在 tumor tissue > normal tissue 37
三、乳癌病人之病理組織切片,IHC stain發現 ENO1在乳癌細胞中之ductal carcinoma表現量較高 38
四、抗癌藥物Tamoxifen使ENO1之表現量上升 39
五、 不同濃度之Tamoxifen對ENO1的表現是有影響性的,濃度越高
ENO1的表現量越高 40
六、Tamoxifen 濃度越高細胞死亡越多 41
七、在相同Tamoxifen濃度下,加藥時間越久 ENO1之表現上升越高
41
八、Chromatin immunoprecipitation 證實在加入Tamoxifen 時,活化NFκB 結合到ENO1的promoter 上,促使ENO1被活化 42
九、Tamoxifen 濃度越高NFκB有被活化 43
十、加入Tamoxifen時,NFκB是Translocation至核裡面 43
十一、加入PDTC抑制NFκB translocation,也可抑制ENO1蛋白之表現 44
十二、PDTC和Tamoxifen co-treat可以使ENO1之表現量也下降 44
十三、利用PDTC抑制NFκB的translocation,使ENO1的表現量下降,在此利用核質分離之技術加以證實PDTC是可以抑制NFκB的Translocation 45
十四、Tamoxifen 活化ENO1 promoter activity 46
十五、MCF-7 和MDA-MB-231對Tamoxifen作用後,ENO1的表現在MCF-7 中隨著濃度增加而增加,但在MDA-MB-231表現比較不明顯 46
十六、加入Tamoxifen之後,NFκB跟ERα形成Complex的量變多了 47
十七、Chromatin immunoprecipitation 證實在加入Tamoxifen 時,活化ERα 結合到ENO1的promoter 上 48

第四章 討論 (Discussion) 49
第五章 圖表 (Figures) 55
第六章 參考文獻 (Reference) 81
附錄 (Appendices)
一、pGL3 vector map and pSM2 vector map 90
二、ENO1 promoter region sequence transcription factor
binding site prediction 91
三、Primer sequences 92
四、The chemical structure of Tamoxifen and
4-hydrotamoxifen 93
五、實驗架構 94
六、Table 1 (PCR & Real time PCR) 94
七、Table 2 (ENO1expression) 95
八、Table 3 (ENO1 expression normal tissue > tumor tissue)
95
九、PDTC co-treated Tamoxifen induces rapid MCF-7 death
faster then PDTC alone、Tamoxifen alone. 96
十、Show difference concentration of Lipofetamine was used
for plasmid transfection into MCF-7 cell. 97

圖目錄 (Figures and Tables lists)
Fig 1. Demonstration the concentration of ENO1 mRNA
expressed in PCR assay from 159 clinical breast
cancer patients 56
Fig 2. Demonstration the general image of ENO1 expression
in Real time PCR assay from 174 clinical breast
cancer patients 57
Fig 3. Immunohistochemical staining of ENO1in breast tumor
tissue 59
Fig 4. Dose dependent treatment of some drugs in Human
breast cancer MCF-7 cell line and the
overexpression of ENO1 protein 62
Fig 5. Dose dependent treatment of Tamoxifen in Human
breast cancer MCF-7 cell line and the
overexpression of ENO1 mRNA 63
Fig 6. Dose dependent treatment of Tamoxifen in Human
breast cancer MCF-7 cell line 65
Fig 7. Tamoxifen induced ENO1 overexpression in Human
breast cancer MCF-7 cell line 66
Fig 8. Chromatin immunoprecipitation (ChIP) assay of the
promoter region of ENO1 gene by NFκB 67
Fig 9. Effects of NFκB inhibitor (PDTC) in MCF-7 cell
line 69
Fig 10.Translocation of NFκB revealed by immunoblotting
70
Fig 11.Effects of NFκB inhibitor (PDTC) in MCF-7 cell
line 71
Fig 12.Effects of Tamoxifen and of NFκB inhibitor (PDTC)
in MCF-7 cell line 72
Fig 13.Translocation of NFκB revealed by immunoblotting
74
Fig 14.Tamoxifen up-regulate the function of ENO1
promoter 75
Fig 15.Dose dependent treatment of Tamoxifen in Human
breast cancer MCF-7 and MDA-MB-231 cell 77
Fig 16.Tamoxifen induced NFκB and ERα association 79
Fig 17.Chromatin immunoprecipitation (ChIP) assay of the
promoter region of ENO1 gene by ER α 80
Alcami J, Lain de Lera T, Folgueira L, Pedraza MA, Jacque JM, Bachelerie F, Noriega AR, Hay RT, Harrich D, Gaynor RB, et al. (1995) Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J 14(7): 1552-1560

Babiker FA, De Windt LJ, van Eickels M, Grohe C, Meyer R, Doevendans PA (2002) Estrogenic hormone action in the heart: regulatory network and function. Cardiovasc Res 53(3): 709-719

Baldwin AS, Jr. (2001) Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest 107(1): 3-6

Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D (1995) Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 376(6536): 167-170

Chang GC, Liu KJ, Hsieh CL, Hu TS, Charoenfuprasert S, Liu HK, Luh KT, Hsu LH, Wu CW, Ting CC, Chen CY, Chen KC, Yang TY, Chou TY, Wang WH, Whang-Peng J, Shih NY (2006) Identification of alpha-enolase as an autoantigen in lung cancer: its overexpression is associated with clinical outcomes. Clin Cancer Res 12(19): 5746-5754

Chang YS, Wu W, Walsh G, Hong WK, Mao L (2003) Enolase-alpha is frequently down-regulated in non-small cell lung cancer and predicts aggressive biological behavior. Clin Cancer Res 9(10 Pt 1): 3641-3644

Ejeskar K, Krona C, Caren H, Zaibak F, Li L, Martinsson T, Ioannou PA (2005) Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death. BMC Cancer 5: 161

Feldman I, Feldman GM, Mobarak C, Dunkelberg JC, Leslie KK (2007) Identification of proteins within the nuclear factor-kappa B transcriptional complex including estrogen receptor-alpha. Am J Obstet Gynecol 196(4): 394 e391-311; discussion 394 e311-393
Feo S, Arcuri D, Piddini E, Passantino R, Giallongo A (2000) ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1). FEBS Lett 473(1): 47-52

Giuliani C, Napolitano G, Bucci I, Montani V, Monaco F (2001) [Nf-kB transcription factor: role in the pathogenesis of inflammatory, autoimmune, and neoplastic diseases and therapy implications]. Clin Ter 152(4): 249-253

Herrero JA, Mathew P, Paya CV (1995) LMP-1 activates NF-kappa B by targeting the inhibitory molecule I kappa B alpha. J Virol 69(4): 2168-2174

Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P (1995) Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270(5241): 1491-1494
Li X, Huang J, Yi P, Bambara RA, Hilf R, Muyan M (2004) Single-chain estrogen receptors (ERs) reveal that the ERalpha/beta heterodimer emulates functions of the ERalpha dimer in genomic estrogen signaling pathways. Mol Cell Biol 24(17): 7681-7694

Miles LA, Dahlberg CM, Plescia J, Felez J, Kato K, Plow EF (1991) Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor. Biochemistry 30(6): 1682-1691

Miles LA, Ellis V (2003) Alpha-enolase comes muscling in on plasminogen activation. Thromb Haemost 90(4): 564-566

Pancholi V (2001) Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci 58(7): 902-920

Pelicano H, Martin DS, Xu RH, Huang P (2006) Glycolysis inhibition for anticancer treatment. Oncogene 25(34): 4633-4646

Sedoris KC, Thomas SD, Miller DM (2007) c-myc promoter binding protein regulates the cellular response to an altered glucose concentration. Biochemistry 46(29): 8659-8668

Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P, Giallongo A (1996) Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem 271(51): 32529-32537

Shang Y, Brown M (2002) Molecular determinants for the tissue specificity of SERMs. Science 295(5564): 2465-2468

Snyder JG, Prewitt R, Campsen J, Britt LD (2002) PDTC and Mg132, inhibitors of NF-kappaB, block endotoxin induced vasodilation of isolated rat skeletal muscle arterioles. Shock 17(4): 304-307
Subramanian A, Miller DM (2000) Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene. J Biol Chem 275(8): 5958-5965

Terrier B, Degand N, Guilpain P, Servettaz A, Guillevin L, Mouthon L (2007) Alpha-enolase: a target of antibodies in infectious and autoimmune diseases. Autoimmun Rev 6(3): 176-182

Yaghmaie F, Saeed O, Garan SA, Freitag W, Timiras PS, Sternberg H (2005) Caloric restriction reduces cell loss and maintains estrogen receptor-alpha immunoreactivity in the pre-optic hypothalamus of female B6D2F1 mice. Neuro Endocrinol Lett 26(3): 197-203

Yavlovich A, Rechnitzer H, Rottem S (2007) Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen. Infect Immun 75(12): 5716-5719
Zivadinovic D, Watson CS (2005) Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells. Breast Cancer Res 7(1): R130-144
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔