樟芝子實體乙酸乙酯層及玫瑰樹鹼對肝癌細胞株HepG2和PLC/PRF/5之活性與其作用機轉之探討

Apoptotic effects of EtOAc extract from Antrodia cinnamomea fruiting bodies and ellipticine in human hepatocellular carcinoma cell line

郭育君(Yu-Chun Kuo)

指導教授 ： 林俊清

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2005年)

摘要

第一篇:
樟芝(Antrodia cinnamomea T.T. Chang & W.N. Chou)為台灣特有種高價值的藥用真菌，又稱為牛樟芝或牛樟菇，僅生長於老齡牛樟樹，依其形態特徵被分類於薄孔菌屬(Antrodia)。有關樟芝之生物活性研究，根據文獻紀載其具有抗發炎、抗氧化、保肝等作用。在抗癌研究方面，則僅侷限於細胞毒殺的作用，對於癌細胞的增生抑制作用機制甚至其分子機轉的探討，都仍屬未知。因此本篇的研究主要是探討樟芝子實體乙酸乙酯層的萃取物(EAC)對肝癌細胞株HepG2與PLC/PRF/5誘發細胞凋亡(apoptosis)的作用機轉。在兩種細胞株中，EAC可以顯著的抑制其細胞增生率，並呈現濃度從屬效應(dose-dependent manner)。藉由分析DNA fragmentation及nucleosome ELISA的方式進一步確認EAC是經由誘發細胞凋亡來抑制兩種不同的肝癌細胞株生長。在HepG2中，EAC可以在不活化p53分子下，直接活化Fas receptor與 Fas ligand，透過death receptor途徑誘發細胞凋亡。另外，EAC在兩種細胞株中都可以透過mitochondrial 途徑誘發細胞凋亡。另一方面，EAC也會影響細胞存活的訊息傳導途徑，刺激細胞質中的IκBα活化，造成細胞核中NF-κB的量降低，活性也受到抑制。故本研究已詳細地探究樟芝子實體乙酸乙酯層的萃取物(EAC)誘發肝癌細胞凋亡(apoptosis)的分子機轉。
第二篇:
玫瑰樹鹼 (ellipticine, 5,11-dimethyl-6H-pyrido[4,3-b]carbazole)是由Ochrosia elliptica Labill (Apocynaceae)所分離出之生物鹼。它被證實具有DNA topoisomerase II的抑制作用因此被認為具有抗癌的發展潛力。但至目前為止，並無任何的研究針對玫瑰樹鹼對肝癌細胞之細胞凋亡誘導作用及相關機轉作探討。因此，本篇研究針對玫瑰樹鹼在肝癌細胞株PLC/PRF/5與HepG2上的抗增生作用及細胞凋亡誘導機制作詳細的探討。在研究結果中發現玫瑰樹鹼可以有效的抑制PLC/PRF/5與HepG2細胞株的細胞生長，並且具有時間與濃度效應 (does and time-dependent manner)。藉由分析DNA fragmentation 的形成及caspase-3的活化作用進一步確認玫瑰樹鹼是經由誘發細胞凋亡(apoptosis)來抑制細胞的生長。在作用機轉的研究方面，PLC/PRF/5與HepG2細胞中，Bcl-2 family與粒腺體膜電位都會受玫瑰樹鹼影響而變化，最後活化caspase-9，因此確認玫瑰樹鹼可以經由mitochondrial apoptotic pathway來誘導肝癌細胞進行細胞凋亡。另外在PLC/PRF/5細胞更發現有大量超氧陰離子 (O2.-) 與過氧化氫(H2O2)的累積，因而造成細胞的氧化壓力(oxidative stress)。在HepG2方面則發現玫瑰樹鹼會活化p53，接下來啟動Fas/Fas ligand的機制，因此產生caspase-8。最後我們在PLC/PRF/5細胞更發現玫瑰樹鹼會活化NF-κB (nuclear factor κB)，當加入NF-κB抑制劑時，可以顯著的增加細胞凋亡，因此抑制NF-κB可以增加玫瑰樹鹼的抗癌作用，減少抗藥性的產生。

關鍵字

細胞凋亡 ； 肝癌細胞株 ； 玫瑰樹鹼 ； 樟芝

並列摘要

Part1:
The fruiting body of Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in two human liver cancer cell lines, HepG2 and PLC/PRF/5. Treatment with EAC decreased the cell growth of HepG2 and PLC/PRF/5 cells in a dose dependent manner. In Fas/APO-1 positive-HepG2 cells, EAC increased the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), in a p53-indenpendent manner. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in HepG2 and PLC/PRF/5 cells. Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of IκBα in cytoplasm and reducing the level and activity of NF-κB in the nucleus, and subsequently attenuated the expression of Bcl-XL in HepG2 and PLC/PRF/5 cells. EAC therefore decreased the cell growth and induced apoptosis both in HepG2 and PLC/PRF/5 cells.
Part2:
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), one of the simplest naturally occurring alkaloids, was isolated from the leaves of the evergreen tree Ochrosia elliptica Labill (Apocynaceae). Here, we reported for the first time that ellipticine inhibited the cell growth of human liver cancer cell lines, HepG2 and PLC/PRF/5, and provided molecular mechanism of this effect. The results showed that ellipticine decreased the cell proliferation of HepG2 and PLC/PRF/5 cells in a dose and time- dependent manner by the XTT assay. Ellipticine initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, alteration of mitochondrial membrane potential, and activation of caspase-9 both in HepG2 and PLC/PRF/5 cells. Treatment of HepG2 cells with ellipticine increased the expression level of Fas/APO-1 and its membrane-bound ligands through p53 activation. In PLC/PRF/5 cells, the earliest oxidative event observed after ellipticine treatment was the increase of production of reactive oxygen species (ROS), including O2.-, and H2O2. Furthermore, ellipticine also inhibited the amount of IκBα in cytoplasm and raised the level and activity of NF-κB in the nucleus, in PLC/PRF/5 cells. Ellipticine therefore decreased the cell growth and induced apoptosis both in HepG2 and PLC/PRF/5 cells.

並列關鍵字

uman hepatocellular carcinoma cell ； ellipticine ； Antrodia cinnamomea ； Apoptosis

參考文獻

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