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研究生:林曉薇
研究生(外文):Hsiao-Wei Lin
論文名稱:PART1:AntrodiaCamphorate純化物抑制人類直腸腫瘤細胞(COLO205)生長及細胞週期G0/G1調控PART2:探討LOX(LysylOxidase)在乳癌細胞中所扮演之角色
論文名稱(外文):PART 1:Studies on the Mechanism of SY-1 Induced Apoptosis and G0/G1 Cell Cycle Arrest in Human Colon Adenocarcinoma CellsPART 2:Studies on the Role of LOX(Lysyl Oxidase) in Breast Cancer
指導教授:何元順
指導教授(外文):Yuan-Soon Ho
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:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:牛樟芝乳癌大腸癌
外文關鍵詞:Antrodia CamphorateLysyl Oxidase
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PART 1:本篇論文研究來自三種不同牛樟芝(Antrodia camphorate;AC) 的子實體中之純化物4,7- Dimethoxy-5- methyl-l,3- benzodioxole (稱做SY-1)。AC是一種藥用傘菌,主要生長在牛樟樹樹幹腐朽之心材內壁,或枯死倒伏之牛樟樹木材潮濕表面,而且可以利用於中藥上,具有抗腫瘤和免疫調節作用。我們證實SY-1能有效抑制人類直腸腫瘤細胞(COLO 205)的生長,濃度在75-225μM 的SY-1的濃度劑量會使癌細胞之細胞週期停留在G0/G1期,而在SY-1 濃度大於 225μM時,則會誘導細胞產生細胞凋亡(apoptosis)。SY-1調控之COLO 205細胞週期停留在G0/G1期時,p53 、 p21/Cip1及p27/Kip1蛋白的表現會增加,而cyclin D1 、cyclin D3及 cyclin A表現量則減少。相較之下,我們使用人類正常結腸黏膜細胞(FHC),給予SY-1後,並沒有引起顯著的影響細胞週期G0/G1期調控蛋白的表現量改變。將COLO 205細胞培養於軟性瓊膠(soft agar)上,給予SY-1之後觀察群落之生成與型態,發現細胞群落聚集的現象有明顯降低的趨勢。根據以上結果證實,我們首先證實SY-1可以抑制COLO 205細胞的增殖,主要是經由抑制細胞生長和群落的聚集。
PART 2:Lysyl oxidase是一種需銅的氨基氧化酶,位於細胞外基質,會與膠原蛋白和彈力蛋白相互作用。最近研究發現,lysyl oixdase會去氧化細胞外基質內的蛋白,lysyl oixdase對組織生長、細胞增生、細胞內訊息傳遞及細胞轉移是扮演重要的角色。由Real time PCR定量mRNA的表現量,發現lysyl oixdase在乳癌腫瘤病人組織表現量比正常組織較多,大約占62%。更進一步利用雷射顯微擷取Laser Capture Microdissection (LCM)和IHC的方法,確認其再現性及表現分布。另一方面,利用實驗室現有乳癌及正常細胞株中lysyl oixdase的表現,發現在MDA-MB-231細胞株中lysyl oixdase被大量表現。在過去研究發現,lysyl oixdase會與鄰近的lysine結合分泌出過氧化氫,會使FAK/Src pathway被活化,進而使癌細胞移動能力增加。因此使用lysyl oixdase抑制劑(β-aminopropionitrile;βAPN),抑制其活性,發現過氧化氫分泌減少和下游FAK/Src pathway磷酸化也降低。接著利用wound healing assay觀察MDA-MB-231細胞移動能力,由結果得知加入lysyl oixdase抑制劑能有效抑制MDA-MB-231細胞移動能力。因此,我們發現lysyl oixdase在乳癌細胞中大量表現,主要功能是促進細胞移動能力。
PART 1:In this study, 4,7-dimethoxy-5-methyl-l,3-benzodioxole ( designated as SY-1 ) was isolated from three different sources of dried fruiting bodies of Antrodia camphorate (AC). AC is a medicinal mushroom that grows on the inner heartwood wall of the endemic species Cinnamomum kanehirai Hay (Lauraceae), and is used in Chinese medicine for its anti-tumor and immunomodulatory activities. We demonstrated that SY-1 profoundly decreased growth of human colon cancer cells (COLO 205) through G0/G1 cell cycle arrest (75-225μM) and induction of apoptosis (> 225μM). Cell cycle arrest induced by SY-1 was associated with a significant increase in protein levels of p53, p21/Cip1, and p27/Kip1, and a decrease in cyclins D1, D3, and A. In contrast, in normal human colonic epithelial cells (FHC), SY-1 treatment did not induce significant changes in G0/G1 phase cell cycle regulatory proteins. The cells were cultured in soft agar for evaluation of anchorage-independent colony formation, and the number of transformed colonies was significantly reduced in the SY-1-treated COLO 205 cells. These findings demonstrate, for the first time, that SY-1 inhibits human colon cancer cell proliferation through inhibition of cell growth and anchorage-independent colony formation in soft agar.
PART 2:Lysyl oxidase (LOX) has been show previously, is an extracellular cupper enzyme that initiates the crosslinking of collagens and elastin. LOX catalyzes the oxidation of the side chain of a peptidyl lysine converting specific lysine and hydroxylysine residues of α–aminoadipic-δ-semialdehydes. Current findings have shown that LOX is required for the development of major organs, and have assigned it highly important roles in development and diseases in terms of migration, invasion, epithelial-mesenchymal transition (EMT) and even intracellular signalling. Form real time PCR data, we can see that LOX mRNA level in breast cancer tissues are higher than in normal tissues. It has been show previously, LOX protein was up-regulated in a lot of cancer tissue, included liver cancer and breast cancer. Laser capture microdissection (LCM) and IHC shows that LOX mRNA was over-expressed in breast cancer tissue. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide–mediated mechanism involving the FAK/Src signaling pathway.Treatment of the invasive breast cancer cell line, MDA-MB-231, with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX catalytic activity,lead to a significant decrease in cell motility/migration. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with βAPN. So, we predict that LOX may play an important role in breast cancer growth.
PART 1:
中文摘要 (Abstract in Chinese)……………..................I
英文摘要 (Abstract in English)…………………………….III
目錄 (Contents)……………………………………………....V
圖目錄 (Figures and Tables lists)………………………...VIII
縮寫表 (Abbreviations)…………………………….………...X

第一章 緒論 (Introduction)…………………..……….……….1
一、 牛樟芝(Antrodia camphorata)……………..….……..2
二、 大腸直腸癌(Colorectal cancer)………….……..…….4
三、 研究目的……………………………...………..………6
第二章 實驗材料與方法 (Materials and Methods)…………..7
一、 藥品試劑……………………………………...………...8
二、 常用溶液…………………………………...………….12
三、 常用儀器…………………………………...………….14
四、 實驗方法………………………………...…………….16
1. Cell culture (細胞培養)
2. Cell growth curve (細胞生長曲線之測定)
3. MTT cell viability assay (細胞存活率之測定)
4. Flow cytometry (流式細胞儀)
5. Western blotting assay (西方墨點法)
6. Soft agar growth assay (軟性瓊膠群落生長試驗)
7. DNA fragmentation analysis (DNA裂片分析)
8. RT-PCR (反轉錄-聚合酶連鎖反應)
第三章 實驗結果 (Results)……………………………………….25
一、 SY-1對人類直腸腫瘤細胞(COLO 205及HT-29)和人類 正常結腸黏膜細胞(FHC)生長的影響
二、 SY-1誘發人類直腸腫瘤細胞(COLO 205)凋亡作用
三、 SY-1誘導人類直腸腫瘤細胞(COLO 205)凋亡作用是透過活化caspase,但人類正常結腸黏膜細胞(FHC)卻不會受到影響
四、 SY-1會使人類直腸腫瘤細胞(COLO 205)的細胞週期停滯於G0/G1時期
五、 SY-1誘發人類直腸腫瘤細胞(COLO 205)和人類正常結腸黏膜細胞(FHC)細胞週期蛋白的變化
六、 SY-1能抑制人類直腸腫瘤細胞(COLO 205)細胞與細胞間聚集的能力
七、 SY-1能抑制人類直腸腫瘤細胞(COLO 205)細胞與細胞間黏著之基因E-cadherin mRNA的變化
第四章 討論 (Discussion)……………………………………...31
第五章 圖表 (Figures)………………………….......……….36
附錄 (Appendices)………………………………..……..………….52
一、 Isolation and chemical structural characterization of 4,7-dimethoxy-5-methyl-l,3-benzodioxole from AC………..53
二、 Dose-dependent effects of SY-1 on cell viability in malignant human cells……………………………………….56
三、 Inhibition of cell proliferation in the SY-1 treated malignant human breast and hepatoma cells…………………………...57
四、 SY-1反應路徑圖……………………………………….…...58
五、 行政院衛生署民國九十六年統計台灣台北市主要死亡原因統計表…………………….…....................................59
六、 行政院衛生署民國九十六年台灣台北市主要癌症死亡原因統計表…………………….…....................................60




圖目錄 (Figures and Tables lists)

Fig 1.Inhibition of cell proliferation in the SY-1 treated malignant and normal human colon cells………………37

Fig 2.The SY-1-induced apoptosis seen in the COLO 205 cells occurs in a cell-specific manner…………...............39

Fig 3.Effects of SY-1 on the levels of cell apoptosis regulatory
proteins………………...…………………………………...….41

Fig 4.Arrest of cell cycle at the G0/G1 phase by SY-1 in human COLO 205 cells…………………..………………..43

Fig 5.Effects of SY-1 on the levels of G0/G1 phase cell cycle regulatory proteins…………………..………………….….46

Fig 6.Anchorage-independent growth of SY-1-treated COLO 205 cells in soft-agar…………………………..….49

Fig 7.Dose dependent treated of SY-1 in Human COLO
205 cells line and the expression of E-cadherin…..…..51

PART 2:
中文摘要 (Abstract in Chinese)…………………….………I
英文摘要 (Abstract in English)…………………...………III
目錄 (Contents)……………………………………...............V
圖目錄 (Figures and Tables lists)………………………….XI

第一章 緒論 (Introduction)………………..……………..……...1
前言………………………………………….………...2
一、 LOX(Lysyl oxidase)………………………………….5
二、 Lysyl oxidase合成…………………………………...6
三、 Lysyl oxidase活化機轉……………………………...7
第二章 實驗材料與方法 (Materials and Methods)….……...9
一、 藥品試劑……………………………………………..10
二、 常用溶液………………………………………..……14
三、 常用儀器……………………………………………..16
四、 實驗方法……………………………………………..18
1. 冷凍切片Frozen sections
2. 蘇木精與伊紅染色法Hematoxylin and eosin stain
3. 雷射顯微擷取Laser Capture Microdissection (LCM)
4. 反轉錄-聚合酶連鎖反應RT-PCR
5. Real time PCR
6. 免疫組織染色Immuo-histochemistry Staining
7. 細胞培養Cell culture
8. 西方墨點法We stern blotting assay
9. Wound healing assay
10. 細胞存活率之測定MTT cell viability assay
11. 流式細胞儀Flow cytometry
第三章 實驗結果 (Results)……………………………………...29
一、 乳癌病人之normal 和tumor tissue,以PCR證實, LOX表現量在 tumor tissue > normal tissue
二、 乳癌病人之normal和tumor tissue,以Real time PCR證實,LOX表現量在tumor tissue > normal tissue
三、 Laser Capture Microdissection (LCM)將normal和tumor cells擷取,以Real time PCR證實,LOX表現量在tumor tissue > normal tissue
四、 乳癌病人之病理組織切片,IHC stain發現 LOX在乳癌細胞中之ductal carcinoma in situ (DCIS)表現量較高,尤其是在invasive ductal carcinoma(IDC)更有被大量表現之情形
五、 利用Western blotting assay 觀察LOX在正常人類乳腺細胞和人類乳癌腫瘤細胞之蛋白表現量之情形
六、 利用LOX 抑制劑β-aminopropionitrile(βAPN)抑制LOX下游FAK/Src pathway之磷酸化情形
七、 利用LOX 抑制劑β-aminopropionitrile(βAPN)抑制LOX分泌之Hydrogen peroxide
八、 LOX 抑制劑β-aminopropionitrile(βAPN)不會影響MDA-MB-231細胞之生長
九、 LOX 抑制劑β-aminopropionitrile(βAPN)會抑制MDA-MB-231細胞之移動能力
第四章 討論 (Discussion)……………………………...………...36
第五章 圖表 (Figures)……………………………………………41
附錄 (Appendices)………………………………………..……….57
一、 實驗架構圖……………………………………………..….58
二、 Table 1(LOX expression)……………………………………59
三、 Table 2(Lymph node)………………………………………..60
四、 Table 3(Stage)………………………………………………..61
參考文獻 (Reference)…………………………………...………..62
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