絞股藍皂甘藉由粒線體活化路徑誘導人類血癌細胞株（HL-60）和小鼠血癌細胞株（WEHI-3）產生細胞凋亡; Gypenoside induces apoptosis in human leukemia HL-60 cells and mouse WEHI-3 cells through mitochondria and caspase-3- dependent pathway

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Title:	絞股藍皂甘藉由粒線體活化路徑誘導人類血癌細胞株（HL-60）和小鼠血癌細胞株（WEHI-3）產生細胞凋亡;Gypenoside induces apoptosis in human leukemia HL-60 cells and mouse WEHI-3 cells through mitochondria and caspase-3- dependent pathway
Authors:	徐卉瑩;Hui-Ying Hsu
Contributors:	中國醫藥大學：生物科技學系
Keywords:	絞股藍皂甘;人類血癌細胞株;小鼠血癌細胞株;Gypenoside;HL-60 cells;WEHI-3 cells
Date:	2008-05-29
Issue Date:	2009-08-11 10:58:34 (UTC+8)
Abstract:	絞股藍皂甘（Gypenoside）是絞股藍（Gynostemma pentaphllum Makino）萃取物的主要活性成份且被廣泛應用在中國醫學研究方面，絞股藍皂甘已被證實能夠導致多種人類癌細胞株產生凋亡，例如：人類大腸癌、子宮頸癌以及肝癌細胞株等。然而目前並沒有相關資訊證明絞股藍皂甘能夠誘導人類血癌細胞（HL-60）和小鼠血癌細胞走向凋亡。本實驗探討絞股藍皂甘是否對於人類血癌細胞株（HL-60）和小鼠血癌細胞株（WEHI-3）誘發抗癌活性機轉，藉由流式細胞儀分析結果發現絞股藍皂甘能夠讓細胞週期停滯於G0/G1期，最後走向細胞凋亡，此結果可經由DNA電泳觀察到DNA斷裂情形得到證實。除此之外，也藉由流式細胞儀分析測得經絞股藍皂甘作用後的人類血癌細胞（HL-60）和小鼠血癌細胞（WEHI-3）裡的活性氧化物（ROS）濃度上升、鈣離子釋放以及粒線體膜電位(MMP)下降等細胞凋亡重要指標，此結果可經由西方墨點法指出絞股藍皂甘能使癌細胞中的抗凋亡蛋白Bcl-2表現量下降；促凋亡蛋白Bax表現量上升，同時也藉由共軛焦顯微鏡觀察到致死蛋白AIF、Endo-G從粒線體釋放至細胞核。根據上述多種實驗結果可證實：絞股藍皂甘能引起細胞中粒線體膜電位(MMP)改變導致凋亡蛋白Cytochrome c從粒線體釋放出而活化了caspase-3，進而誘發人類血癌細胞株（HL-60）和小鼠血癌細胞株（WEHI-3）走向凋亡路徑 Gypenosides (Gyp) are extracted from Gynostemma pentaphyllum Makino that had been used as folk medicine in Chinese population. Gyp have been shown to induce cell death and apoptosis in many human cancer cell lines, such as colon, cervical and liver cancers. However, there is no available information to address Gyp induced apoptosis in HL-60 and WEHI-3 cells. We examined the effects of Gyp on human leukemia HL-60 and mouse WEHI-3 cells. Therefore, the results from flow cytomertic analysis indicated that Gyp arrest cell cycle in G0/G1 and induced apoptosis in both examined cell lines. We also used DAPI stain and DNA gel electrophoresis to confirm Gyp induced apoptosis in both cell lines. Flow cytometric also used for analysis the levels of reactive oxygen species, Ca2+ and mitochondrial membrane potential (MMP) in both cell lines. The results showed Gyp promoted ROS and Ca2+ production and decreased the MMP level. Western blotting show that Gyp treatment gradually decreased the levels of anti-apoptotic protein (Bcl-2), but increased the levels of the pro-apoptotic protein (Bax). Gyp promoted the release of cytochrome c from mitochondia based on the changes of MMP and the activation of caspase-3 before leading to apoptosis. We also used confocal laser microscope to show AIF and Endo-G are released from mitochondrial then move to nuclei
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