University of Macau Abstract PROAPOPTOTIC ACTIVITY OF EZHU OIL AND FURANODIENE ON CULTURED HUMAN HEPG2 CELL by Xiao Yu Thesis Supervisor: Dr. Shaoping Li Prof. YiTao Wang Chinese Medicinal Sciences Ezhu, one of traditional Chinese medicines, is derived from three species of Curcuma including Curcuma phaeocaulis Val., C. kwangsiensis S. G. Lee et C. F. Liang and C. wenyujin Y. H. Chen et C. Ling according to Chinese Pharmacopoeia. In this study, we evaluate the cytotoxicity of ezhu oil (C.wenyujin) and a pure compound furanodiene isolated from ezhu oil to human HepG2 cells and investigate the underlying mechanism of the antiproliferative effect. Ezhu oil has been found to be toxic to HepG2 cell and to induce cellular apoptosis. Ezhu oil causes cell cycle S/G2 phase arrest and the antiproliferative mechanism has been focused particularly on mitochondrial changes, the active caspase-3 enzyme expression, release of cytochrome c, the activation of caspases-3 and the cleavage of PARP. Furthermore, to detect the toxicity of furanodiene on HepG2 cell, we attempted to characterize in detail the cell signaling cascades resulting in furanodiene-induced human HepG2 cell apoptosis, which caused G2/M arrest, focused particularly on mitochondrial changes, the role of p38 and ERK mitogen-activated protein kinase (MАРK) activation. We found that furanodiene-induced mitochondrial changes also included the release of cytochrome c, the activation of caspases-3 and the cleavage of PARP leading to the activation of p38 and suppression of ERK in HepG2. This is the first study reporting the proapoptotic activities and mechanisms of ezhu oil and furanodiene in human HepG2 cells. 5 Key words: Ezhu oil, furanodiene, HepG2, apoptosis, caspase-3, mitochondria, cytochrome c, MAPK, ERK, P38.

摘要 莪術為溫郁金(C. Wenyujin Y. H. Chen et C. Ling) 義術(C. kwangsiensis S. G. Lee et C. F. Liang)以及廣西莪術(Curcuma. phaeocaulis Val.)的乾燥根莖。據 報導,我術油具有抗腫瘤及抗病毒等功效。為此,我們將 術油和從莪術油中提 取分離出來的單體成分味喃二烯作用於人肝癌細胞 HepG2 中,檢測其毒性並對 其殺傷肝癌細胞的機制進行探討。 在莪術油的實驗當中,我們發現我術油對 HepG2 細胞有明顯的殺傷作用,能夠 抑制其生長並誘導其發生凋亡。我們主要測定了装術油作用 HepG2 細胞后,能 夠使其細胞週期阻滯 G2/M期,主要是表現在G1期細胞數目的減少以及S期明 顯增加,伴隨著 G2期的升高。之後,還檢測出了細胞色素C的釋放,線粒體膜 電勢的升高,半胱氨酸蛋白酶-3 活化以及 ADP 核糖聚合酶裂解。 在喃二烯的檢測中,檢測了所有在莪術油中檢測的指標,且得出的結果都是相 似的。同樣檢測到了細胞色素 C 的釋放,線粒體膜電勢的升高,半胱氨酸蛋白 酶-3 活化以及ADP 核糖聚合酶裂解。不同的是,味喃二烯對細胞週期的影響 主要表現在使其阻滯在G2/M期。此外,我們還檢測出咲喃二烯誘導的P38 磷酸 化增加以及細胞外資訊調節激酶磷酸化減少。 本次實驗,爭對莪術油及味喃二烯對 HepG2 細胞促凋亡作用的研究,還是第一 次。研究的結果顯示,我術油及吠喃二烯對於臨床治療肝癌非常有研究價值。不 仅可能是潜在的有效的抗癌药物,也可以和抗癌药物联合运用以减低化疗药物的 剂量,减低对人体的毒性,从而提高疗效。 關鍵字:莪術油,味喃二烯,人肝癌細胞,凋亡,半胱氨酸蛋白酶-3,線粒體, 細胞色素C,絲裂原活化蛋白激酶,細胞外資訊調節激酶, P38.