- 學位論文
Baicalein影響小鼠黑色素瘤細胞株產生ROS之作用機轉探討
Mechanism of the Baicalein induced Reactive Oxygen Species Formation in Mouse B16F10 Melanoma Cell
摘要
漢方草藥黃芩根部萃取物之黃芩素(baicalein; 5,6,7,-trihydroflavone)為一種類黃酮物質，被認為具有抗發炎抗癌的特性。在過去的文獻中說明baicalein在血小板會透過12-lipoxygenase去引發氫氧自由基 (hydroxyl radical)和透過PGHS peroxidase去引發半醌自由基 (semiquinone radical)的生成。本次研究我們利用電子順磁共振的技術，發現baicalein可以在B16F10小鼠黑色素瘤細胞株以濃度相關性方式去引發hydroxyl radical和 superoxide訊號產生。利用phospholipase A2 (cPLA2) 抑制劑，AACOCF3 和quinacrine和高濃度的 arachidonic acid可以有效的減弱hydroxyl radical訊號的強度，因此認為和AA代謝的酵素有關。而利用mannitol和catalase這兩種ROS的清除劑，也可以有效的抑制氫氧自由基的生成。 (-)-Epicatechin 和 (-)-epicatechin gallate兩者屬於benzene-1, 3-diol類的flavonoids也同樣被作為12-LOX的抑制劑，但是在ESR study卻無法在B16F10小鼠黑色素瘤細胞偵測到任何的hydroxyl radical訊號。利用siRNA方式抑制12-LOX蛋白表現可以減弱高濃度的baicalein對細胞所刺激生成的hydroxyl radical。另外在western blotting實驗中我們發現baicalein可以刺激細胞內active caspase-3蛋白的活性，但是卻無法有效的抑制12-LOX蛋白的表現。在MTT實驗中baicalein可以讓細胞生長受到抑制但是在投與ROS 清除劑後卻可以讓細胞存活率顯著的回復。因此在眾多實驗數據顯示中我們認為baicalein刺激B16F10小鼠黑色素瘤細胞生成的superoxide會經由superoxide dismutase (SOD) 和fenton reaction去生成對細胞具有傷害性的hydroxyl radical。Baicalein可以做為一個強力的電子提供者去和脂質過氧化自由基的中間產物透過12-LOX去做co-oxidation動作進而造成細胞產生hyroxyl radical。同時baicalein也可以刺激caspase-3的活化，這些可能就是造成baicalein造成細胞存活率下降和細胞凋亡的重要證據
關鍵字
黑色素瘤並列摘要
Baicalein (5,6,7,-trihydroflavone) as flavonoid originated from the root of Chinese medical herd Scutellaria baicalensis, has been shown to exert anti-inflammatory and anti-cancer effects. In previous study demonstrated that baicalein induced hydroxyl radical formation via 12-lipoxygenase (12-LOX) and induced semiquinone radical formation via PGHS-peroxidase in human platelets. Here we found that baicalein also induces hydroxyl radical and superoxide formation in B16F10 melanoma cell line in dose dependent manner by using electron spin resonance (ESR) technique. Two phospholipase A2 inhibitors (cPLA2), AACOCF3 and quinacrine and a high concentration of arachidonic acid (AA) attenuates the intensity of hydroxyl radical signal indicating that baicalein induced hydroxyl radical formation is associated with AA metabolite enzymes. Two ROS scavengers, mannitol and catalase, also significantly inhibit the intensity of hydroxyl radical signal in ESR study. (-)-Epicatechin and (-)-epicatechin gallate which are flavonoids contain benzene-1,3-diol structure are also used as 12-LOX inhibitors, but they showed rarely detectable signal of hydroxyl radical after incubated with B16F10 melanoma cell suspension in ESR study. Cell transfected with 12-LOX siRNA could attenuate hydroxyl radical signal which induced by high concentration of baicalein. According to western blotting results, we found that baicalein had the activated effect in caspase-3 activity but had no effect in 12-LOX expression. In MTT assay, we also found that baicalein caused reduction of cellular viability and were reverse by addition of ROS scavengers. Present data indicate that superoxide induced by baicalein could promptly convert to hydroxyl radical through superoxide dismutase (SOD) and fenton reaction in B16F10 melanoma suspension. Baicalein pose as a strong electron donor which co-oxidation with fatty acid peroxy radical intermediate through 12-LOX to induce hydroxyl radical formation. These may be the evidence that baicalein caused cell growth retardation and cell apoptosis.
參考文獻
延伸閱讀
- 聞國軒（2011）。Baicalein誘發人類前骨髓白血病細胞產生ROS之作用探討〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://doi.org/10.6831/TMU.2011.00067
- Lee, J. W., & Lee, I. S. (2008). Rubus coreanus Miquel萃取物對小鼠之類造骨MC3T3-E1細胞增殖與分化的影響. 藥物食品分析, 16(1), 80-87. https://www.airitilibrary.com/Article/Detail?DocID=10219498-200803-16-1-80-87-a
- 林若蓓（2016）。探討洛神花萼發酵萃取物對抑制小鼠B16F10黑色素瘤細胞生成黑色素之影響〔碩士論文，國立宜蘭大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0046-3107201617183000
- 林依萱（2010）。醣化終極產物在大鼠心肌細胞(H9c2)中對MEK-ERK訊息路徑影響之研究〔碩士論文，長榮大學〕。華藝線上圖書館。https://doi.org/10.6833/CJCU.2010.00030
- 黃建勳（2017）。Inhibitory Mechanisms of Hinokitiol, a Natural Tropolone Derivative, on Mouse Melanoma B16-F10 Cells〔博士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1701201722525900