α-水芹烯對人類肝癌細胞株之生長抑制作用

Inhibition of cell growth in human liver tumor cells by α-phellandrene

李怡蓁(Yi-Chen Li)

指導教授 ： 張文正

長榮大學/健康科學學院/醫學研究所/碩士(2010年)

https://doi.org/10.6833/CJCU.2010.00099

摘要

本研究擬以香辛料及香草植物中常見之活性成份- α-水芹烯（α-phellandrene, α-PA）為實驗材料，配合人類肝癌細胞株（J5）之細胞培養為實驗模式，探討α-PA對於J5細胞株細胞生長之影響及其作用機轉。由研究結果顯示，以不同濃度α-PA處理J5細胞株24小時後，其細胞生存力有顯著下降的現象（p＜0.05），且呈現劑量依賴（dose-dependent manner）之關係。配合流式細胞儀分析發現，α-PA並無造成J5細胞株細胞週期滯留之現象，而且α-PA並不會影響J5細胞株內調控細胞凋亡（apoptosis）之內、外在路徑中的caspase 8以及caspase 3的表現。利用西方墨點法分析亦發現，α-PA與控制組比較，並不會改變J5細胞中Bax及Bcl-2蛋白質之含量。因此，初步推論α-PA造成J5細胞株生存力的下降，可能並非經由誘發細胞凋亡之路徑。另外，本研究發現α-PA具有增加肝癌細胞株內活性氧（reactive oxygen species, ROS）之含量以及降低粒線體膜電位（mitochondrial membrane potential, △Ψm）之作用；由細胞內ATP含量之變化狀況發現，α-PA會破壞細胞膜的完整性而造成細胞內ATP之耗損，在α-PA處理J5細胞株48小時後，其ATP耗損有顯著增加之現象（p＜0.05）；由細胞內NO產生量分析發現，J5細胞株以50 μM α-PA處理後，ㄧ氧化氮的含量有顯著高於控制組之現象（p＜0.05）。因此，本研究推論α-PA造成J5細胞株死亡之方式可能是與其造成細胞壞死（necrosis）之機制有關。另外，本研究進ㄧ步利用西方墨點法分析造成細胞自噬（autophagy）之指標蛋白- PI3K III、Beclin-1及LC3 II等蛋白質含量變化，結果發現50 μM α-PA會造成J5細胞株細胞內PI3K III、Beclin-1及LC3 II蛋白質表現顯著增加（p＜0.05）。因此，本研究推論α-PA造成J5細胞株細胞死亡之機轉亦可能與其誘發肝癌細胞株之細胞自噬有關。綜合上述結果發現α-PA可抑制人類肝癌細胞株之細胞生長，其作用機轉可能與其誘發J5細胞株之細胞壞死與細胞自噬有關，而非經由誘發細胞凋亡作用。但是，更深入之分子調控機制及其預防癌症之相關動物實驗仍需進一步之研究。

關鍵字

α-水芹烯 ； 細胞壞死 ； 細胞凋亡 ； 細胞自噬 ； 人類肝癌細胞株

並列摘要

To investigated the inhibition of cell growth in human liver tumor cells by α-phellandrene (α-PA). α-PA is a common active principle from essential oil of dietary specie or herb. And, the human liver tumor cells (J5) cultured was as an experimental model in this study. According to the results, all of the various concentrations of α-PA were significantly decrease the cell viability of J5 cells in this study (P<0.05), with a dose-dependent manner. From FACs analysis, the cell cycle distribution were not significantly change after various concentrations α-PA treatment for 24 hours. In addition, the activities of caspase 8 and caspase 3, important regulate moleculars on induce pathway of apoptosis, were not change after α-PA treatment for 24 hours in J5 cells. From Western blot examination, the expression of Bax and Bcl-2 protein was not change after α-PA treatment for 24 hours in J5 cells. Therefore, the decline of cell viability of J5 cells by α-PA may be not through the induction of apoptosis pathway. In this study, α-PA increases the content of reactive oxygen species and reducing mitochondrial membrane potential of J5 cells after 24 hours treatment. And, α-PA was significantly increase the ATP deplation of J5 cells after 48 hour treatment (P<0.05) and their NO level was significantly increased after 24 hours treatment (P<0.05). On the other hands, 50 μM α-PA were significantly increased the protein level of PI3K III, Beclin-1 and LC3 II on J5 cells (P<0.05). In these results showed that, α-PA can inhibit cell proliferation of human liver tumor cells. The mechanisms of cell death maybe involve in necrosis or autophagy, but not apoptosis.

並列關鍵字

α-phellandrene ； necrosis ； apoptosis ； autophagy ； human liver tumor cells.

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