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Title: | 苦橙果皮萃取物誘導人類肝癌細胞自體吞噬達到抑癌效果 Bitter Orange Peel Extract Induces Autophagy In Human Hepatoma Cells |
Authors: | Huey-Yin Lee 李慧穎 |
Advisor: | 沈立言(Lee-Yan Sheen) |
Keyword: | 肝癌,苦橙果皮萃取物,多甲氧基黃酮,自體吞噬,合併功效, liver cancer,bitter orange peel extract,polymethoxyflavones,autophagy,combination effect, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 肝癌於全球人類癌症致死率排名第二位,由於現行的治療方法效力不佳,目前肝癌患者的五年相對存活率僅16%,因此許多患者嘗試以天然成分作為佐劑,希望能提高傳統療法的療效並減緩其副作用。柑橘類水果為全球重要的農產品之一,除作為新鮮水果食用之外,剩餘的果皮則可開發成為副產品。柑橘類果皮含有多種黃酮類成分,而多甲氧基黃酮為其特有的成分,過去研究發現此類化合物具有抗氧化、抗發炎及減緩脂肪堆積等生理活性,近來其抗癌效果亦受到重視。本研究利用苦橙果皮經超臨界萃取所得之果皮萃取物(bitter orange peel extract, BOPE),探討其對人類肝癌Hep 3B細胞的抑癌機制,及其主要成分的合併作用。細胞存活率結果顯示在BOPE的處理下可顯著抑制人類肝癌細胞的增殖,形態上可觀察到細胞內產生空泡化現象。細胞免疫染色結果發現BOPE使自噬小體形成的重要指標蛋白LC3在細胞中有聚集的現象,且西方墨點法的結果亦顯示LC3-II表現量隨BOPE的處理有顯著性上升,故判定BOPE可經由誘導細胞產生自噬作用(autophagy)而達到抑制效果。深入探討其分子機制發現,BOPE首先可促進Hep 3B細胞大量產生ROS及降低ATP水平而導致內質網壓力的發生。內質網壓力的增加促使內質網中的鈣離子大量釋放到細胞質中,進一步活化下游的AMPK、TSC2,抑制mTOR活性,並且活化DAPK及Beclin-1,最終導致自體吞噬。而HPLC分析結果顯示BOPE中主要成分為橘皮素及川陳皮素,為了進一步了解橘皮素及川陳皮素兩者間的作用方式,將兩者進行單獨處理及共同處理之下發現後者對於細胞的抑制效果更佳。以不同比例將橘皮素與川陳皮素共同培養觀察其對細胞存活率的影響,並透過合併指數(combination index, CI)的換算發現兩者的確發揮協同效果(synergistic effect)。綜合上述結果,苦橙果皮萃取物可藉由誘導人類肝癌細胞產生內質網壓力調節之自體吞噬達到抑癌效果,且其主成分橘皮素與川陳皮素扮演者協同性作用,具有輔助抗癌治療之潛力。 Liver cancer is the second major cancer related death in the world. There are only 16% of 5-year relative survival rate among liver cancer patients because of the poor prognosis and cancer therapy side effects. To relieve the uncomfortable symptoms, natural adjuvant has become an alternative trend instead of conventional treatment. Polymethoxyflavones (PMFs) are a group of flavones found in citrus fruits peel. Tangeretin and nobiletin are major compounds in PMFs which their potential biological activities have been reported, such as anti-inflammation, anti-diabetes and anti-cancer. The objectives of this study are to investigate the anti-liver cancer activity of bitter orange peel extract (BOPE) and its molecular mechanisms in human hepatoma Hep 3B cells. The results showed that BOPE effectively reduced Hep 3B cell viability with the IC50 of 48.95 μg/mL at 24 hours. In addition, BOPE induced LC3 aggregation and conversion of LC3-I to LC3-II, which required for autophagosome formation, without increasing hypodiploid proportion and plasma membrane leakage. These results demonstrated that BOPE induced cell death of Hep 3B cells by autophagy rather than apoptosis and necrosis. In this study, we also investigated the molecular mechanisms of BOPE-induced cell autophagy. Results showed that BOPE promoted ROS generation and ATP depletion in Hep 3B cells, leading to ER stress, following by elevatation of cytosolic calcium ion concentration and activation of AMPK/TSC2/mTOR and DAPK/Beclin-1 pathways. Furthermore, major components of BOPE, tangeretin and nobiletin, exhibited synergistic effect to inhibit cell viability in Hep 3B cells. In conclusion, tangeretin and nobiletin from BOPE can synergistically induce ER-stress mediated cell death by autophagy in human hepatoma Hep 3B cells and it could be used as a potential complementary agent for liver cancer therapy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53111 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 食品科技研究所 |
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