- 學位論文
α-次亞麻油酸對SK-Hep-1人類肝癌細胞基質金屬蛋白酶-2/9活化與Twist1調控的上皮間質轉化之影響
Effect of alpha-linolenic acid on matrix metalloproteinase-2/9 activation and Twist1-mediated epithelial-mesenchymal transition in SK-Hep-1 human hepatocellular carcinoma cells
摘要
根據GLOBOCAN 2018全球癌症統計資料報告，肝癌在全球癌症發生率位居第六，每年約有85萬個新增病例。轉移是導致肝細胞癌(hepatocellular carcinoma, HCC) 死亡最常見的因素，死亡率高居癌症死亡第四位。因此，如何降低肝癌轉移成為各國積極探討的主要議題。上皮間質轉化(epithelial-mesenchymal transition, EMT)是上皮細胞轉變成間質細胞的重要步驟，ㄧ旦EMT被活化，腫瘤上皮細胞失去細胞極性和細胞間黏附作用，並進一步促使細胞移行和侵襲。Twist1促癌基因是調節EMT相關基因表現的關鍵轉錄因子，在乳癌、大腸癌、肺癌和肝癌等多種癌細胞中皆發現Twist1大量表現，其已被證實與啟動EMT、導致腫瘤發展和癌症患者預後不佳有關。在眾多轉錄因子中，信號傳導及轉錄激活因子3 (signal transducer and activator of transcription, STAT3)被證實可上調Twist1基因轉錄作用。先前研究顯示omega-3脂肪酸具有抑制癌症進展功效，然而大部分研究多聚焦於二十碳五烯酸(eicosapentaenoic acid, EPA)及二十二碳六烯酸(docosahexaenoic acid, DHA)，鮮少討論關於α-次亞麻油酸(alpha-linolenic acid, ALA)的抗癌效果。因此，本研究以SK-Hep-1人類肝癌細胞為模式，探討ALA對Twist1調控EMT相關蛋白表現及基質金屬蛋白酶(matrix metalloproteinase)活化之機制。結果顯示，ALA抑制肝癌細胞移行及侵襲能力，同時增加E-cadherin表現，但α-SMA、MMP-2、MMP-9的蛋白表現及Twist1、Snail 2和ZEB-1等EMT相關轉錄因子蛋白表現則被ALA下調；Gelatin Zymography分析證實ALA亦可抑制MMP-2、MMP-9酵素活性；細胞核內轉錄因子累積分析結果顯示，ALA處理1小時後可顯著降低STAT3α磷酸化表現，並同時減少STAT3核內累積，處理4小時後，顯著減少Twist1核內累積。以ALA及si-STAT3α RNA靜默處理可顯著減少Twist1及α-SMA蛋白表現；ALA及si-Twist1 RNA 處理亦可抑制α-SMA蛋白表現。綜合上述實驗結果說明ALA下調Twist1蛋白表現是透過減少STAT3α磷酸化及STAT3α核累積，此可進一步抑制α-SMA表現並增加E-cadherin表現。本研究結論說明ALA可透過調控Twist1介導的α-SMA、E-cadherin表現及MMP-2、MMP-9活化，達抑制SK-Hep-1人類肝癌細胞轉移能力。
並列摘要
According to the report from GLOBOCAN 2018, liver cancer is sixth commonly diagnosed cancer around the world, and the number of new cases is approximately 850,000 people annually. Metastasis is the major concern that causes death in hepatocellular carcinoma (HCC), which is the fourth most common cause of cancer-related death worldwide. How to reduce the metastasis of liver cancer has been a major issue in various countries. Epithelial-mesenchymal transition (EMT) is a critical process for the initial transformation from epithelial cells become mesenchymal cells. Upon activation of EMT, tumor epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties in cancer cells. Twist1 oncogene is a crucial transcription factor that regulates the expression of EMT-related gene. High level of Twist1 expression can be found in a variety of cancer cells, including breast cancer, colorectal cancer, lung cancer and liver cancer. Overexpression of Twist1 has been demonstrated to be associated with the initiation of EMT program, which contributes to tumor progression and a poor prognosis of cancer patients. Moreover, signal transducer and activator of transcription (STAT3) was found to up-regulate Twist1 gene transcription. Previous studies showed that omega-3 fatty acids was observed to inhibit cancer progression, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), however, the anticancer activity of alpha-linolenic acid (ALA) is still unclear. Therefore, the aim of this study is to investigate the mechanism of α-linolenic acid on Twist1 regulating EMT-related protein expression and matrix metalloproteinase (MMP) activation in SK-Hep-1 human liver cancer cells. Wound-healing assay and boyden chamber assay demonstrated that ALA significantly inhibited cell migration and invasion. Results from Western blotting showed that ALA significantly increased E-cadherin and decreased α-SMA, MMP-2 and MMP-9 protein expression, and the level of EMT-related transcription factors, such as Twist1, Snail 2 and ZEB-1 were decreased as well. Gelatin zymography results showed ALA reduced MMP-2 and MMP-9 enzyme activity. Results of nuclear accumulation of transcription factors showed treatment with ALA for 1 hour significantly inhibited the phosphorylation of STAT3α, simultaneously reduced the accumulation of STAT3α in nucleus. Moreover, the nuclear accumulation of Twist1 was significantly decreased after treatment with ALA for 4 hours. Treatment with ALA and si-STAT3α RNA significantly reduced Twist1 and α-SMA protein expression. Treatment with ALA and si-Twist1 RNA, the protein expression of α-SMA protein inhibited as well. These results suggest that ALA downregulates Twist1 protein expression through inhibition of phosphorylation of STAT3α, and also directly reduces nuclear accumulation of STAT3α, which subsequently decreased α-SMA and increased E-cadherin protein expression. Moreover, MMP-2 and MMP-9 protein expression and enzyme activity were decreased by ALA treatment. In conclusion, ALA inhibited SK-Hep-1 human liver cancer cell metastasis via modulation of Twist1-mediated α-SMA and E-cadherin protein expression as well as MMP-2/-9 activation.
參考文獻
延伸閱讀
- 劉淑惠（2016）。次亞麻油酸對調控Twist1表現和Twist1啟動Hs578T人類三陰性乳腺癌細胞間質化和轉移之研究〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0003-0908201611310900
- Kao, Y. W. (2010). 基質金屬蛋白酶-7最小功能片段(6 kDa)參與調控αvβ3整合蛋白在膜上富含膽固醇硫酸鹽微域的胞吞作用之研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2010.10408
- 鄭方茹（2013）。二十二碳六烯酸對表皮生長因子誘發SK-BR3人類乳腺癌細胞尿激酶型血纖維蛋白溶解酶原活化因子及基質金屬蛋白酶-1/-9表現機制之探討〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2013.00082
- 吳沛樺（2019）。吲哚胺2,3-雙加氧酶活化多環芳香烴感受器路徑和β-連環蛋白訊息傳遞路徑促進肝細胞癌惡化〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201903174
- Liu, Y., Wang, J., Xia, Y., Mu, J., Sun, J., & Wang, J. (2017). Inhibitory Effect of Polypeptide from Argopecten irradias on Mice with Transplanted H_(22) Hepatocarcinoma and Its Mechanism. Advance Journal of Food Science and Technology, 13(2), 59-67. https://doi.org/10.19026/ajfst.13.3766