絞股藍皂苷誘發人類肺癌細胞株（A549）的細胞週期停止在G0/G1期與計畫性死亡; Gypenosides induces in vitro G0/G1 phase arrest of cell cycle and apoptosis in human lung A549 cells

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Title:	絞股藍皂苷誘發人類肺癌細胞株（A549）的細胞週期停止在G0/G1期與計畫性死亡;Gypenosides induces in vitro G0/G1 phase arrest of cell cycle and apoptosis in human lung A549 cells
Authors:	陳宜珊;Yi-Shan Chen
Contributors:	中國醫藥大學醫學研究所
Keywords:	絞股藍皂;細胞週期;計劃性死亡;G0/G1期;抗藥性基因;Gypenosides;cell cycle;apoptosis;G0/G1 phase;MRP
Date:	1993
Issue Date:	2009-12-24 10:57:58 (UTC+8)
Abstract:	中草藥的研究在近年來成為非常熱門的研究主題。葫蘆科植物絞股藍（Gynostemma pentaphyllum MAKINO）是一種在亞洲治療肝炎以及心臟血管性疾病常使用的中草藥，絞股藍皂苷（Gypenosides）為由葫蘆科植物絞股藍經由n-butanol層中所萃取出來的主要藥效成分。然而絞股藍皂苷引起的細胞週期抑制與誘導計畫性死亡的機制到現在仍然不是非常的清楚。在本篇研究中，藉由探討細胞增生（proliferation）、細胞週期（cell cycle）及細胞凋亡（apoptosis）等機制，評估絞股藍皂苷對於人類肺癌細胞株（A549）抗癌的作用機轉。由實驗數據發現，絞股藍皂苷對人類肺癌細胞株會抑制細胞增生、細胞週期停止在G0/G1期及誘導細胞凋亡。藉由反轉錄聚合酶反應（Reverse-Transcriptase Polymerase chain reaction；RT-PCR）與西方點墨法(Western Blotting) 分析細胞週期素，發現p21、p27皆有上升的趨勢。因而引發細胞週期停止在G0/G1期之上游因子p21、p27的表現量也會隨著加入絞股藍皂苷劑量增加而表現量增加，故推論p21、p27也許是調控促使人類肺癌細胞株發生細胞週期停止在G0/G1期的主要因子。流式細胞計數儀分析與DNA裂解實驗指出，絞股藍皂苷會誘發人類肺癌細胞株產生凋亡現象(apoptosis)。藉由RT-PCR與Western Blotting的方法可以發現，Bax有些許的上升，Bcl-2與p53的量並沒有變化，而caspase-3與caspase-9的量也是明顯上升，NF-κB也有些許上升的趨勢。因此我們認為，絞股藍皂苷可以藉由NF-κB的路徑，導致Bax的上升，而促使caspase-9活化，再使下游的caspase-3活化而導致計畫性死亡，而這樣的死亡路徑是不經由p53這條路徑的。; Recently, Herbal medicines are increasingly becoming a popular project. Gynostemma pentaphyllum MAKINO is noted for its functions of treating hepatitis and cardiovascular diseases in Asia. Gypenosides are the major components that are extracted from Gynostemma pentaphyllum MAKINO. However, the molecular mechanism underlying the gypenosides-induced cell cycle arrest and apoptotic process is unclear. In this study, we have evaluated the chemopreventive role of gypenosides in human lung cancer (A549) cells in vitro by studying the regulation of proliferation, cell cycle and apoptosis. Gypenosides inhibited cell proliferation, induced G0/G1 arrest and apoptosis in A549 cells. Investigation on the levels of CDKIs (p21 and p27) by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) and Western Blotting showed that p21 and p27 were increased with the increasing doses of gypenosides in A549 cells. The levels of p21 and p27 increased after A549 cells were cotreated with various concentrations of gypenosides. The increase of the levels of p21 and p27 may be the major factor for gypenosides to cause G0/G1 arrest in the examined cells. Flow cytometric assay and gel electrophoresis of DNA fragmentation also confirmed that gypenosides induced apoptosis in A549 cells. Our data demonstrated that gypenosides-induced apoptotic cell death was accompanied by up-regulation of Bax, NF-κB, caspase-3 and caspase-9, while it had no effect on the levels of Bcl-2 and p53. Taken together, gypenosides therefore appears to exert its anticarcinogenic properties by inhibiting proliferation, inducing G0/G1 phase arrest and apoptosis underwent activation of NF-κB, Bax and caspase-3 in human lung A549 cancer cell line.
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