Evodiamine造成人類結直腸癌細胞凋亡及G2/M期停留之探討

大腸結直腸癌一直為癌症死因前幾位，隨著飲食西化，大腸結直腸癌罹患率節節上升。Evodiamine (EVO)為天然中草藥吳茱萸Evodiae fructus所萃取之具活性之生物鹼，在先前研究中已發現EVO對癌細胞具有顯著之細胞毒性，然而，EVO對於抑制癌細胞生長之作用機制至今尚未釐清。於本實驗結果中發現，給予EVO 對於人類結直腸癌細胞COLO205、HT29具有明顯抑制細胞存活率之效果，並且呈現劑量及時間之依存性。COLO205及HT29在給予EVO後可明顯觀察到細胞凋亡之特徵，例如DNA ladder 的產生，染色質濃縮的現象，caspase 3蛋白及其上游粒線體caspase 9蛋白有明顯cleavage form增加，利用caspase activity assay實驗結果發現，EVO處理下caspase 3及caspase 9活性有顯著上升，粒線體內cytochrome c及AIF蛋白釋放， Bax蛋白表現增加， Bcl-xL蛋白表現降低，由流式細胞儀觀察到粒線體膜電位下降。使用流式細胞儀，利用DCHF-DA分析細胞處理EVO後ROS的產生，發現EVO並未產生ROS，前處理抗氧化劑N-acetylcystein (NAC)亦無法抑制EVO所造成的細胞凋亡及DNA斷裂，由流式細胞儀觀察細胞週期變化，發現細胞給予EVO後細胞週期G2/M期明顯增加並降低G1期，在其他研究發現，當G2/M期增加時會促使細胞tubulin聚集，於本研究中發現EVO會誘導tubulin聚集，增加cyclin B1、cdc2、cdc25蛋白之表現。此外也發現EVO會透過誘導JNKs磷酸化而造成細胞凋亡，前處理JNKs抑制劑SP600125可明顯回復細胞存活率，並降低由EVO所造成的Bax蛋白增加，回復Bcl-xL蛋白表現。綜合以上結果推論，在COLO205及HT29細胞中，EVO所具有的抗癌作用主要藉由JNKs蛋白質的磷酸化，影響Bax及Bcl-xL蛋白的表現使粒線體膜電位改變，誘發粒線體途徑細胞凋亡，並造成細胞週期停留於G2/M期進而誘導人類結直腸癌細胞凋亡，由本研究結果認為EVO對於人類結直腸癌的治療具有相當大的開發潛力。

關鍵字

人類結直腸癌細胞；細胞凋亡

並列摘要

Colorectal carcinoma is one of the major leading causes of cancer mortality, as westernized diet, morbidity of colorectal carcinoma were increased. EVO derived from Chinese herb Evodiae fructus with a significant cytotoxicity cancer cells was identified, however the mechanism of anti-tumor effects are still undefined. In the present study, EVO significantly reduced the viability of colorectal carcinoma cells COLO205 and HT29, and dose and time-dependent responses elicited by EVO is identified. Induction of apoptosis characterized such as DNA fragment and chromatin condensation in EVO-induced colorectal cancer cells was observed. Increase caspase 3 and its upstream mitochondria caspase 9 cleavage form protein expression, used caspase activity assay, activation of caspase 3 and caspase 9 activity were observed, cytochrome c and AIF protein release, Bax protein increased and Bcl-xL protein decreased, and decreased mitochondria membrane potential was detected by flow cytometry. Used DCHF-DA analysis ROS production in EVO-treated cells were not detected by flow cytometry, pretreat with anti-oxidant N-acetylcystein (NAC) can not inhibit apoptosis and DNA fragmentation in EVO-induced cells. An increase in G2/M phase cells and a decrease in G1 phase cells were detected in EVO-treated COLO205 and HT29 cells with inducing tubulin aggregation and cyclin B, cdc2, cdc25 protein expression. In addition, our study presents apoptosis through JNKs phosphorylation, pretreat JNKs inhibitor SP600125 reverse cell viability significantly, reduced Bax and reversed Bcl-xL protein expression in EVO-treated cells. The above results suggest that the anti-tumor of EVO in vitro through JNKs phosphorylation, affect Bax and Bcl-xL protein expression, change mitochondria membrane potential, induction mitochondria apoptosis and cell cycle arrest at G2/M in colon carcinoma cells was demonstrated. In our study, EVO for the treatment of colorectal carcinoma has considerable development potential.

並列關鍵字

Evodiamine ； apoptosis ； colorectal carcinoma cells

參考文獻

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Evodiamine造成人類結直腸癌細胞凋亡及G2/M期停留之探討

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