進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-0502201310052700
論文名稱(中文) 研究超表現γ-穀氨醯水解酶誘發的葉酸缺乏對細胞的影響
論文名稱(英文) Studies on the cellular responses to the folate deficiency induced by γ-glutamyl hydrolase overexpression
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 1
出版年 102
研究生(中文) 王姿雅
研究生(英文) Tzu-Ya Wang
學號 t36991058
學位類別 碩士
語文別 英文
論文頁數 56頁
口試委員 指導教授-傅子芳
口試委員-蔡振寧
召集委員-徐麗君
中文關鍵字 葉酸  穀氨醯水解酶  細胞爬行 
英文關鍵字 folate  γ-glutamylhydrolase  cell migration 
學科別分類
中文摘要 先前已有結果顯示,葉酸的多寡和癌症的進程有很大的關係,包含原位癌或是轉移。然而,對於葉酸在影響細胞的爬行這方面其機制還不是非常清楚。我們的目標是在研究在葉酸缺乏時,細胞機能到底是如何被影響的,尤其在細胞的附著與爬行能力這方面。為了達成我們的目標,我們利用超表現γ-穀氨醯水解酶(γ-glutamyl hydrolase, GGH)在具有高度爬行能力的細胞株(老鼠黑色素瘤細胞)上。GGH是位於溶體內的半胱胺酸肽酶,主要是去水解多谷胺酸鏈的葉酸至單谷胺酸的葉酸,而單谷胺酸葉酸較容易被細胞吸收和送出細胞膜外。利用微生物學的方式去偵測細胞內的葉酸含量,我們發現藉由在細胞內超表現γ-穀氨醯水解酶可以降低細胞內約40%的葉酸總量,降地細胞內的葉酸量影響了細胞週期及阻止細胞的生長能力,但卻不會造成細胞凋亡。而且降低細胞含量還會抑制其爬行的能力以及降低細胞爬行標的分子的蛋白質表現量,包含N-cadherin, vimentin和twist。在動物模式上我們發現超表現γ-穀氨醯水解酶會降低斑馬魚的N-cadherin表現量,還有延遲了epiboly的發育。另外,在人類肺腺癌細胞株上我們意外的發現,超表現γ-穀氨醯水解酶反而促進了細胞爬行的能力,而且提升了間質細胞標的分子的蛋白表現量包含α-SMA 和twist,而上皮細胞標的分子E-cadherin的表現量則是下降。從我們的結果顯示出,超表現γ-穀氨醯水解酶導致細胞內葉酸缺乏而且改變了黑色素瘤細胞的爬行能力,其可能是藉由調控一些分子的表現進而影響細胞的附著與移動。
英文摘要 Folate status has been shown to affect cancer progression, including in situ neoplasia and metastasis. However, the effect of folate on cell migration and its underlying mechanism remains unclear. The aim of this study is to investigate how folate deficiency affects cell physiology, especially adhesion and migration. To achieve our goal, we overexpress γ-glutamyl hydrolase (GGH) in B16F10 cell line, a highly mobile melanoma cell line. GGH, a lysosomal cysteine peptidase, hydrolyzes polyglutamyl folates to monoglutamyl folates which are more efficiently absorbed and exported through cell membrane. With microbiological assay, we show that transiently overexpressing GGH decreases 40% of intracellular total folate. Decrease in intracellular folate level affect cell cycle and retard cell growth but not cause apoptosis. Decreased folate content inhibits cell mobility and decreases the expression of several migration markers, N-cadherin, vimentin, and twist. In vivo studies show that GGH-overexpressing decreases zebrafish N-cadherin expression and leads to epiboly delay. Unexpectedly, the migration ability in A549 cells is opposite to that in B16F10 cells. Overexpression GGH in A549 increases the expression of mesenchymal markers, such as α-SMA and twist but decreases epithelial marker, E-cadherin. These results suggest that GGH overexpression cause folate deficiency and alter the mobility of B16F10 melanoma cells possibly via modulating the expression of molecules involving in adhesion and migration.
論文目次 摘要 I
Abstract II
Acknowledgement III
Abbreviations IV
Contents V
Contents of Figures VIII
I. Introduction 1
1.1 Folate 1
1.2 Folate metabolism and role of folate 1
1.3 Folate deficiency 2
1.4 Folate deficiency and cancer progression 3
1.5 γ-Glutamyl hydrolase (GGH) 4
1.6 Zebrafish (Danio rerio) 5
II. Specific aim 7
III. Materials and methods 8
3.1 Cell culture 8
3.2 Plasmids 8
3.3 Transfection 9
3.4 Electroporation 9
3.5 Intracellular folate content measurement by microbiology assay 10
3.5.1. Sample collection 10
3.5.2. Sample conversion 11
3.5.3. Sample folate measurement 11
3.6 Cell growth assay 11
3.7 Cell cycle analysis 12
3.8 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay 12
3.9 Cell adhesion strength measurement 13
3.10 Cell migration assay 13
3.10.1. Wound healing assay 13
3.10.2. Transwell assay 14
3.11. Western blot 14
3.12 RNA extraction and RT-PCR 15
3.12.1 Total RNA extraction 15
3.12.2 Reverse transcription 16
3.13 Zymography assay 17
3.14 Zebrafish maintenance and transgenic fish 17
3.15 Transient microinjection in zebrafish embryos 18
3.16 Statistical anaylsis 18
IV. Results 19
4.1 GGH-overexpression in B16F10 resulted in folate deficiency. 19
4.2 GGH-overexpression effected cell growth and cell cycle of B16F10 but did not cause apoptosis and autophagy. 19
4.3 GGH-overexpression enhanced cell adhesion and inhibited cell migration. 20
4.4. GGH-overexpression altered expression of adhesion molecules, cytoskeleton and transcription factor. 21
4.5 GGH-overexpression decreased matrix metalloproteinases(MMPs) enzyme activity. 21
4.6 GGH-overexpression did not affect actin polymerization. 21
4.7 GGH-overexpression in zebrafish embryos resulted in abnormal development and altered embryonic adhesion molecule. 21
4.8 GGH-overexpression altered adhesion molecules expression and promoted migration in A549. 22
4.9 GGH-overexpression cause A549 autophagy. 22
V. Discussion 24
5.1 The strategies to induce intracellular folate deficiency. 24
5.2 The possible pathway to regulate B16F10 migration ability as GGH overexpress. 25
5.3 The expression pattern of migration-related markers 26
5.4 GGH-overexpression affect the cell cycle but not cell apoptosis and autophagy. 26
5.5 GGH-overexpression resulted in different cellular responses in B16F10 and A549. 27
VI. Future experiments 28
VII. References 29
VIII. Figures 33
IX. Appendices 49
參考文獻 1. Branda, R.F., et al., Effects of folate deficiency on the metastatic potential of murine melanoma cells. Cancer Res, 1988. 48(16): p. 4529-34.
2. Birn, H., The kidney in vitamin B12 and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins. Am J Physiol Renal Physiol, 2006. 291(1): p. F22-36.
3. Barry, S., Folate Chemistry and Metabolism, in Folate in Health and Disease, Second Edition. 2009, CRC Press. p. 1-24.
4. Schirch, V. and W.B. Strong, Interaction of folylpolyglutamates with enzymes in one-carbon metabolism. Archives of Biochemistry and Biophysics, 1989. 269(2): p. 371-380.
5. Allegra, C.J., B.A. Chabner, and J.C. Drake, Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates. Journal of Biological Chemistry, 1985. 260(17): p. 9720-9726.
6. Yao, R., et al., Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro. Proceedings of the National Academy of Sciences, 1996. 93(19): p. 10134-10138.
7. Miller, J.W., et al., Folate, DNA methylation, and mouse models of breast tumorigenesis. Nutrition Reviews, 2008. 66: p. S59-S64.
8. Fox, J.T. and P.J. Stover, Chapter 1 Folate‐Mediated One‐Carbon Metabolism. 2008. 79: p. 1-44.
9. O'Brien, M.M., et al., The North/South Ireland Food Consumption Survey: vitamin intakes in 18-64-year-old adults. Public Health Nutr, 2001. 4(5A): p. 1069-1079.
10. Iyer, R. and S.K. Tomar, Folate: A Functional Food Constituent. Journal of Food Science, 2009. 74(9): p. R114-R122.
11. Intakes, A.R.o.t.S.C.o.t.S.E.o.D.R., et al., Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. 1998: The National Academies Press.
12. Halsted, C.H., et al., Metabolic Interactions of Alcohol and Folate. The Journal of Nutrition, 2002. 132(8): p. 2367S-2372S.
13. Cornelia, U., Genetic Variability in Folate-Mediated One-Carbon Metabolism and Cancer Risk, in Nutrient-Gene Interactions in Cancer. 2006, CRC Press. p. 75-91.
14. Blount, B.C., et al., Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implications for cancer and neuronal damage. Proceedings of the National Academy of Sciences, 1997. 94(7): p. 3290-3295.
15. Jacob, R.A., et al., Moderate Folate Depletion Increases Plasma Homocysteine and Decreases Lymphocyte DNA Methylation in Postmenopausal Women. The Journal of Nutrition, 1998. 128(7): p. 1204-1212.
16. Brattstrom, L. and D.E. Wilcken, Homocysteine and cardiovascular disease: cause or effect? Am J Clin Nutr, 2000. 72(2): p. 315-23.
17. Kruman, II, et al., Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease. J Neurosci, 2002. 22(5): p. 1752-62.
18. Duan, W., et al., Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease. Journal of Neurochemistry, 2002. 80(1): p. 101-110.
19. Le Leu, R.K., G.P. Young, and G.H. McIntosh, Folate deficiency reduces the development of colorectal cancer in rats. Carcinogenesis, 2000. 21(12): p. 2261-2265.
20. Bistulfi, G., et al., Mild folate deficiency induces genetic and epigenetic instability and phenotype changes in prostate cancer cells. BMC Biol, 2010. 8: p. 6.
21. Kim, Y.I., Will mandatory folic acid fortification prevent or promote cancer? Am J Clin Nutr, 2004. 80(5): p. 1123-8.
22. Wang, T.P., et al., Folate deprivation enhances invasiveness of human colon cancer cells mediated by activation of sonic hedgehog signaling through promoter hypomethylation and cross action with transcription nuclear factor-kappa B pathway. Carcinogenesis, 2012. 33(6): p. 1158-68.
23. Koury, M.J., et al., Folate deficiency delays the onset but increases the incidence of leukemia in Friend virus-infected mice. Blood, 1997. 90(10): p. 4054-61.
24. Hayashi, I., et al., Folate deficiency induces cell-specific changes in the steady-state transcript levels of genes involved in folate metabolism and 1-carbon transfer reactions in human colonic epithelial cells. J Nutr, 2007. 137(3): p. 607-13.
25. Fiskerstrand, T., P.M. Ueland, and H. Refsum, Folate depletion induced by methotrexate affects methionine synthase activity and its susceptibility to inactivation by nitrous oxide. J Pharmacol Exp Ther, 1997. 282(3): p. 1305-11.
26. Wang, X., et al., Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. The FASEB Journal, 2012.
27. Yao, R., et al., Identification, Cloning, and Sequencing of a cDNA Coding for Rat -Glutamyl Hydrolase. Journal of Biological Chemistry, 1996. 271(15): p. 8525-8528.
28. Esaki, T., et al., Cloning of mouse gamma-glutamyl hydrolase in the form of two cDNA variants with different 5' ends and encoding alternate leader peptide sequences. Gene, 1998. 219(1-2): p. 37-44.
29. Kao, T.-T., et al., Recombinant Zebrafish γ-Glutamyl Hydrolase Exhibits Properties and Catalytic Activities Comparable with Those of Mammalian Enzyme. Drug Metabolism and Disposition, 2009. 37(2): p. 302-309.
30. Huangpu, J., et al., Purification and Molecular Analysis of an Extracellular γ-Glutamyl Hydrolase Present in Young Tissues of the Soybean Plant. Biochemical and Biophysical Research Communications, 1996. 228(1): p. 1-6.
31. Chave, K.J., et al., Molecular Modeling and Site-directed Mutagenesis Define the Catalytic Motif in Human γ-Glutamyl Hydrolase. Journal of Biological Chemistry, 2000. 275(51): p. 40365-40370.
32. Chave, K.J., J. Galivan, and T.J. Ryan, Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human gamma-glutamyl hydrolase. Biochem. J., 1999. 343(3): p. 551-555.
33. Galivan, J., et al., Glutamyl hydrolase. pharmacological role and enzymatic characterization. Pharmacol Ther, 2000. 85(3): p. 207-15.
34. Baggott, J.E., et al., Folate conjugase activity in the plasma and tumors of breast-cancer patients. Am J Clin Nutr, 1987. 46(2): p. 295-301.
35. Schneider, E. and T.J. Ryan, Gamma-glutamyl hydrolase and drug resistance. Clinica Chimica Acta, 2006. 374(1–2): p. 25-32.
36. Cole, P.D., et al., Effects of Overexpression of {{gamma}}-Glutamyl Hydrolase on Methotrexate Metabolism and Resistance. Cancer Res, 2001. 61(11): p. 4599-4604.
37. McGuire, J.J., Anticancer antifolates: current status and future directions. Curr Pharm Des, 2003. 9(31): p. 2593-613.
38. Kim, S., et al., EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line. Cellular Signalling, 2009. 21(6): p. 892-898.
39. Kuo, L., et al., Src oncogene activates MMP-2 expression via the ERK/Sp1 pathway. J Cell Physiol, 2006. 207(3): p. 729-34.
40. Lopez-Bergami, P., et al., Rewired ERK-JNK signaling pathways in melanoma. Cancer Cell, 2007. 11(5): p. 447-60.
41. Huntington, J.T., et al., Overexpression of Collagenase 1 (MMP-1) Is Mediated by the ERK Pathway in Invasive Melanoma Cells: ROLE OF BRAF MUTATION AND FIBROBLAST GROWTH FACTOR SIGNALING. Journal of Biological Chemistry, 2004. 279(32): p. 33168-33176.
42. Weiss, M.B., et al., TWIST1 Is an ERK1/2 Effector That Promotes Invasion and Regulates MMP-1 Expression in Human Melanoma Cells. Cancer Research, 2012. 72(24): p. 6382-6392.
43. Chuderland, D. and R. Seger, Calcium regulates ERK signaling by modulating its protein-protein interactions. Commun Integr Biol, 2008. 1(1): p. 4-5.
44. Suzuki, K., et al., Structure, Activation, and Biology of Calpain. Diabetes, 2004. 53(suppl 1): p. S12-S18.
45. Mayanil, C.S., et al., Maternal intake of folic acid and neural crest stem cells. Vitam Horm, 2011. 87: p. 143-73.
46. Chi, D.D., et al., Molecular detection of tumor-associated antigens shared by human cutaneous melanomas and gliomas. Am J Pathol, 1997. 150(6): p. 2143-52.
47. Lal, S., et al., Calpain 2 is required for the invasion of glioblastoma cells in the zebrafish brain microenvironment. J Neurosci Res, 2012. 90(4): p. 769-81.
48. Gottardi, C.J., et al., The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc Natl Acad Sci U S A, 1996. 93(20): p. 10779-84.
49. Li, C.X. and M.J. Poznansky, Characterization of the ZO-1 protein in endothelial and other cell lines. J Cell Sci, 1990. 97 ( Pt 2): p. 231-7.
50. Blakley, R.L., The biochemistry of folic acid and related pteridines. 1969, Amsterdam: North-Holland Publishing Co.
51. Oppeneer, S.J., et al., Genetic variation in folylpolyglutamate synthase and gamma-glutamyl hydrolase and plasma homocysteine levels in the Singapore Chinese Health Study. Molecular Genetics and Metabolism, 2012. 105(1): p. 73-78.
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2023-01-22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