頭頸癌因容易早期轉移、對化學放射治療具抗性，使患者五年存活率低於50%，其中患者癌組織之NME1 (NME/NM23 nucleoside diphosphate kinase 1; Nm23-H1) 表現量與預後呈正相關性；以口腔鱗狀上皮癌SAS細胞建立NME1低表現量之穩定轉殖株 (SASshRNAnm23) 與其控制組 (SASshRNA)，證實NME1可抑制癌細胞遷徙及提升化學放射治療敏感性，並降低對糖解作用之依賴。本研究目的為評估水產活性成分抑制頭頸癌惡化之潛力並探討可能的作用機制。
本研究先將臺灣蜆 (Corbicula fluminea) 以乙醇萃取脂溶性成分，利用氣相層析儀 (gas chromatography) 分析其脂肪酸組成發現含有eicosapentaenoic acid (EPA, 20:5; 1.86%) 及docosahexaenoic acid (DHA, 22:6; 1.42%)。首先以人類肺纖維母細胞為正常細胞模式，確認EPA或DHA (0~100 μM) 處理48小時之細胞存活率高於80%。再將兩株SAS細胞分別處理EPA或DHA於24~72小時以trypan blue exclusion assay觀察細胞存活率與輔助化學治療之效果，並以流式細胞儀分析細胞週期變化、以西方墨點法分析細胞週期調節蛋白之表現。脂質代謝重整現象則以細胞中性脂質含量、細胞之脂肪酸合成酶 (fatty acid synthase, FAS) 活性及細胞內脂肪酸組成變化為指標。癌症幹細胞 (cancer stem cells) 特徵則以球體形成能力 (sphere formation) 及乙醛去氫酶 (aldehyde dehydrogenase, ALDH) 活性為指標。
結果發現以EPA或DHA (0~100 μM)處理SASshRNAnm23及SASshRNA細胞24、48與72小時，可抑制細胞存活率並呈現濃度效應。同時，2 µM cisplatin與3~6 µM EPA或DHA共處理可降低SASshRNAnm23之細胞存活率與SASshRNA組相近，說明EPA或DHA可提升SASshRNAnm23細胞之化學治療效果，並增加細胞週期S期 (33.7%±0.9%增至35.0%±1.5%) 及G2M期 (58.4%±2.3%增至61.1%±1.6%)、促進細胞週期蛋白B (cyclin B)及週期蛋白依賴激酶2 (cyclin dependent kinase 2，CDK2)表現量 (SASshRNAnm23經1 µM DHA處理其cyclin B及CDK2為SASshRNA之1.8及1.5倍)，因此EPA或DHA可能經由阻滯細胞週期而提升SASshRNAnm23細胞化療敏感性。同時，SASshRNAnm23之中性脂質含量顯著高於SASshRNA，但其FAS活性無顯著差異。此外，SASshRNAnm23之球體形成與ALDH活性皆高於SASshRNA，而處理6及12 µM EPA或DHA均可降低兩株細胞之球體數量及ALDH活性。
綜合上述，EPA或DHA透過調節細胞週期及幹細胞特徵以抑制SAS細胞增生、提升低NME1細胞之化療敏感性，因此EPA及DHA為化療抗性頭頸癌之潛力輔助治療成分。

Owing to early metastasis and poor response to chemo-radiation, overall 5 year survival rates in Head and neck squamous cell carcinoma (HNSCC) have not improved significantly. NME1 (NME/NM23 nucleoside diphosphate kinase 1; Nm23-H1) expression is positively associated with clinical prognosis of OSCC patients. By the established in vitro models of stable clones of NME1-knockdown (SASshRNANnm23) and NME1-knockdown control (SASshRNA) cells which derived from a primary oral squamous cell carcinoma SAS cells, previous studies revealed that NME1 inhibit cell mobility, improve chemosensetivity and reduce dependence of glycolysis. This study aimed to evaluate the therapeutic potential of aquatic bioactive component on cancer progression of HNSCC, and to investigate the anti-proliferative mechanisms.
Lipid-soluble components of Corbicula fluminea was obtained by ethanol-extracted muscles of fresh water clam, which contain eicosapentaenoic acid (EPA, 20:5; 1.86%) or docosahexaenoic acid (DHA, 22:6; 1.42%). Fristly, human primary lung fibroblasts were use to evaluate the cytotoxicity of EPA or DHA on human normal cells. The viability of fibroblasts was above 80% after treatment with EPA or DHA (0~100 μM) for 48 hr. The chemosensitivity of SAS cells were observed by trypan blue exclusion assay and cell cycle analysis. The changes of cyclins were measured by Western blotting. Sphere formation and aldehyde dehydrogenase (ALDH) were examined as markers of cancer stemness. Lipid metabolism reprogramming was evaluated by neutral lipid content, fatty acid synthase (FAS) activity, and fatty acid profile of cells.
Treatment with EPA or DHA was resulted in declined cell viability in SASshRNAnm23 and SASshRNA cells in a concentration dependent manner. Besides, co-treatment cisplatin (2 µM) with EPA or DHA (3~6 µM) significantly decreased cell viability, and increased cell population of S phase (33.7%±0.9% to 35.0%±1.5%) and G2M phase (58.4%±2.3% to 61.1%±1.6%), as well as elevated expression levels of cyclin B and cyclin dependent kinase 2 (CDK2) (1.8- and 1.5-folds of SASshRNA after cisplatin treatment in 1 µM DHA group) in SASshRNAnm23 cells. These results imply that EPA/DHA regulate cell cycle rendering chemosensitivity of SASshRNAnm23. Moreover, neutral lipid content of SASshRNAnm23 is significantly higher than SASshRNA. However, the FAS activity has no difference in SASshRNA and SASshRNAnm23 cells. Additionally, sphere formation and ALDH activity are significantly augmented in SASshRNAnm23; EPA or DHA (6~12 µM) inhibited sphere formation and decreased ALDH activity in SASshRNA and SASshRNAnm23 cells.
Taken together, EPA or DHA inhibit cell proliferation and increase cisplatin sensitivity by regulation of cell cycle and stemness properties in NME1 knockdown SAS cells. Therefore, EPA and DHA are potent adjuvant therapeutic agents in chemoresistant HNC.

第一章 前言 6
第二章 文獻回顧 7
第一節、癌症惡化特徵 7
第二節、頭頸癌 9
第三節、NME1基因 11
第四節、臺灣蜆之生理活性 12
第五節、多元不飽和脂肪酸Omega-3與Omega-6 13
第六節、EPA及DHA之生理活性 14
第三章、研究目的 16
第四章、研究設計 17
第一節、評估蜆肉脂溶性萃取物及EPA或DHA對SAS細胞生長的影響 18
第二節、探討EPA或DHA作用於SAS細胞化療抗性影響 18
第三節、探討EPA或DHA對SAS細胞癌症幹細胞特性之影響 18
第四節、探討EPA或DHA對SAS細胞內生性脂肪酸生成之影響 18
第五章 研究方法 19
第一節、蜆肉脂溶性物質之製備 19
第二節、細胞來源 19
第三節、錐蟲藍排除法(trypan blue exclusion assay) 20
第四節、MTT assay 21
第五節、分析細胞週期 (cell cycle analysis) 21
第六節、球體形成 21
第七節、ALDH 活性測定 22
第八節、細胞內中性脂質含量測定 22
第九節、脂溶性物質萃取與脂肪酸衍生化 23
第十節、脂肪酸氣相層析與鑑定 23
第十一節、脂肪酸合成酶(fatty acid synthase, FAS)活性測定 23
第十二節、細胞蛋白質萃取及定量 24
第十三節、西方墨點法(Western blotting) 25
第十四節、統計分析 26
第六章 結果 27
第一節、評估食物成分應用於具有化療抗性頭頸癌之潛力 27
第二節、EPA或DHA於頭頸癌輔助治療之潛力 27
第三節、EPA或DHA調整頭頸癌細胞脂肪酸之生合成 29
第四節、EPA或DHA對頭頸癌癌症幹細胞之影響 30
第七章 討論 32
第一節、蜆肉脂溶性萃取物、EPA或DHA抑制SAS細胞增生 32
第二節、EPA或DHA提升NME1低表現之SAS細胞化療敏感性 32
第三節、EPA或DHA增加SAS細胞內脂質之含量 33
第四節、EPA及DHA抑制SAS細胞癌症幹細胞特徵 35
第八章 結論 36
參考文獻 37
圖
圖一、EPA、DHA抑制SAS細胞之存活 45
圖二、EPA或DHA抑制SASSHRNA及SASshRNAnm23細胞存活 46
圖三、EPA或DHA作用36小時提升低NME1細胞之化療敏感性 47
圖四、EPA或DHA作用48小時提升低NME1細胞之化療敏感性 48
圖五、EPA或DHA增加cisplatin處理下SASshRNA細胞之週期蛋白cyclin A表現量 49
圖六、DHA增加cisplatin處理下SASshRNA細胞週期蛋白cyclin B及NME1表現量 50
圖七、DHA促進低NME1細胞cisplatin處理下之週期蛋白cyclin B及CDK2表現量，但對NME1表現量無影響 51
圖八、EPA增加SASshRNAnm23細胞內中性脂質之含量 52
圖九、EPA或DHA對SASshRNA和SASshRNAnm23細胞中的飽和脂肪酸組成之影響 53
圖十、 EPA或DHA對SASshRNA和SASshRNAnm23細胞中不飽和脂肪酸組成之影響 54
圖十一、 EPA或DHA對SASshRNA和SASshRNAnm23細胞中omega-3脂肪酸組成之影響 55
圖十二、EPA或DHA對SASshRNA和SASshRNAnm23細胞中FAS活性之影響 56
圖十三、EPA或DHA抑制SASshRNA和SASshRNAnm23細胞之球體形成能力 57
圖十四、EPA或DHA抑制SASshRNAnm23細胞ALDH活性 58
表
表一、EPA或DHA處理36小時阻滯低NME1細胞之細胞週期於S及G2M期 59
表二、EPA或DHA處理48小時阻滯低NME1細胞之細胞週期於G2M期 60
表三、EPA對SASshRNA及SASshRNAnm23細胞內脂肪酸組成比例之影響 61
表四、DHA對SASshRNA及SASshRNAnm23細胞內脂肪酸組成比例之影響 62
附錄
附錄一、食物成分對SAS細胞存活之影響 63
附錄二、蜆肉固醇萃取物及脂肪酸萃取物抑制SAS細胞之存活 64
附錄三、顯微鏡觀察EPA、DHA對SAS細胞球體生成能力之影響 65
附錄四、1 µM EPA或DHA作用下SASshRNA和SASshRNAnm23細胞脂肪酸分析之GC圖譜 66
附錄五、10 µM EPA或DHA作用下SASshRNA和SASshRNAnm23細胞脂肪酸分析之GC圖譜 67
附錄六、100 µM EPA或DHA作用下SASshRNA和SASshRNAnm23細胞脂肪酸分析之GC圖譜 68
附錄七、植物固醇β-Sitosterol或stigmasterol之抑癌作用 69

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