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

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Title:	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
Other Titles:	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
Authors:	連秀屘 Hsiu-Man Lien
Contributors:	楊定亞 Ding-Yah Yang 化學系
Keywords:	樟芝、分離純化、COLO205 細胞 Antrodia camphorate、Purification、COLO 205 cells
Date:	2011
Issue Date:	2015-03-06T07:36:54Z (UTC)
Abstract:	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?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?直腸腫瘤細胞株COLO 205及人?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?直腸腫瘤COLO 205細胞的生長，濃?在50-150μM的SY-1的濃?劑?會使癌細胞之細胞週期停?在G0/G1期，而在SY-1濃?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?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?則減少。而以相同濃度的SY-1在人?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?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?之生成與型態，發現細胞群?聚集的現象有明顯?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?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?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?直腸腫瘤細胞(COLO 205)的生長，濃?在75-225μM的apiole的濃?劑?會使癌細胞之細胞週期停?在G0/G1期，而在apiole濃?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?在G0/G1期，p53、p21/Cip1及p27/Kip1蛋白的表現會增加，而cyclin D1的表現?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?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.
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