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標題: | 大蒜素有效抑制動物實驗中肝癌腫瘤之生長且可經由p53調控人類肝癌細胞株誘導產生不同死亡模式之機制研究 Allicin Inhibits the Tumor Growth in vivo and Induces Different Modes of Cell Death via p53 Modulating in Human Liver Cancer Cell Lines |
作者: | Yung-Lin Chu 朱永麟 |
指導教授: | 沈立言(Lee-Yan Sheen) |
關鍵字: | 大蒜素,p53,自體吞噬,LC3-II,人類肝癌細胞株, allicin,p53,autophagy,LC3-II,human liver cancer cells, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 大蒜是一種歷史悠久的食物與藥物,其主要活性成分為大蒜素(Allicin),可從新鮮的大蒜破碎而取得,然而大蒜素具有對光、熱敏感的特性且容易降解成為diallyl sulfide、diallyl disulfide與diallyl trisulfide,故必須儲存在低溫的環境下。目前肝癌病患在台灣的癌症死亡率位居第二名,儘管有許多研究指出大蒜對於癌症有很好的治療效果,但目前針對大蒜對肝癌治療的相關研究並不明確。在本研究中,我們將著重於探討大蒜素經由p53之調控,誘導Hep G2(p53wild type)與Hep 3B(p53mutation)細胞產生不同細胞死亡模式。實驗結果顯示大蒜素不僅可以誘導Hep G2 cells產生p53所調控之自體吞噬性死亡,亦導致Hep 3B細胞產生細胞凋亡。由西方墨點法可以觀察到大蒜素降低了Hep G2細胞質中的p53、PI3K/AKT與mTOR等蛋白質的表現量;同時增加了AMPK/TSC2與LC3-II等訊息傳遞路徑蛋白質的表現。此外,利用共軛焦顯微鏡觀察之結果顯示,大蒜素造成Hep G2細胞吞噬小體之形成以及p53、AIF與Endo G蛋白質在Hep G2與Hep 3B細胞內的表現與分布。然而大蒜素也可誘導Hep 3B細胞產生大量ROS,進而造成DNA損傷、粒線體膜電位下降、活化caspase依賴性與非依賴性凋亡途徑造成細胞凋亡。進一步利用了siRNA-TP53使Hep G2細胞的p53表現量降低,再將大蒜素添加於細胞培養液中,發現Hep G2細胞產生細胞凋亡之現象,此結果與Hep 3B細胞之結果相同。綜合以上實驗,證明大蒜素的確能誘導人類肝癌細胞產生細胞凋亡或自體吞噬性死亡,且對於具細胞凋亡抗性的肝癌細胞可能具有輔助性基因治療的潛力。 Garlic has been used throughout history for both culinary and medicinal purpose. Allicin is a major component of crushed garlic. Although it is sensitive to heat and light, and easily degraded into various compounds such as diallyl sulfide, diallyl disulfide, and diallyl trisulfide, allicin is still a major bioactive compound of crushed garlic. Hepatocellular carcinoma is the top two causes of cancer-related death in Taiwan. Although numerous studies have shown the cancer preventive properties of garlic and its components, there is no study on the effect of allicin on the growth of human liver cancer cells. In this study, we focused on allicin-induced different modes of cell death in human liver cancer Hep G2 (p53wild type) and Hep 3B (p53mutation) cells through p53-regulation. Our results indicated that allicin induced p53-mediated autophagic cell death in Hep G2 cells and caused Hep 3B cells apoptosis. Using western blotting, we observed that allicin decreased the level of cytoplasmic p53, PI3K/mTOR signaling pathway, Bcl-2 and increased the expression of AMPK/TSC2, and LC3-II signaling pathways in Hep G2 cells. In addition, the image of LC3-II punctate with mitotracker-red (labeling mitochondria) resulting in allicin-induced Hep G2 cells autophagosome formation and the protein expression of p53, AIF and Endo G in Hep G2 and Hep 3B cells could be observed by confocal laser microscope. On the other hand, allicin could induce ROS production in Hep 3B cells, caused DNA damage, decreased mitochondria membrane potential (ΔΨm) and activated caspase-dependent and -independent apoptosis pathways. To gain insight into the cell death mechanism in p53 knocked down Hep G2, we silenced p53 gene using siRNA mediated silencing. Allicin treatment induced apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells. These results suggest that allicin induced cell death in human hepatoma cells either through autophagy or apoptosis and might be a potential novel complementary gene therapeutic agent for the treatment of apoptosis resistant cancer cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58762 |
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顯示於系所單位: | 食品科技研究所 |
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