進階搜尋


 
系統識別號 U0026-0812200915174011
論文名稱(中文) ACSL1在乳癌細胞中所扮演的角色
論文名稱(英文) The Novel Role of Acyl-CoA Synthetase Long-chain Family Member 1 in Breast Cancer
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 皇甫建安
研究生(英文) Chien-an HuangFu
電子信箱 s1696105@mail.ncku.edu.tw
學號 s1696105
學位類別 碩士
語文別 中文
論文頁數 92頁
口試委員 口試委員-張文粲
指導教授-賴明德
口試委員-吳昭良
中文關鍵字 乳癌  內皮生長因子受器  脂質代謝  內質網壓力  長鏈型醯基輔酵素A合成酵素 
英文關鍵字 breast cancer  EGFR  ER stress  ACSL  lipid metabolism 
學科別分類
中文摘要 內質網(endoplasmic reticulum, ER)是真核細胞內最主要負責新合成蛋白質與蛋白質修飾的胞器。當細胞遭受到環境影響,使它無法正確的折疊蛋白質,就會累積許多未摺疊的蛋白質(unfolded proteins),造成內質網壓力(ER stress)。許多研究指出很多癌細胞都會誘導內質網壓力的上升,例如:乳癌與肝癌。此外也有文獻報導指出不正常活化脂質代謝會造成乳癌細胞的發生率上升。但較少研究顯示到底內質網壓力是如何去調控脂質代謝。在我們實驗室學長與學姊的研究發現內質網壓力可調控長鏈型醯基輔酵素A合成酵素3(acyl-CoA synthetase long-chain family member 3, ACSL3),這個酵素為脂質進入代謝的第一個步驟,最主要是將長鏈脂肪酸接上CoA。產生的acyl-CoA可參與生合成新的脂質(anabolic)與將它當作能量消耗掉(catabolic)兩個途徑。ACSL家族共含有5個成員,分別為ACSL1, ACSL3, ACSL4, ACSL5,和ACSL6。我的研究目標則是去觀察乳癌細胞中是否ACSL也同樣扮演重要的角色?首先我們將內質網壓力誘導物處理兩種乳癌細胞,可造成ACSL1的上升與脂質的累積。我們也使用Triacsin C (ACSL1, 3, and 4的抑制劑) 和C75 (脂質合成的抑制劑,inhibitor of Fatty acid synthase)處理細胞,發現Triacsin C可有效的去抑制脂質累積。接著也去利用Triacsin C處理MDA-MB-231細胞,可觀察到Triacsin C會抑制生長與爬行。為了確認ACSL1扮演重要的角色,我們在乳癌細胞中建立了專一降低ACSL1的stable clones。這些細胞株中我們發現細胞生長速度沒有明顯的差異,但細胞爬行則是有明顯的變慢。接著也進一步去分析到底哪些爬行因子受到改變,可觀察到磷酸化的paxillin, 磷酸化的FAK與上皮生長因子接受體(EGFR)皆有下降的情形。由結果告訴我們ACSL1未來可做為目標去治療乳癌細胞。
英文摘要 The endoplasmic reticulum (ER) is a major organelle for folding and maturation of newly synthesized proteins. Excess of unfolded proteins induce ER stress, when cells encounter adverse physiological conditions that impact on protein folding in ER. Several studies have showed that ER stress was induced in various tumors, such as breast tumors and hepatocellular carcinomas. Besides, many evidences indicated that dysregulation of lipid metabolism increases risk of developing cancer. However, relatively little information links ER stress to dysregulation of lipid metabolism. Our previous studies have showed that ER stress can induce lipid accumulation via acyl-CoA synthetase long-chain family member 3 (ACSL3) which catalyzes the first step in fatty acid metabolism by converting long-chain fatty acid into acyl-CoA thioesters and enters both anabolic and catabolic pathways. The family of mammalian ACSLs include five members, ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. Aim of my research is to investigate which ACSL member plays an essential role in breast cancer. Our results showed that ER stress could induce lipid accumulation and expression of ACSL1 in several breast cancer cell lines. Triacsin C (an inhibitor of ACSL1, 3, and 4) repressed lipid accumulation under ER stress. However, C75 (an inhibitor Fatty acid synthase, the rate-limiting step for the biosynthesis) could not repress it. Then, we investigated whether downregulation of ACSLs could impair cell growth and migration in MDA-MB-231 cells. Therefore we established ACSL1 knockdown stable clones in breast cancer cell line. We further studied whether ACSL1 is an essential role in tumorigenesis. We determined cell growth and migration with MTT assay and boyden chamber respectively. The migration rate decreased but growth rate had no significant change in ACSL1 knockdown stable clones. We also analyzed the expression of migration-associated molecules such as phospho-paxillin, phospho-FAK and EGFR. Those molecules were downregulated in stable clones, but lipid accumulation was not attenuated in those cells. Our results implied that ACSL1 will become a therapeutic agent in breast cancer.
論文目次 緒論
一、脂質與腫瘤細胞的相關性 1
二、內質網壓力與癌細胞的相關性 2
三、ACSL在脂質合成中的角色 3
四、研究目標與策略 6
材料與方法
一、細胞培養與藥物處理 7
二、質體製備 12
三、抽取total RNA 15
四、反轉錄聚合連鎖反應 17
五、基因殖入轉染(Transfection)與細胞株挑選 20
六、油紅染色 22
七、細胞生長分析計算 23
八、細胞移動分析 24
九、西方墨點法 26
十、構築FLAG-ACSL3與可對抗shRNA的FLAG-ACSL3質體(附圖實驗)32
十一、免疫組織化學染色法(Immunohistochemistry)(附圖實驗)36
十二、繪圖軟體與統計分析 38
結果
一、內質網壓力下可造成乳癌細胞的脂質累積 39
二、內質網壓力下會造成乳癌細胞ACSL1表現量上升 39
三、利用ACSL抑制劑可使ER stress的脂質累積下降以及惡性乳癌細胞的生長和爬行下降 41
四、乳癌細胞中降低ACSL1的表現可抑制爬行,而生長與細胞型態則沒有太大差異 42
五、降低ACSL1會影響乳癌細胞爬行有關的訊息因子 44
六、降低ACSL1並不會造成乳癌細胞受到刺激下的脂質累積 45
討論 46
結論 52
附圖結果 53
參考文獻 57
圖 60
附圖與附表 77
自述 92
參考文獻 1.Garber K. Energy deregulation: licensing tumors to grow. Science 2006;312:1158-1159.
2.Kuhajda FP. Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 2000;16:202-208.
3.Menendez JA, Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer 2007;7:763-777.
4.Kuhajda FP. Fatty acid synthase and cancer: new application of an old pathway. Cancer Res 2006;66:5977-5980.
5.Menendez JA, Lupu R, Colomer R. Targeting fatty acid synthase: potential for therapeutic intervention in her-2/neu-overexpressing breast cancer. Drug News Perspect 2005;18:375-385.
6.Tong L, Harwood HJ, Jr. Acetyl-coenzyme A carboxylases: versatile targets for drug discovery. J Cell Biochem 2006;99:1476-1488.
7.Migita T, Narita T, Nomura K, Miyagi E, Inazuka F, Matsuura M, Ushijima M, et al. ATP citrate lyase: activation and therapeutic implications in non-small cell lung cancer. Cancer Res 2008;68:8547-8554.
8.Calle EE, Kaaks R. Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms. Nat Rev Cancer 2004;4:579-591.
9.Hardy S, Langelier Y, Prentki M. Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. Cancer Res 2000;60:6353-6358.
10.Hardy S, St-Onge GG, Joly E, Langelier Y, Prentki M. Oleate promotes the proliferation of breast cancer cells via the G protein-coupled receptor GPR40. J Biol Chem 2005;280:13285-13291.
11.Menendez JA, Vellon L, Oza BP, Lupu R. Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia-inducible factor-1alpha (HIF-1alpha)-related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her-2/neu oncogene. J Cell Biochem 2005;94:857-863.
12.Kleizen B, Braakman I. Protein folding and quality control in the endoplasmic reticulum. Curr Opin Cell Biol 2004;16:343-349.
13.Blais JD, Filipenko V, Bi M, Harding HP, Ron D, Koumenis C, Wouters BG, et al. Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol Cell Biol 2004;24:7469-7482.
14.Hochachka PW, Buck LT, Doll CJ, Land SC. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci U S A 1996;93:9493-9498.
15.Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 2004;14:20-28.
16.Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res 2007;67:10631-10634.
17.Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 2004;4:966-977.
18.Aguirre-Ghiso JA, Liu D, Mignatti A, Kovalski K, Ossowski L. Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo. Mol Biol Cell 2001;12:863-879.
19.Ozawa K, Tsukamoto Y, Hori O, Kitao Y, Yanagi H, Stern DM, Ogawa S. Regulation of tumor angiogenesis by oxygen-regulated protein 150, an inducible endoplasmic reticulum chaperone. Cancer Res 2001;61:4206-4213.
20.Breckenridge DG, Germain M, Mathai JP, Nguyen M, Shore GC. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 2003;22:8608-8618.
21.Lee AH, Scapa EF, Cohen DE, Glimcher LH. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 2008;320:1492-1496.
22.Oyadomari S, Harding HP, Zhang Y, Oyadomari M, Ron D. Dephosphorylation of translation initiation factor 2alpha enhances glucose tolerance and attenuates hepatosteatosis in mice. Cell Metab 2008;7:520-532.
23.Soupene E, Kuypers FA. Mammalian long-chain acyl-CoA synthetases. Exp Biol Med (Maywood) 2008;233:507-521.
24.Achouri Y, Hegarty BD, Allanic D, Becard D, Hainault I, Ferre P, Foufelle F. Long chain fatty acyl-CoA synthetase 5 expression is induced by insulin and glucose: involvement of sterol regulatory element-binding protein-1c. Biochimie 2005;87:1149-1155.
25.Fujino T, Kang MJ, Suzuki H, Iijima H, Yamamoto T. Molecular characterization and expression of rat acyl-CoA synthetase 3. J Biol Chem 1996;271:16748-16752.
26.Soupene E, Kuypers FA. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains. BMC Mol Biol 2006;7:21.
27.Mashima T, Sato S, Sugimoto Y, Tsuruo T, Seimiya H. Promotion of glioma cell survival by acyl-CoA synthetase 5 under extracellular acidosis conditions. Oncogene 2009;28:9-19.
28.Lewin TM, Kim JH, Granger DA, Vance JE, Coleman RA. Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently. J Biol Chem 2001;276:24674-24679.
29.Coleman RA, Lewin TM, Van Horn CG, Gonzalez-Baro MR. Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? J Nutr 2002;132:2123-2126.
30.Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005;6:56-68.
31.McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer 2005;5:505-515.
32.Richards MR, Harp JD, Ory DS, Schaffer JE. Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes. J Lipid Res 2006;47:665-672.
33.Kondoh M, Tsukada M, Kuronaga M, Higashimoto M, Takiguchi M, Himeno S, Watanabe Y, et al. Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice. Toxicol Lett 2004;148:133-139.
34.Hansson MD, Rzeznicka K, Rosenback M, Hansson M, Sirijovski N. PCR-mediated deletion of plasmid DNA. Anal Biochem 2008;375:373-375.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2014-07-17起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2014-07-17起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw