咖啡酸苯乙酯誘發C6神經膠質瘤細胞凋亡與生長停滯之機制探討

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 博碩士論文 > Item 310902500/1317

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题名:	咖啡酸苯乙酯誘發C6神經膠質瘤細胞凋亡與生長停滯之機制探討 The mechanistic study of caffeic acid phenethyl ester induced apoptosis and growth arrest in C6-glioma cells
作者:	郭星君 Hsing Chun Kuo
贡献者:	中山醫學院：生物化學研究所;曾翠華
关键词:	Rb;MAPK;CAPE Retinoblastoma protein;Mitogen-activated protein kinase;caffeic acid phenethyl ester
日期:	2001
上传时间:	2010-05-04T02:28:05Z (UTC)
摘要:	Caffeic acid phenethyl ester（CAPE）是蜂膠的組成分之一。蜂膠具多功能的生物與藥理作用:有抗菌、抗發炎、抗黴菌、抗病毒、抗腫瘤等性質。目前蜂膠中抑制癌症細胞的成分部分已被鑑定出來，主要是Caffeic acid (CA)類的衍生物。過去我們發現CAPE and ethyl caffeicate (EC)對於口腔癌化細胞具有毒殺能力，所以我們針對C6 glioma cells以及H3B cells先以CAPE與其相關衍生物CA及EC作處理，發現CAPE投以C6 glioma cells之毒殺能力最強。當處理CAPE（50μM）36小時可觀察到細胞核濃染和DNA片段化現象；接著同一時間利用流式細胞儀分析細胞週期，得知有37 ﹪細胞DNA呈現凋亡片段，55 ﹪的細胞停滯在G0/G1期，而且只有8 ﹪細胞處於S期，G2/M期消逝為0 ﹪。以CAPE為化學治療劑所導致C6 glioma cells的細胞凋亡路徑之研究，發現處理CAPE 3小時後，p53與Fas/Apo旋即上昇，Cytochrome C自粒腺體釋出至細胞質，且伴隨p53的磷酸化而致使活化下游相關基因表達，而anti-apoptotic Bcl-2表現減少和pro-apoptotic Bax、Bak、Bcl-Xs表現增加。在藉由MEK抑制劑（PD98059）與p38 抑制劑（SB203580）交叉作用，得知p38 和ERK 參與C6 glioma cells的凋亡訊息路徑。另外，之前細胞週期分析CAPE（50μM）處理24小時，細胞週期停滯在G0/G1期；利用IB分析相關細胞週期蛋白：RB, cyclins, cdks, and CDKIs，發現果然Rb phosphorylation, cyclin D, cyclin E蛋白表現減少，且CDKIs p16, p21, p27表現大增。除此之外，利用MEK抑制劑（PD98059）作用下，ERK 主導CAPE對於C6 glioma cells的細胞週期G0/G1停滯。經由以上實驗推論，當C6 glioma cells 處理CAPE之下，ERK與p38分別調控著細胞生長停滯與凋謝死亡不同路徑；除此p38 kinase調控著因CAPE所誘發的細胞凋亡路徑是由於影響p53及caspase 3的作用。 More recently, work on the anti-inflammatory, anti-bacteria, anti-fungus, anti-virus and anti-tumor activity of propolis has concentrated on caffeic acid phenethyl ester（CAPE） which is one of components. As our previous studies, we had found CAPE and ethyl caffeicate (EC) exhibited significant cytotoxicity on oral cancer cells. Herein we further investigated CA（caffeic acid）, EC, and CAPE by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay on the growth of H3B hepatocytes and C6 glioma cells. CAPE showed significant cytotoxicity on C6 glioma cells. Further investigation showed that C6 glioma cells underwent internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after 36-hr treatment with CAPE (50 μM). Flow cytometric analysis observed that the hypodiploid phase (apoptosis peak, 37%), DNA content analysis exhibited 55 % of cells in the G0/G1 phase, 8 % in the S phase, and 0 % in the G2/M phase after 36-hr treatment with CAPE. C6 cells treatment with CAPE for 3 hrs could increase tumor suppressor-p53 and regulate Fas/APO receptor in response to chemotherapy leads to activation of apoptotic signaling pathway with activation of caspase 3, proteolytic cleavage of PARP, downregulation of Bcl-2 and upregulation of Bax, Bcl-Xs and, Bak that mediated by extracellular signal—regulated protein kinase and p38 kinase. Neverthless, CAPE on C6 glioma cells inhibited the cell cycle progression arrested in G0/G1 by Flow cytometric analysis. According to IB, we examined expression of cell cycle-related proteins including RB, cyclins, cdks, and CDKIs. It demonstrated that CAPE decrease Rb phosphorylation, cyclin D, cyclin E and increased expression of p16, p21, p27 significantly in C6 glioma cells. In addition, inhibition of CAPE-induced ERK1/2 activation by PD98059 resulted in a reduced G0/G1 phase percent by DNA content assay. CAPE on C6 glioma cells inhibited the cell cycle arrest in G0/G1 phase that mediated by extracellular signal—regulated protein kinase（ERK）. We report here that ERK-1/2 and p38 kinase oppositely regulate CAPE-induced cell growth arrest and apoptosis of C6-glioma cells and the reverse effects of the two MAP kinases are triggered by the converse regulation of p53 and caspase-3 in response to apoptotic signal pathway.
URI:	http://140.128.138.153:8080/handle/310902500/1317
显示于类别:	[生化微生物免疫研究所] 博碩士論文

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