頭頸部癌 (Head and neck squamous carcinoma cell , HNSCC) 範圍涵蓋顏面、鼻咽腔、鼻腔、口腔、咽喉及頸部，同時為全球癌症死亡原因排名第六名。當頭頸癌細胞發生轉移會造成癌症難以治療，並造成癌症的復發。過去研究指出HNSCC的Twist過度表現會增加EMT (epithelial-mesenchymal transitions, 上皮-間質轉變) 的作用，抑制E-cadherin，促進β-catenin入核，使癌細胞轉移到別處，EMT不僅賦予腫瘤細胞的轉移性，而且還使這些細胞與增殖及產生耐藥性與癌症幹細胞的特徵是造成頭頸癌難以治癒的原因之一。FaDu為人類的咽喉部鱗狀細胞癌細胞，被選擇的原因是Twist表達非常低。轉染Twist會明顯增加EMT現象，能發現細胞型態有顯著的改變，與原本FaDu細胞大不相同，此細胞為FaDu-pCDH -Twist，在此簡稱為FaDu-Twist。AC-0為樟芝萃取物的主成分之一，在研究上有抗癌效果，能促進癌細胞凋亡，所以本實驗希望能夠用AC-0來抑制FaDu-Twist的生長。實驗結果顯示AC-0會抑制FaDu-Twist對其IC50為7.12 μM ，另外AC-0對FaDu細胞的IC50為24.47 μM。利用西方墨點法可以發現AC-0在5、7.5、10 μM可以使FaDu-Twist促凋亡蛋白增加，如caspase-3和Bax。抗轉移的部分，發現給予AC-0濃度1.25 μM的時候，migration的情況有明顯抑制，且Twist的表現量也有下降，E-cadherin有回升。其中當給予AC-0的時候FaDu-twist細胞內有ROS的產生，並經由預處理NAC後發現細胞存活率恢復，得到ROS可能為造成細胞死亡的重要原因。同時在裸鼠異位移植腫瘤實驗中也發現隨著AC-0濃度提高，FaDu-twist腫瘤也有被抑制的趨勢，並已提前給予AC-0的組別最為明顯。結果表明AC-0確實造成FaDu-twist產生ROS並造成細胞凋亡，並抑制癌細胞轉移。

Head and neck squamous cell carcinoma (HNSCC) is a major global health problem, and the sixth most common cause of cancer-related mortality worldwide. The main cause of mortality may be due to the development of metastases and recurrences that are increasingly resistant to the radiation or chemotherapy. Recent studies have shown that the over-expression of TWIST, a master EMT (epithelial–mesenchymal transition) regulator, promotes metastasis in HNSCC cells. TWIST can inhibit E-cadherin and promote β-catenin into the nucleus, so that cancer cells transferred elsewhere and resistant, even make the cells acquire the characteristics of stem cells. FaDu is human squamous cell carcinoma of the hypopharynx that was studied for ectopic TWIST expression because of its intrinsically low TWIST expression and epithelial phenotype. Transfection of TWIST and over-expression of TWIST-induced EMT in FaDu cells has been shown to alter the morphology of FaDu cells, and those cells referred as FaDu-TWIST cells. AC-0, the main bioactive compound of fermented mycelia of Antrodia camphorat has been identified as a key pharmacological agent. However, whether AC-0 can induce apoptosis in Head and neck squamous cell carcinoma is not yet well studied. The present study explored the anticancer properties of AC-0 against FaDu-TWIST cell lines in vitro and in vivo. Results from in vitro studies demonstrated that AC-0 treatment significantly decreased the cell viability of FaDu (IC50 = 24.47 μM ) and FaDu-TWIST cells (IC50 = 7.12 μM ). In addition to this, AC-0 is able to induce apoptosis, which was evidenced by dysregulation of caspase-3 and Bax expressions in the cells. Furthermore, AC-0 significantly inhibited the cancer cell migration, and restored the E-cadherin expression. This data imply potent anti-metastatic activity of AC-0. In contrast, incubation of FaDu-TWIST cells with AC-0 showed significantly increased generation of reactive oxygen species (ROS). Confirmatory results showed that pretreatment of cells with ROS inhibitor (NAC) resulted significant decrease of cancer cell death that was induced by AC-0. In vivo data further supported that AC-0 treatment significantly decreased the growth of FaDu-TWIST derived tumors development in xenograft athymic nude mice. This study concludes that AC-0 can induce apoptosis and inhibit the metastasis, which can be used in the development of novel anticancer drugs.

縮寫表……………………………...………………………………………IV
中文摘要……………………………………..……………………………VII
Abstract…………………………………………………………………VIII
第一章 前言………………………………………………………………1
第二章 文獻探討………………………………...…………………………3
第一節 樟芝…………………………………………………………….4
第二節 頭頸癌-下咽癌………………….……………………………6
第三節 上皮變間質型的轉換(Epithelial-mesenchymal transition, EMT)……………………………...……………………………10
第四節 細胞凋亡……………………...………………………………12
第五節 Twist與癌細胞的關係………………………………………..15
第三章 研究動機與實驗設計…………………….………………………17
第一節 研究動與假說………………………………………..………18
第二節 實驗設計……………………………………………………….18
第四章 材料與方法……………………….………………………………19
第一節 實驗儀器……………………...………………………………20
第二節 實驗材料……………………………………………………21
第三節 實驗方法………………………...……………………………24
一、 細胞培養 (cell culture)………………………………………24
二、 細胞型態 (morphology)觀察…………..……………………26
三、 細胞存活率 (cell viability)測定……………..………………27
四、 細胞總蛋白質萃取( total protein extraction)……..…………28
五、 蛋白質定量…………………………………………………31
六、 西方墨點法(Western Blotting) ……………..………………33
七、 細胞遷移試驗 (Migration assay) ………………...…………40
八、 ROS(reactive oxygen species)測定……………………41
九、 腫瘤異種移植(Tumor Xenograft Study) ……………………42
第五章 實驗結果與圖表………………….………….…..……………43
第一節 樟芝萃取物(AC-0)對FaDu和FaDu-twist之存活率與生長情形的影響………………………………..…………………………44
第二節 樟芝萃取物(AC-0)對FaDu-twist的轉移之影響…………44
第三節 樟芝萃取物(AC-0)對FaDu-twist凋亡的影響…………45
第四節 樟芝萃取物(AC-0)誘發FaDu-twist細胞活性氧自由基(ROS)的產生……………………………………………………………45
第五節 AC-0抑制裸鼠皮下異位移植FaDu-twist腫瘤的生長...45
第六章 討論……………………….........…………………………………55
第七章 結論……………………………….………………………………58
第八章 參考文獻………………………….…………………………..60

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