唾液酸轉移酶主要是催化唾液酸鍵結到細胞表面醣蛋白或醣脂質上醣質部分的末端產生所謂唾液酸化的反應，在癌症中常過度表現並伴隨癌症的轉移。目前為止的唾液酸轉移酶抑制劑雖然在體外有良好抑制唾液酸轉移酶的效果但並不適用於臨床上的應用，主要是因為在細胞膜穿透率相當的差。在本論文中我們與中研院合作利用lithocholic acid-based的唾液酸轉移酶抑制劑AL10並測試其抗轉移的效果。在高度轉移的A549與CL1-5肺癌細胞株可以發現過度表現α-2,3-唾液酸轉移酶。利用共軛焦顯微鏡顯示AL10具有良好的細胞膜穿透性且有效降低肺癌細胞的唾液酸化。AL10沒有顯著細胞毒性並可明顯在體外抑制肺癌細胞的貼附、遷移、肌動蛋白的聚合以及侵犯。藉由AL10抑制貼附與遷移是伴隨抑制beta1 integrin的唾液酸化。此外beta1 integrin下游活化的訊息分子FAK也同樣被抑制。更重要的是，AL10在in vivo中抑制肺癌細胞的轉移，而此效果可能是藉由抑制趨化受體CXCR4的唾液酸化，其在特定器官的轉移上扮演重要的角色。血清生化檢驗也顯示AL10不會造成任何肝臟與腎臟功能上明顯的改變。綜合以上的實驗結果顯示在in vivo中AL10是相當有效的唾液酸轉移酶抑制劑並經由抑制beta1 integrin與CXCR4的唾液酸化來具抗轉移的效果。

Sialyltransferases (STs), which catalyze the sialylation reaction by
adding sialic acids to the terminal positions of oligosaccharide of
glycoproteins and glycolipids, are over-expressed in cancer cells
and associated with cancer metastasis. Until now, ST inhibitors
are not applicable for clinical use because of poor cell
permeability, although showing potent effect in vitro. In this study,
we synthesize a lithocholic acid-based ST inhibitor AL10 and test
its anti-metastatic effect. Overexpression of α-2,3-ST is found in
highly metastatic A549 and CL1-5 lung cancer cells. Confocal
microscopy demonstrates that AL10 is cell permeable and may
attenuate total sialylation on cell surface. AL10 has no cytotoxicity
but inhibits adhesion, migration, actin polymerization and invasion
of A549 and CL1-5 cells in vitro. Inhibition of adhesion and
migration by AL10 is associated with reduced sialylation of beta1
integrin. In addition, activation of the beta1 integrin downstream
signaling molecule focal adhesion kinase is also attenuated. More
importantly, AL10 suppresses lung metastasis in vivo and this
effect may be linked with reduced sialylation of the chemokine
receptor CXCR4 which has been found to play a critical role in
organ-specific metastasis. Serum biochemical assay indicates that
AL10 does not affect liver and kidney functions of experimental
animals. Taken together, we conclude that AL10 is an effective
sialyltransferase inhibitor and exerts anti-metastatic effect in vivo
via suppression of sialylation of beta1 integrin and CXCR4.

縮寫語------------------------------4
中文摘要----------------------------5
英文摘要----------------------------6
前言--------------------------------7
研究目的---------------------------11
實驗材料---------------------------12
實驗方法及步驟---------------------14
實驗結果---------------------------25
討論-------------------------------39
結論圖表---------------------------46
附圖-------------------------------47
參考文獻---------------------------54

1. Fuster MM, Esko JD. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 2005;5(7):526-42.
2. Varki A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 2007;446(7139):1023-9.
3. Harduin-Lepers A, Mollicone R, Delannoy P, Oriol R. The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology 2005;15(8):805-17.
4. Harduin-Lepers A, Recchi MA, Delannoy P. 1994, the year of sialyltransferases. Glycobiology 1995;5(8):741-58.
5. Tsuji S. Molecular cloning and functional analysis of sialyltransferases. J Biochem 1996;120(1):1-13.
6. Schauer R. Achievements and challenges of sialic acid research. Glycoconj J 2000;17(7-9):485-99.
7. Ashwell G, Morell AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol Relat Areas Mol Biol 1974;41(0):99-128.
8. Jancik J, Schauer R. Sialic acid--a determinant of the life-time of rabbit erythrocytes. Hoppe Seylers Z Physiol Chem 1974;355(4):395-400.
9. Bratosin D, Mazurier J, Tissier JP, et al. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie 1998;80(2):173-95.
10. Durocher JR, Payne RC, Conrad ME. Role of sialic acid in erythrocyte survival. Blood 1975;45(1):11-20.
11. Smith H. Questions about the behaviour of bacterial pathogens in vivo. Philos Trans R Soc Lond B Biol Sci 2000;355(1397):551-64.
12. Corfield T. Bacterial sialidases--roles in pathogenicity and nutrition. Glycobiology 1992;2(6):509-21.
13. Kelm S, Schauer R. Sialic acids in molecular and cellular interactions. Int Rev Cytol 1997;175:137-240.
14. Hedlund M, Ng E, Varki A, Varki NM. alpha 2-6-Linked sialic acids on N-glycans modulate carcinoma differentiation in vivo. Cancer Res 2008;68(2):388-94.
15. Dall'Olio F, Malagolini N, di Stefano G, Minni F, Marrano D, Serafini-Cessi F. Increased CMP-NeuAc:Gal beta 1,4GlcNAc-R alpha 2,6 sialyltransferase activity in human colorectal cancer tissues. Int J Cancer 1989;44(3):434-9.
16. Sata T, Roth J, Zuber C, Stamm B, Heitz PU. Expression of alpha 2,6-linked sialic acid residues in neoplastic but not in normal human colonic mucosa. A lectin-gold cytochemical study with Sambucus nigra and Maackia amurensis lectins. Am J Pathol 1991;139(6):1435-48.
17. Gessner P, Riedl S, Quentmaier A, Kemmner W. Enhanced activity of CMP-neuAc:Gal beta 1-4GlcNAc:alpha 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett 1993;75(3):143-9.
18. Recchi MA, Harduin-Lepers A, Boilly-Marer Y, Verbert A, Delannoy P. Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells. Glycoconj J 1998;15(1):19-27.
19. Ogawa JI, Inoue H, Koide S. alpha-2,3-Sialyltransferase type 3N and alpha-1,3-fucosyltransferase type VII are related to sialyl Lewis(x) synthesis and patient survival from lung carcinoma. Cancer 1997;79(9):1678-85.
20. Ogawa J, Tsurumi T, Yamada S, Koide S, Shohtsu A. Blood vessel invasion and expression of sialyl Lewisx and proliferating cell nuclear antigen in stage I non-small cell lung cancer. Relation to postoperative recurrence. Cancer 1994;73(4):1177-83.
21. Sewell R, Backstrom M, Dalziel M, et al. The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer. J Biol Chem 2006;281(6):3586-94.
22. Kaneko Y, Yamamoto H, Kersey DS, Colley KJ, Leestma JE, Moskal JR. The expression of Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase and alpha 2,6-linked sialoglycoconjugates in human brain tumors. Acta Neuropathol 1996;91(3):284-92.
23. Wang PH, Li YF, Juang CM, et al. Altered mRNA expression of sialyltransferase in squamous cell carcinomas of the cervix. Gynecol Oncol 2001;83(1):121-7.
24. Liang PH, Wu CY, Greenberg WA, Wong CH. Glycan arrays: biological and medical applications. Curr Opin Chem Biol 2008;12(1):86-92.
25. Wang X, Zhang LH, Ye XS. Recent development in the design of sialyltransferase inhibitors. Med Res Rev 2003;23(1):32-47.
26. Cambron LD, Leskawa KC. Inhibition of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase by nucleotides, nucleotide sugars and nucleotide dialdehydes. Biochem Biophys Res Commun 1993;193(2):585-90.
27. Chang KH, Lee L, Chen J, Li WS. Lithocholic acid analogues, new and potent alpha-2,3-sialyltransferase inhibitors. Chem Commun (Camb) 2006(6):629-31.
28. Chang WW, Yu CY, Lin TW, Wang PH, Tsai YC. Soyasaponin I decreases the expression of alpha2,3-linked sialic acid on the cell surface and suppresses the metastatic potential of B16F10 melanoma cells. Biochem Biophys Res Commun 2006;341(2):614-9.
29. Raftopoulou M, Hall A. Cell migration: Rho GTPases lead the way. Dev Biol 2004;265(1):23-32.
30. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer 2002;2(2):91-100.
31. Gu J, Taniguchi N. Regulation of integrin functions by N-glycans. Glycoconj J 2004;21(1-2):9-15.
32. Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer 2008;8(8):604-17.
33. Zhao Y, Sato Y, Isaji T, et al. Branched N-glycans regulate the biological functions of integrins and cadherins. FEBS J 2008;275(9):1939-48.
34. Lin S, Kemmner W, Grigull S, Schlag PM. Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells. Exp Cell Res 2002;276(1):101-10.
35. Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, Bellis SL. Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 2005;65(11):4645-52.
36. Christie DR, Shaikh FM, Lucas JAt, Lucas JA, 3rd, Bellis SL. ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function. J Ovarian Res 2008;1(1):3.
37. Zhuo Y, Chammas R, Bellis SL. Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis. J Biol Chem 2008;283(32):22177-85.
38. Seales EC, Jurado GA, Singhal A, Bellis SL. Ras oncogene directs expression of a differentially sialylated, functionally altered beta1 integrin. Oncogene 2003;22(46):7137-45.
39. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet 1993;342(8878):1024-5.
40. Owens LV, Xu L, Craven RJ, et al. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 1995;55(13):2752-5.
41. Raman D, Baugher PJ, Thu YM, Richmond A. Role of chemokines in tumor growth. Cancer Lett 2007;256(2):137-65.
42. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4(7):540-50.
43. Wagner PL, Hyjek E, Vazquez MF, et al. CXCL12 and CXCR4 in adenocarcinoma of the lung: association with metastasis and survival. J Thorac Cardiovasc Surg 2009;137(3):615-21.
44. Phillips RJ, Burdick MD, Lutz M, Belperio JA, Keane MP, Strieter RM. The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non-small cell lung cancer metastases. Am J Respir Crit Care Med 2003;167(12):1676-86.
45. Otsuka S, Bebb G. The CXCR4/SDF-1 chemokine receptor axis: a new target therapeutic for non-small cell lung cancer. J Thorac Oncol 2008;3(12):1379-83.
46. Frommhold D, Ludwig A, Bixel MG, et al. Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation. J Exp Med 2008;205(6):1435-46.
47. Bannert N, Craig S, Farzan M, et al. Sialylated O-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokine receptor 5 contribute to high affinity binding of chemokines. J Exp Med 2001;194(11):1661-73.
48. Cardones AR, Murakami T, Hwang ST. CXCR4 enhances adhesion of B16 tumor cells to endothelial cells in vitro and in vivo via beta(1) integrin. Cancer Res 2003;63(20):6751-7.