黃芩素誘導人類肝癌細胞（J5）細胞凋亡及抑制細胞轉移之分子機轉; The molecular mechanisms of baicalein-induced apoptosis and metastasis in human hepatoma J5 cells

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题名:	黃芩素誘導人類肝癌細胞（J5）細胞凋亡及抑制細胞轉移之分子機轉;The molecular mechanisms of baicalein-induced apoptosis and metastasis in human hepatoma J5 cells
作者:	蔡宏劼;Hung-Chieh Tsai
贡献者:	中國醫藥大學：醫學研究所碩士班
关键词:	黃芩素;肝癌;細胞凋亡;轉移;baicalein;J5;apoptosis;metastasis
日期:	2007-06-28
上传时间:	2009-08-13 14:50:57 (UTC+8)
摘要:	黃芩素是一種抗氧化劑，並且已被證明可引起人類癌細胞株的凋亡。然而，黃芩素對人類肝癌細胞（J5 cell line）引起的凋亡，其確切的分子機轉並不清楚。本研究中，我們利用流式細胞儀分析偵測細胞週期變化及細胞凋亡（apoptosis），並利用DAPI螢光染色法、彗星試驗、共軛焦顯微鏡與西方墨點法等技術探究其分子機制。結果顯示細胞週期停滯G2/M期是經由cdc25c與cdc2進行磷酸化阻滯cdc2-cycin B1複合物。細胞活性氧化物（ROS）、鈣離子濃度的上升與粒腺體膜電位下降。在凋亡的探討也證實，黃芩素促使粒線體膜上凋亡誘導因子（AIF）及核酸內切酶G（Endo G）釋放出來，並造成DNA的斷裂。且細胞色素（cytochrome c）釋放，啟動粒線體凋亡路徑，下游caspase-9與caspase-3隨之被活化。在本文中，NAC（ROS抑制劑）可以降低ROS的產生，降低細胞的死亡率，證明細胞凋亡與ROS產生有關。黃芩素也造成細胞的氧化壓力（oxidative stress），促使鈣離子從內質網釋放出來，導致細胞內鈣離子平衡遭受破壞，產生內質網壓力，且GRP78與GADD153蛋白表現上升，活化下游路徑造成細胞凋亡。癌細胞轉移侵襲過程中會透過基質金屬蛋白水解酶（matrix metalloproteinase，MMPs）降解細胞基質extracellular matrix（ECM）。由研究結果中指出，黃芩素抑制人類肝癌細胞的轉移，透過降低MMP-9的表現，可以抑制細胞的移動及細胞侵入，而達到抑制腫瘤轉移的目的。 Baicalein is an antioxidant and had been demonstrated to induce apoptosis in human cancer cell lines. However, the exact molecular mechanism of apoptosis induced by baicalein in human liver cancer J5 cells is unclear. In this study, we investigated the cell cycle arrest and apoptosis occurrence in J5 cells after exposed to baicalein by flow cytometry. We also used DAPI stain、comet assay、confocal and western blot to detect the mechanisms. The result from flow cytometric analysis demonstrated that caused G2/M-phase arrest by phosphorylation cdc25c and cdc2 inhibit activity of cdc2-cycin B1 complex. Flow cytometric analysis indicated that baicalein promoted the increase of reactive oxygen species (ROS) and Ca2+ in J5 cells. Baicalein also decreased the levels of mitochondrial membrane potential from J5 cells. We also detect cytochrome c、AIF and endo G release from mitochondria, indicated that baicalein elicited a significant increase of DNA fragmentation in J5 cells and promote the cell apoptosis. We also detect increase expression of caspase-9 and caspase-3. In this study, we also used NAC (ROS inhibitor) to decrease ROS and apoptosis generation. Baicalein cause oxidative stress production and Ca2+ release from ER and promote GADD153 and GRP78 expression. Matrix metalloproteinase (MMPs), one of the families of enzymes that degrade the extracellular matrix (ECM), are considered to play an important role of tumor invasion and spread. We have demonstrated that baicalein inhibits the invasion of human liver cancers by suppressing MMP-9 expressions.
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