- 學位論文
抹草萃取物促進乳癌細胞週期停滯及細胞凋亡之研究
Anti-proliferative effects of Desmodium caudatum extracts on breast cancer cells by inducing cell cycle arrest and apoptosis
摘要
乳癌(breast cancer)發生在乳房腺上皮組織，其乳管細胞異常增生(proliferation)被視為最初主要的致癌原因[1]，因此，抑制細胞增生是早期抗乳癌之關鍵。近年來尋求天然植萃物或成份來抑制或延遲癌症的發展成為研究的重點，抹草(Desmodium caudatum)在臺灣坊間為廣泛使用於驅邪去煞之草本植物，在中醫應用於治療發熱性疾病[2]，已有研究顯示抹草萃取物(Desmodium caudatum extract, DCE)富含類黃酮等多酚(polyphenol)成分，具有抗發炎和抗氧化功效[3]；然而至今尚未有DCE對於抗癌之相關醫學研究。本篇目的為利用體內外之試驗，探討DCE抑制乳癌細胞生長之作用及釐清其分子機制。首先，利用裸鼠皮下移植人類乳腺癌細胞株MCF-7腫瘤試驗模式，經腫瘤切片染色及西方墨點法分析，發現DCE可藉由調控細胞週期(cell cycle)運行以及促進細胞凋亡(apoptosis)來抑制腫瘤生長。於斑馬魚試驗中，初步顯示DCE具有抑制血管新生(angiogenesis)之功效。在體外試驗中，以不同DCE濃度處理MCF-7及正常人類乳腺細胞H184B5F5/ M10並進行細胞毒性測試，經流式細胞儀分析研究顯示DCE對於正常細胞無明顯毒性，而對MCF-7細胞生長具有抑制作用且呈現濃度及時間依賴性效應。在較高劑量的DCE可促進MCF-7細胞週期G0/G1期停滯(arrest)，其分子調控可能藉由活化p53路徑，進而抑制轉錄因子E2F所致。此外，較低劑量之DCE亦可調節粒線體途徑中抗凋亡蛋白Bcl-2，並活化caspase 3及其下游蛋白CAD (caspase activated DNase)之表現，以導致細胞凋亡。綜合上述，經由體外與體內試驗顯示DCE能夠抑制乳腺癌細胞生長作用，藉以證實抹草具抗乳癌之潛力，而能應用於醫藥食品的開發。
並列摘要
Breast cancer has become the most frequently diagnosed and second leading cause of mortality among women in the world. Chemoprevention is one of the predominant attractive strategies for anti-breast cancer. The use of phytonutrient as anticancer agents has gained considerable importance in recent years. The aim of the study is to examine the anti-proliferative effects of Desmodium caudatum, used as a traditional Chinese herbal medicine with anti-inflammatory and antioxidant activities, on breast cancer. First, D. caudatum extracts (DCE), rich in polyphenol, was evidenced by its inhibition on the growth of human breast adenocarcinoma MCF-7 cells in xenograft tumor studies. The histological and molecular data showed that DCE exhibited the anti-proliferative effect by inducing cell cycle arrest and apoptosis. In the Danio rerio (zebrafish) test, DCE has also an inhibitory effect on angiogenesis. In vitro, flow cytometric data revealed that DCE dose-dependently inhibited the growth of MCF-7 cells, but not that of normal mammary epithelial H184B5F5/ M10 cells. The higher doses of DCE can block cell cycle in G0/G1 phase, and down-regulate E2F expression and Rb phosphorylation in MCF-7 cells. Furthermore, the DCE-induced cell cycle arrest might be exerted by inducing the expressions of p53, p21 and p27 that, in turn, repressed the activities of cyclin D1/cdk4 and/or cyclin E/cdk2, preventing the phosphorylation of Rb and the subsequent dissociation of Rb/E2F complex. DCE was also evaluated for apoptotic activity in MCF-7 cells. Molecular data showed the apoptotic effect of DCE in the cells might be mediated via mitochondrial pathway. Taken together, this is the first report to demonstrate that DCE exhibited anti-proliferative effect in breast cancer cells by inducing cell cycle arrest and apoptosis, and these findings may open interesting perspectives to the strategy in human breast cancer treatment.