The Molecular Mechanism of Anticancer Activities of Curcuma Longa Essential Oils

癌症目前是國人十大死因之首位，腦癌雖不在其中之前位，但其致死率卻很高，值得研究探討。薑黃屬(Curcuma)為薑黃多年生草本植物，許多文獻都指出薑黃植物具有抗發炎、降血糖、保肝、抗氧化、抗微生物及病毒等生物活性。本研究選用了國產的薑黃植物萃取出的低沸點?油處?人?腦癌細胞株，探討薑黃精油抗腫瘤活性及調控之分子機制。在MTT測試下薑黃精油TCLR-1對於人?腦癌GBM 8401細胞的毒殺效果相對於人?腦癌GBM 8901細胞較佳。因此後續研究針對人類腦癌 GBM 8401細胞。而單染實驗中薑黃精油TCLR-1對人?腦癌GBM 8401細胞之細胞週期並沒有顯著的停留，反而是在短時間內快速走向凋亡。由此可推測薑黃精油TCLR-1致死人?腦癌GBM 8401細胞是走細胞凋亡路徑；在雙染實驗中，偵測Annexin V/PI 和DNA fragmentation，證實是藉由細胞凋亡而非細胞壞死途徑。而經由西方墨點法來推測細胞凋亡途徑的探討中，薑黃精油?激GBM 8401細胞走Caspase-3路徑，之後會再進一步驗證其是否繼續走Caspase-8及Caspase-9路徑。透過本研究，除了提供?油抗腦癌的生物活性及其抗癌的分子機制，也期望將其發展成生物製劑提供腦癌症病患者有效的治?的選擇或成為抗癌藥物的輔助試劑。希望藉由小分子精油的特性可以達到治療腦癌的有效成果。Cancer is the leading cause of death among people. The brain cancer, though not one of the prevalent diseases, is worth of investigation because of its high death rate. Curcuma (Curcuma) is a perennially herbal turmeric. Many papers have pointed out that turmeric plant has anti-inflammatory, reducing the biological activity of blood glucose, liver protection, anti-oxidation, anti-microbial and anti-virus activities. We applied the essential oils TCLR-1, extracted from the local turmeric plant, to human brain cancer cell lines to explore and elucidate the molecular mechanism of the anti-tumor activity. With the MTT test of turmeric oil TCLR-1 for toxicity applied to human brain cancer cells, GBM 8401 is more sensitive compared with GBM 8901, the other human brain cancer cell line. Therefore, we applied GBM 8401 for further experiments. The single-transfection experiments in turmeric oil TCLR-1 to study cell cycle of human brain cancer GBM 8401 cells did not show the stage arrest significantly, however, it is turned to apoptosis in a short time. The double staining experiment was confirmed that turmeric oil TCLR-1 forced the cells to apoptosis rather than necrosis. Western blotting experiment was used to confirm the apoptosis pathway judged from the accumulation of Caspase-3 in both time- and dose- dependent manners with the treatment of turmeric essential oils in GBM 8401 cells. We thus have a clear shoot that the turmeric oil TCLR-1 showed the antitumor activity through the induction of cell apoptosis in brain cancer cells.致謝.........................Ⅰ 中文摘要.......................Ⅱ 英文摘要......................Ⅲ 目錄……………………………………………………………………………...…..Ⅳ 圖表目錄................................VI 第一章 前言............1 一、 癌症(惡性腫瘤)......................1 二、 腦癌…….......………………………….........2 (一)腦瘤定義及分類...............................2 (二)致病因子…………………………………….4 (三)癥狀與診斷.......................................4 (四)盛行率.............................5 (五)腦瘤的症狀...................................5 (六)腦瘤的診斷................................5 (七)腦瘤的治療............................6 三、細胞週期.......................................9 (一)細胞週期分期..............................9 四、細胞凋亡(Apoptosis).................11 (一) 細胞凋亡之定義.............................12 (二) 細胞凋亡的特徵. .............................11 (三) 細胞凋亡訊號路徑..........................13 1.外在途徑—Extrinsic pathway...................14 2.內在途徑— Intrinsic pathway................14 四、薑黃植物來源與應用..........................16 (一)薑黃屬(Curcuma)...................16 (二) 薑黃種…………………………………………………………………….16 (三) 薑黃的化學成分及藥理作用...................17 (四)薑黃素的常見副作用...............................18 五、研究動機與目的...................................20 第二章 材料與方法.....................................21 一、實驗材料........... ………………………………………………………………..21 (一)細胞株.................... ......21 (二)實驗藥物............................21 (三)藥品試劑................. ...21 (四)抗體.....................................22 (五)主要儀器與耗材.................................23 二、實驗用溶液配方.........................25 三、實驗方法...............................28 (一)薑黃精油成分分析與鑑定..........................28 (二)細胞培養與繼代....................28 (三) 細胞冷凍保存........................29 (四) 冷凍細胞活化.................................29 (五) 細胞增生抑制分析(MTT Assay)...............29 (六) 細胞週期分析........................30 (七) Annexin V-Propidium iodide assay...........30 (八) 蛋白質萃取....................31 (九) 蛋白質濃度測定與蛋白質樣品製備............31 (十) 蛋白質電泳分析(SDS-PAGE) .........32 (十一) 西方墨點法(Western blotting) ........32 第三章 結果..................................34 一、薑黃精油成分分析………………………………………………………..34 二、薑黃植物之精油藥物對於人類腦癌細胞GBM 8401及GBM 8901之細胞毒性………………..35 三、薑黃精油對腦癌GBM 8401之細胞週期影響...........37 四、薑黃精油誘導人類腦癌(GBM 8401)細胞之細胞凋亡.........39 五、薑黃精油對非小對腦癌GBM 8401之之caspase蛋白影響.....41 第四章 討論與結論...........................43 第五章 參考資料...........................4