|Abstract: ||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.