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標題: 普萊氏月桃萃取純化物-Flavokawain B引發人類大腸癌細胞凋亡機制之探討
Involvement of reactive oxygen species and GADD153 upstream of mitochondrial apoptosis by flavokawain B, a novel chalcone from Alpinia pricei Hayata, in HCT116
作者: 郭瑜芬
Kuo, Yu-Feng
關鍵字: 類黃酮;flavonoid;普萊氏月桃;活性氧分子;細胞凋亡;flavokawain B;Alpinia pricei Hayata;ROS;GADD153;apoptosis
出版社: 生物醫學研究所
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Flavokawain A, a novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice. Cancer Res 65, 3479-3486.
Flavokawain B is a member of flavonoids which are found in a variety plants. Previous studies indicated that flavonoids act as anti-oxidants to inhibit oxidative stress generated within cells. However, others demonstrated that flavonoids trigger generation of reactive oxygen species (ROS) leading to cell death in cancer cells. Flavokawains are mainly isolated from kava plants, and have been associated with low-cancer incidences. In particular, flavokawain A is shown to induce Bax-dependent apoptosis in bladder cancer cells. However, the biological effect of other structurally-related flavokawain B and flavokawain C is not understood.
Flavokawain B used in this study was from Alpinia pricei Hayata, a native plant in Taiwain. We found that flavokawain B suppressed human colon cancer cell (HCT116) growth by inducing apoptosis through mitochondrial apoptosis pathway. Moreover, we observed an increase in protein expression of ER stress inducible gene-GADD153 in flavokawain B treated group. A decrease in Bcl-2 was accompanied with an increase in Bim expression in flavokawain B-treated cells. These alternations affected mitochondria membrane potential, coupling with the translocation of Bak and cytochrome c release and led to apoptosis. However, p53 was not essential to flavokawain B-mediated GADD153 up-regulation.
Furthermore, we detected an increase in ROS generation upon flavokawain B treatment. The anti-oxidant NAC inhibited ROS production as well as apoptosis and GADD153 up-regulation induced by flavokawain B. Therefore, we postulated that ROS was the upstream signaling molecule involved in flavokawain B-induced apoptosis. Taken together, our results demonstrated that flavokawain B triggered ROS generation and GADD153 up-regulation, leading to mitochondria-induced apoptosis in human colon cancer.

Flavokawain B存在於許多植物中,屬於類黃酮家族成員之一。在先前的研究中指出類黃酮可藉由其抗氧化能力清除細胞內所產生之氧化壓力;然而,有其他的文獻則指出類黃酮可促使腫瘤細胞產生過量的活性氧分子並進一步導致細胞死亡。迄今,研究報告中所提及之flavokawains大多是從卡瓦椒萃取而來,且此家族中部分的化合物被指出有抗癌的功效,如:flavokawain A被發現可造成膀胱癌細胞凋亡,在這過程中BAX有著非常重要的角色。除了flavokawain A之外,其餘之結構相似物-flavokawain B及flavokawain C功能目前還不是很清楚。
本實驗所使用之flavokawain B則由台灣特有種-普萊氏月桃 (Alpinia pricei Hayata)之地下莖以乙醇萃取後純化所得。經由體外細胞株實驗後,我們發現flavokawain B可引發人類大腸癌細胞之細胞凋亡,並與粒腺體路徑活化路徑有關。更進一步的實驗證實flavokawain B可使ER stress inducible gene-GADD153 mRNA及蛋白表現量上升。GADD153的角色為轉錄因子,可調控下游Bim蛋白表現量上升,與引發Bcl-2的蛋白表現量下降,這些Bcl-2家族蛋白進一步地調控粒腺體之膜電位,同時我們也觀察到Bak位置改變伴隨著cytochrome c釋放到細胞質中,也伴隨著細胞凋亡;然而GADD153表現量的上升與p53無關。
利用流式細胞儀分析,我們偵測到HCT116細胞因flavokawain B之處理而產生大量之活性氧分子,且利用抗氧化劑NAC可抑制flavokawain B所產生的活性氧分子,也可同時抑制flavokawain B造成的細胞凋亡,並抑制因flavokawain B處理而上升的 GADD153蛋白表現量。由我們的實驗結果可得知flavokawain B是經由活性氧分子導致GADD153活化,最終造成粒腺體路徑形式的細胞凋亡產生。
其他識別: U0005-0607200914465900
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