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研究生:連秀屘
研究生(外文):Hsiu-Man Lien
論文名稱:樟芝純化物4,7-二甲氧基-5-甲基-1,3-二苯并間二氧雜戊烷及其第五取代衍生物抑制人類直腸腫瘤細胞增生及細胞週期G0/G1調控之研究
論文名稱(外文):Study of the Anti-Proliferative and G0/G1 Cell Cycle Arrest Activities of 4,7-Dimethoxy-5-methyl-l,3-benzodioxole from Antrodia camphorate and 5-Substituted Derivatives on Human Colorectal Cancer Cells
指導教授:楊定亞
指導教授(外文):Ding-Yah Yang
口試委員:何元順王應然喬長誠賴雅韻
口試委員(外文):Yuan-Soon HoYing-Jan WangCharng-Cherng ChyauYa-Yun Lai
口試日期:2011-11-07
學位類別:博士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:102
語文別:中文
論文頁數:93
中文關鍵詞:樟芝、分離純化、COLO205 細胞
外文關鍵詞:Antrodia camphorate、Purification、COLO 205 cells
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樟芝僅生長於台灣特有的牛樟樹樹幹中,生長極為緩慢,子實體於牛樟腐朽樹幹的中空內部長出,有強烈的黃樟香味,具有保肝及抑制腫瘤細胞增生等多種生理功能。本研究的目的乃欲分離純化來自三種不同來源的樟芝(Antrodia camphorate; AC) 萃取物都存在之活性純化物,以製備型高效液相層析分離純化,並將純化所得之化合物進行紫外光譜、質譜及核磁共振分析;鑑定發現其主要活性成分為非三萜化合物SY-1 (4,7-dimethoxy-5- methyl-l,3- benzodioxole),之後我們並進一步探討其對人類直腸腫瘤細胞株COLO 205及人類正常結腸表皮黏膜細胞(FHC)生長的抑制作用,同時也進行其生物活性機轉的相關實驗。經實驗結果證實SY-1能有效抑制人類直腸腫瘤COLO 205細胞的生長,濃度在50-150μM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150μM 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。而以相同濃度的SY-1在人類正常結腸表皮黏膜細胞(FHC),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,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。
另從樟芝萃取物分離得到的純化合物SY-1,我們以SY-1為先導化合物,以4,7-dimethoxy-l,3-benzodioxole為骨架,第5位的甲基(-CH3)取代為其他長度的碳鏈,在眾多化合物中挑選10個類緣化合物並進行活性篩選,進行對人類直腸腫瘤細胞株COLO 205生長的抑制作用。其中以第5位取代基為-CH2CH=CH2 (apiole)活性最好。我們證實apiole能有效抑制人類直腸腫瘤細胞(COLO 205)的生長,濃度在75-225μM的apiole的濃度劑量會使癌細胞之細胞週期停留在G0/G1期,而在apiole濃度大於225μM時,則會誘導細胞產生細胞凋亡(apoptosis)。apiole調控之COLO 205細胞週期停留在G0/G1期,p53、p21/Cip1及p27/Kip1蛋白的表現會增加,而cyclin D1的表現量則減少。而細胞凋亡相關蛋白,活化caspases3、8、9蛋白的表現量增加;且bax蛋白有增加的趨勢,bcl-2蛋白有減少的現象,而細胞凋亡指標 bax/bcl-2比值有明顯的增加,且產生DNA片段化現象。根據以上結果,證實apiole可抑制COLO 205細胞的增殖,其誘發人類直腸腫瘤細胞株COLO 205凋亡作用是透過活化caspas
家族蛋白路徑。
由以上實驗結果證實,樟芝萃取物之純化合物(SY-1)及其第五取代衍生物apiole,可有效之抑制人類直腸腫瘤細胞COLO 205的生長,而從這些觀察表示,SY-1及其衍生物apiole可以作為一種新的治療癌症的化療藥物。
Antrodia camphorata (AC) is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), an endemic species that is used in Chinese medicine for its anti-tumor and immunomodulatory properties. In this study, we concentrated that a possible active compound SY-1 which contained in all three different sources of Antrodia camphorata was further collected using preparative high performance liquid chromatography. The purified compound was confirmed as 4,7-dimethoxy-5-methyl-l,3-benzodioxole using ultraviolet, mass spectroscopy and nuclear magnetic resonance analyses. SY-1 profoundly decreased the proliferation of human colon cancer cells (COLO 205) through G0/G1 cell-cycle arrest (50–150 mM) and induction of apoptosis (>150 mM). Cell-cycle arrest induced by SY-1 was associated with a significant increase in levels of p53, p21/Cip1 and p27/Kip1, and a decrease in cyclins D1, D3 and A. In contrast, SY-1 treatment did not induce significant changes in G0/G1 phase cell-cycle regulatory proteins in normal human colonic epithelial cells (FHC). Thecells were cultured in soft agar to evaluate anchorage-independent colony formation, and we found that 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.
A set of ten 4,7-dimethoxy-l,3-benzodioxole derivatives based on a lead compound previously discovered by our group, SY-1, which was isolated from Antrodia camphorata, were evaluated for their in vitro inhibitory activity on human colorectal carcinoma cells (COLO 205). Structure–activity relationship studies of the 10 compounds indicated the importance of the chain length of thealkyl group at the 5-position, and the 2-propenyl substituent named ‘apiole’ exhibited the most potent inhibitory activity. In the present study, we demonstrate that the SY-1 analogue ‘apiole’ decreased the proliferation of COLO 205 cells, but not that of normal human colonic epithelial cells (FHC). The G0/G1 cell cycle arrest induced by apiole (75–225 mM) was associated with significantly increased levels of p53, p21 and p27 and decreased levels of cyclin D1. Concerning COLO 205 cell apoptosis, apiole (>150 mM) treatment significantly increased the levels of cleaved caspases 3, 8, 9 and bax/bcl-2 ratio and induced ladder formation in DNA fragmentation assay and sub-G1 peak in flow cytometry analysis.
These findings suggest that SY-1 and apiole can suppress COLO 205 cell growth; however, the detailed mechanism of these processes require further investigation.
目 錄
誌謝................................................................................... I
摘 要.................................................................................. II
Abstract ............................................................................. IV
目 錄.................................................................................. VI
縮 寫 表.................................................................................X
一、前言 ................................................................................ 1
二、大腸直腸癌簡介 ....................................................................... 3
三、樟芝簡介 ............................................................................. 6
貳、實驗材料與方法(Materials and Methods) ................................................ 15
一、藥品試劑 ............................................................................ 15
二、溶液配置 ............................................................................ 18
三、儀器設備 ............................................................................ 19
四、實驗方法 ............................................................................ 21
1. 樟芝來源及平板(plate)之培養方法 ....................................................... 21
2. SY-1之HPLC分析條件 ................................................................... 21
3. 分離純化(Isolation) ................................................................ 22
4. 實驗抽提流程圖 ....................................................................... 23
5. 細胞培養(Cell culture) .............................................................. 24
6. 細胞存活率之測定(MTT assay)........................................................... 24
7. 細胞週期分析 (cell cycle analysis) ................................................... 25
8. 西方墨點法 (immunoblotting) .......................................................... 26
9. DNA片段化之分析(DNA fragmentation analysis) .......................................... 26
參、實驗結果(Results) ................................................................... 29
一、樟芝純化物SY-1之分離純化及結構鑑定 ..................................................... 29
二、SY-1及其衍生物對人類直腸腫瘤細胞COLO 205生長的影響 ...................................... 30
三、SY-1和apiole對人類直腸腫瘤細胞COLO 205細胞週期的影響 .................................... 30
四、SY-1和apiole對人類直腸腫瘤細胞株COLO 205的細胞週期調節蛋白和凋亡相關蛋白之影響 ............. 32
五、SY-1和apiole誘發人類直腸腫瘤細胞COLO 205 DNA片段化現象(DNA fragmentation).............. 35
肆、討論(Discussion) .................................................................... 35
伍、結論(Conclusion) .................................................................... 42
圖表(Figures and Tables) ................................................................ 51
附錄 (Appendices) ....................................................................... 67
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