- 學位論文
山葵萃取物抗大腸癌功效與分子機制之探討
Study of anti-colorectal cancer functions and molecular mechanisms of Wasabia japonica extract
摘要
大腸癌是導致全世界癌症死亡的最主要的原因之一，且其發生率始終高居不下，因此針對大腸癌之預防與治療的研究不斷受到重視。山葵(Wasabia japonica)俗稱wasabi，是日本和台灣常使用的嗆辣調味料。山葵地下莖含豐富的機能性成分，如維生素C與A、礦物質、微量元素以及異硫氫酸酯(isothiocyanates：ITCs)的成份。研究顯示山葵主要成分之一6-甲基硫己基異硫氰酸鹽(6-methylsulfinylhexyl isothiocyanate；6-MITC)，具有解毒、抗發炎以及誘導癌細胞凋亡的能力，為一種有效的化學防癌物質。過去有關山葵的研究除了以6-MITC進行化學防癌的研究外，鮮少有文獻以山葵萃取物作為研究對象。因此探討山葵萃取物對大腸癌細胞的抗癌作用及機轉的調控，並評估其開發成抗癌保健食品或藥物的潛力勢必是重要的。毒性分析結果發現，山葵萃取物的確會對Colo 205大腸癌細胞產生毒性。進一步研究發現，山葵萃取物除了可活化TNF-α、Fas-L和caspase 8/caspase3調控細胞凋亡，亦可透過調控Bid、cytochrome c (Cyt c)和caspase 9蛋白誘發粒線體所導致的細胞凋亡機制。此外，我們也發現山葵萃取物會促進自噬作用的相關蛋白表現活化(Beclin-1、Atg5和LC3)，導致細胞產生自噬作用。在利用自噬作用的促進劑RAD001 (mTOR抑制劑)合併山葵萃取物後結果發現，細胞的毒性作用與自噬作用的現象明顯的增加，證實山葵萃取物誘發的自噬作用主要是促進細胞的死亡。最後，利用異種移植動物實驗進一步的驗證，發現山葵萃取物確實亦會降低腸腫瘤的生長速率。綜合以上的結果我們可以知道山葵萃取物可透過促進大腸癌細胞凋亡以及自噬死亡的作用進而達到抑制大腸癌的效果。另外，我們在利用1,2-二甲基聯胺(1,2-dimethylhydrazine；DMH)/葡聚糖硫酸鈉(dextran sodium sulfate；DSS)大腸癌誘發之動物實驗中發現高劑量山葵萃取物(100 mg/kg)可以有效的降低老鼠的死亡率以及腸毒性，並且可以透過促進caspase 3的表現進而導致細胞凋亡現象。這些結果顯示山葵萃取物對於抗大腸癌上面的研究有著更進一步的發展，並且在未來抗大腸癌和抗惡病質的保健食品研發上面也有很大的幫助。
並列摘要
Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide. Thus, new treatment strategies and protections for CRC have become an important focus. Wasabia japonica, usually called wasabi, is used as spicy sauce in Japan and Taiwan. The rhizome of wasabi is rich in functional ingredients, such as vitamin C and A, minerals, trace elements and isothiocyanates (ITCs). Studies showed that one of the major ingredients of wasabi, 6-(methylsulfinyl) hexyl isothiocyanate (6-MITC), possessed the activities of detoxification, anti-inflammation, and inducing apoptosis of cancer cells. Up to the present, most of the studies of wasabi were focused on the function of 6-MITC instead of crude extract, and there is no study reporting the cytotoxicity and mechanism of wasabi against CRC. This study aimed to investigate the molecular mechanism of the anti-cancer of wasabi extract (WE) in colon cancer cells to evaluate the potential of wasabi as a functional food for chemoprevention. We demonstrated that WE could induce a strong cytotoxicity in Colo 205 cells. First, we found that WE induced extrinsic apoptosis and mitochondrial death machinery by extracellular stimuli pathways through activation of TNF-α, Fas-L, caspase 3, caspase 8, caspase 9, cleaved-Bid and release of mitochondrial cytochrome c (Cyt c). Second, we also proved the induction of autophagy by WE via promoting LC3-II in Colo 205 cells. RAD001 (mTOR inhibitor) is an autophagy agonist, indicated the autophagic cell death by increasing AVO level and cytotoxicity in WE-treated Colo 205 cells. These data suggested that autophagy played a pivotal role for the induction of CRC cell death. An in vivo xenograft model verified that tumor growth was delayed by WE treatment. Taken together, our studies revealed that WE exhibits anti-CRC properties through the induction of apoptosis and autophagic cell death. Furthermore, the induction of CRC mice by 1,2-dimethylhydrazine (DMH) and dextran sodium sulfate (DSS) showed that mortality and intestinal toxicity were decreased, and the apoptosis-related protein caspase 3 level was increased in WEH (high dose WE; 100 mg/kg) treatment. These results provide support for the application of WE as a chemopreventive functional food and as a prospective treatment and anti-cachexia of CRC in the future.