簡易檢索 / 詳目顯示

回結果列表
研究生： 徐子凡
Hsu, Zih-Fan
論文名稱： 探討雙高石膽酸衍生物對於唾液酸轉移酶及癌細胞轉移的影響
Bishomolithocholic Acid: A Unique Template for Human Sialyltransferase Inhibitor, Optimized to Suppress Cancer Metastasis
指導教授： 李文山
Li, Wen-Shan
林文偉
Lin, Wen-Wei
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 96
中文關鍵詞： 唾液酸 、唾液酸轉移酶 、癌症轉移 、雙高石膽酸
英文關鍵詞： sialic acid, sialyltransferase, metastasis, bishomolithocholic acid
DOI URL： http://doi.org/10.6345/NTNU201900320
論文種類： 學術論文
相關次數： 點閱：187  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 在台灣，癌症為十大死因之首，其中癌細胞轉移為九成癌症病人之死因。癌細胞表面具有過度表現的唾液酸是細胞癌化的重要指標，此現象不僅會促進癌細胞的生長，亦可增加其侵犯周邊組織與轉移之能力。已有實驗證實，藉由調控唾液酸轉移酶能影響細胞表面之唾液酸表現。因此，抑制唾液酸轉移酶的活性會是一個有潛力的癌症轉移治療方式。
    我們合成雙高石膽酸衍生物HZF01-04，並進行初步生物試驗的研究與探討。HZF01-04具有極高的選擇性唾液酸轉移酶抑制能力，針對ST6Gal I有良好的抑制效果，其IC50數值介於7.8-12.2 μM之間。此外，HZF01-04對於人類三陰性乳癌細胞株MDA-MB-231的轉移能力具有抑制效果，其IC50數值之範圍介於7.5-10 μM之間。進一步的生物活性測試正在進行中，包括物化性質與動物實驗。
    此研究結果有助於選擇性唾液酸轉移酶抑制劑的開發，希望能應用於N-醣鏈過度唾液酸化的癌症病人之治療。

    Cancer is the major cause of death and metastasis is the greatest contributor to cancer deaths. Tumor cells display elevated expression of sialic acid on membranes, which promote cancer cell metastasis and progression. Aberrant sialylation is correlated with poor patient prognosis. Therefore, interfering hypersialylation through inhibition of sialytransferase activity may be a therapeutic target to metastatic cancer.
    In this investigation, we synthesized a series of bishomolithocholic acid derivatives HZF01-04 with various length linkers. HZF01-04 demonstrated significant inhibitory selectivity toward N-glycan sialyltransferase, β-galactoside α-2,6-sialyltransferase (ST6Gal I). The selectivity ratio of N-glycan vs O-glycan is over 80-fold. The findings show that HZF01-04 suppress serum-induced cancer cell migration in a dose dependent manner. Further studies for their mechanism of action and animal model are in progress.
    These efforts pave the way for the development of selective sialyltransferase inhibitors as effective anti-metastatic agents for the treatment of patients with aberrant overexpression of N-glycan sialylation.

    中文摘要 I Abstract II 謝誌 III Contents IV Scheme Contents VII Table Contents VIII Figure Contents IX List of Abbreviation X Awards & Honors XII I. Introduction 1 1.1. Background 1 1.2. Sialic Acid 2 1.3. Sialyltransferase 4 1.4. Sialylated Antigen 7 1.4.1 Sialyl Lewis antigens (SLea and SLex) 7 1.4.2 Truncated O-glycans 8 1.4.3 Highly branched N-glycan 8 1.4.4 Polysialic acid (polySia) 8 1.4.5 Glycosphingolipid (GSL) 9 1.5. Sialylation in Cancer Metastasis 9 1.5.1 Metastatic process 9 1.5.2 Role of glycoconjugates in metastasis 10 1.6. Sialyltransferase Inhibitors 12 1.6.1 Donor analogous inhibitors 12 1.6.2 Transition-state analogous inhibitors 13 1.6.3 Acceptor analogous inhibitors 13 1.6.4 Other sialyltransferase inhibitors 14 1.7. Previous Study 15 1.8. Research Motivation 19 II. Results and Discussion 21 2.1. Synthesis of Bishomolithocholic Acid Derivatives HZF01-04 21 2.1.1. Retrosynthesis 21 2.1.2. Synthesis of PEG linker 1a-1d 22 2.1.3. Synthesis of bishomolithocholic diacid 10 23 2.1.4. Synthesis of bishomolithocholic acid derivatives 25 2.2. In Vitro Studies of HZF01-04 27 2.2.1. Cytotoxicity of HZF01-04 in MDA-MB-231 27 2.2.2. Inhibition of ST3Gal I and ST6Gal I 28 2.2.3. Antimigration activity of HZF01-04 29 2.2.4. Cytotoxicity of HZF01-04 in MDA-MB-231 (SFM) 32 2.3. Preliminary Structure-Activity Relationship Studies 33 III. Conclusions 37 IV. Material and Methods 39 4.1. General Experimental 39 4.2. Synthetic Procedures 40 4.2.1. Preparation of PEG linker 40 4.2.2. Preparation of bishomolithocholic acid 43 4.2.3. Preparation of HZF01 46 4.2.4. Preparation of HZF02 49 4.2.5. Preparation of HZF03 52 4.2.6. Preparation of HZF04 55 4.3. Cell Line Used and Cell Culture 58 4.4. MTT Cytotoxicity Analysis 58 4.5. Sialyltransferase Assay 58 4.6. Transwell Migration Assay 59 V. References 61 Spectra 69

    1. 衛生福利部統計處, (2018), 106年主要死因統計結果分析, Retrieved May, 30, 2019, from https://dep.mohw.gov.tw/DOS/cp-3960-41756-113.html.
    2. Chaffer, C. L.; Weinberg, R. A. A perspective on cancer cell metastasis, Science 2011, 331, 1559-1564.
    3. Steeg, P. S. Targeting metastasis, Nat. Rev. Cancer 2016, 16, 201-218.
    4. Vajaria, B. N.; Patel, P. S. Glycosylation: A hallmark of cancer?, Glycoconj. J. 2017, 34, 147-156.
    5. Pinho, S. S.; Reis, C. A. Glycosylation in cancer: Mechanisms and clinical implications, Nat. Rev. Cancer 2015, 15, 540-555.
    6. Bull, C.; Stoel, M. A.; den Brok, M. H.; Adema, G. J. Sialic acids sweeten a tumor's life, Cancer Res. 2014, 74, 3199-3204.
    7. Rodrigues, J. G.; Balmana, M.; Macedo, J. A.; Pocas, J.; Fernandes, A.; de-Freitas-Junior, J. C. M.; Pinho, S. S.; Gomes, J.; Magalhaes, A.; Gomes, C.; Mereiter, S.; Reis, C. A. Glycosylation in cancer: Selected roles in tumour progression, immune modulation and metastasis, Cell. Immunol. 2018, 333, 46-57.
    8. Lu, J.; Gu, J. Significance of beta-galactoside alpha2,6 sialyltranferase 1 in cancers, Molecules 2015, 20, 7509-7527.
    9. Wang, L.; Liu, Y.; Wu, L.; Sun, X. L. Sialyltransferase inhibition and recent advances, Biochim. Biophys. Acta 2016, 1864, 143-153.
    10. Yarema, K. J.; Goon, S.; Bertozzi, C. R. Metabolic selection of glycosylation defects in human cells, Nat. Biotechnol. 2001, 19, 553-558.
    11. Li, F.; Ding, J. Sialylation is involved in cell fate decision during development, reprogramming and cancer progression, Protein Cell 2018, doi:10.1007/s13238-13018-10597-13235.
    12. Wang, X.; Zhang, L. H.; Ye, X. S. Recent development in the design of sialyltransferase inhibitors, Med. Res. Rev. 2003, 23, 32-47.
    13. Szabo, R.; Skropeta, D. Advancement of sialyltransferase inhibitors: Therapeutic challenges and opportunities, Med. Res. Rev. 2017, 37, 219-270.
    14. Rodrigues, E.; Macauley, M. S. Hypersialylation in cancer: Modulation of inflammation and therapeutic opportunities, Cancers (Basel) 2018, 10, 207.
    15. Pearce, O. M.; Laubli, H. Sialic acids in cancer biology and immunity, Glycobiology 2016, 26, 111-128.
    16. Amano, M.; Galvan, M.; He, J.; Baum, L. G. The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death, J. Biol. Chem. 2003, 278, 7469-7475.
    17. Zhuo, Y.; Chammas, R.; Bellis, S. L. Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis, J. Biol. Chem. 2008, 283, 22177-22185.
    18. Joy Burchell, R. P., Andrew Hanby, Caroline Whitehouse, Lucienne Cooper, Henrik Clausen, David Miles and Joyce Taylor-Papadimitriou An α 2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas, Glycobiology 1999, 9, 1307-1311.
    19. Glavey, S. V.; Manier, S.; Natoni, A.; Sacco, A.; Moschetta, M.; Reagan, M. R.; Murillo, L. S.; Sahin, I.; Wu, P.; Mishima, Y.; Zhang, Y.; Zhang, W.; Zhang, Y.; Morgan, G.; Joshi, L.; Roccaro, A. M.; Ghobrial, I. M.; O'Dwyer, M. E. The sialyltransferase ST3Gal6 influences homing and survival in multiple myeloma, Blood 2014, 124, 1765-1776.
    20. Gomes, C.; Osorio, H.; Pinto, M. T.; Campos, D.; Oliveira, M. J.; Reis, C. A. Expression of ST3Gal4 leads to SLe(x) expression and induces c-met activation and an invasive phenotype in gastric carcinoma cells, PLoS One 2013, 8, e66737.
    21. Perez-Garay, M.; Arteta, B.; Pages, L.; de Llorens, R.; de Bolos, C.; Vidal-Vanaclocha, F.; Peracaula, R. Alpha2,3-sialyltransferase ST3Gal III modulates pancreatic cancer cell motility and adhesion in vitro and enhances its metastatic potential in vivo, PLoS One 2010, 5, e12524.
    22. Julien, S.; Adriaenssens, E.; Ottenberg, K.; Furlan, A.; Courtand, G.; Vercoutter-Edouart, A. S.; Hanisch, F. G.; Delannoy, P.; Le Bourhis, X. ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity, Glycobiology 2006, 16, 54-64.
    23. Gong, L.; Zhou, X.; Yang, J.; Jiang, Y.; Yang, H. Effects of the regulation of polysialyltransferase ST8SiaII on the invasiveness and metastasis of small cell lung cancer cells, Oncol. Rep. 2017, 37, 131-138.
    24. Kedmi, R.; Peer, D. Zooming in on selectins in cancer, Sci. Transl. Med. 2016, 8, 345fs311.
    25. Borsig, L. Selectins in cancer immunity, Glycobiology 2018, 28, 648-655.
    26. Kannagi, R.; Izawa M Fau - Koike, T.; Koike T Fau - Miyazaki, K.; Miyazaki K Fau - Kimura, N.; Kimura, N. Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis, Cancer Sci. 2004, 95, 377-384.
    27. Munkley, J. The glycosylation landscape of pancreatic cancer, Oncol. Lett. 2019, 17, 2569-2575.
    28. Fu, C.; Zhao, H.; Wang, Y.; Cai, H.; Xiao, Y.; Zeng, Y.; Chen, H. Tumor-associated antigens: Tn antigen, STn antigen, and T antigen, HLA 2016, 88, 275-286.
    29. Munkley, J. The role of sialyl-Tn in cancer, Int J Mol Sci 2016, 17, 275.
    30. Munkley, J.; Oltean, S.; Vodak, D.; Wilson, B. T.; Livermore, K. E.; Zhou, Y.; Star, E.; Floros, V. I.; Johannessen, B.; Knight, B.; McCullagh, P.; McGrath, J.; Crundwell, M.; Skotheim, R. I.; Robson, C. N.; Leung, H. Y.; Harries, L. W.; Rajan, P.; Mills, I. G.; Elliott, D. J. The androgen receptor controls expression of the cancer-associated STn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer, Oncotarget 2015, 6, 34358-34374.
    31. Beatson, R.; Maurstad, G.; Picco, G.; Arulappu, A.; Coleman, J.; Wandell, H. H.; Clausen, H.; Mandel, U.; Taylor-Papadimitriou, J.; Sletmoen, M.; Burchell, J. M. The breast cancer-associated glycoforms of muc1, muc1-Tn and sialyl-Tn, are expressed in cosmc wild-type cells and bind the C-type lectin MGL, PLoS One 2015, 10, e0125994.
    32. Elkashef, S. M.; Allison, S. J.; Sadiq, M.; Basheer, H. A.; Ribeiro Morais, G.; Loadman, P. M.; Pors, K.; Falconer, R. A. Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment, Sci. Rep. 2016, 6, 33026.
    33. Colley, K. J.; Kitajima K Fau - Sato, C.; Sato, C. Polysialic acid: Biosynthesis, novel functions and applications, Crit. Rev. Biochem. Mol. Biol. 2014, 49, 1549-7798.
    34. Hamamura, K.; Furukawa, K. Glycosylation is involved in malignant properties of cancer cells, Cancer Transl. Med. 2017, 3, 209-213.
    35. Gomez-Cuadrado, L.; Tracey, N.; Ma, R.; Qian, B.; Brunton, V. G. Mouse models of metastasis: Progress and prospects, Dis. Model. Mech. 2017, 10, 1061-1074.
    36. Mazzocca, A.; Carloni, V. The metastatic process: Methodological advances and pharmacological challenges, Curr. Med. Chem. 2009, 16, 1704-1717.
    37. Bull, C.; Heise, T.; Adema, G. J.; Boltje, T. J. Sialic acid mimetics to target the sialic acid-siglec axis, Trends Biochem. Sci. 2016, 41, 519-531.
    38. Bull, C.; Boltje, T. J.; van Dinther, E. A.; Peters, T.; de Graaf, A. M.; Leusen, J. H.; Kreutz, M.; Figdor, C. G.; den Brok, M. H.; Adema, G. J. Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastatic spread, ACS Nano 2015, 9, 733-745.
    39. Rillahan, C. D.; Antonopoulos, A.; Lefort, C. T.; Sonon, R.; Azadi, P.; Ley, K.; Dell, A.; Haslam, S. M.; Paulson, J. C. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome, Nat. Chem. Biol. 2012, 8, 661-668.
    40. Macauley, M. S.; Arlian, B. M.; Rillahan, C. D.; Pang, P. C.; Bortell, N.; Marcondes, M. C.; Haslam, S. M.; Dell, A.; Paulson, J. C. Systemic blockade of sialylation in mice with a global inhibitor of sialyltransferases, J. Biol. Chem. 2014, 289, 35149-35158.
    41. Pauling, L. Molecular architecture and biological reactions, Chem. Eng. News 1946, 24, 1375-1377.
    42. Guo, J.; Li, W.; Xue, W.; Ye, X. S. Transition state-based sialyltransferase inhibitors: Mimicking oxocarbenium ion by simple amide, J. Med. Chem. 2017, 60, 2135-2141.
    43. Li, W.; Niu, Y.; Xiong, D. C.; Cao, X.; Ye, X. S. Highly substituted cyclopentane-CMP conjugates as potent sialyltransferase inhibitors, J. Med. Chem. 2015, 58, 7972-7990.
    44. Kajihara, Y.; Kodama H Fau - Wakabayashi, T.; Wakabayashi T Fau - Sato, K.; Sato K Fau - Hashimoto, H.; Hashimoto, H. Characterization of inhibitory activities and binding mode of synthetic 6'-modified methyl N-acetyl-beta-lactosaminide toward rat liver CMP-D-Neu5Ac: D-galactoside-(2-->6)-alpha-d-sialyltransferase, Carbohydr Res. 1993, 247, 179-193.
    45. Lin, T. W.; Chang, W. W.; Chen, C. C.; Tsai, Y. C. Stachybotrydial, a potent inhibitor of fucosyltransferase and sialyltransferase, Biochem. Biophys. Res. Commun. 2005, 331, 953-957.
    46. Wu, C. Y.; Hsu, C. C.; Chen, S. T.; Tsai, Y. C. Soyasaponin I, a potent and specific sialyltransferase inhibitor, Biochem. Biophys. Res. Commun. 2001, 284, 466-469.
    47. Hsu, C. C.; Lin, T. W.; Chang, W. W.; Wu, C. Y.; Lo, W. H.; Wang, P. H.; Tsai, Y. C. Soyasaponin-I-modified invasive behavior of cancer by changing cell surface sialic acids, Gynecol. Oncol. 2005, 96, 415-422.
    48. Chang, K. H.; Lee, L.; Chen, J.; Li, W. S. Lithocholic acid analogues, new and potent alpha-2,3-sialyltransferase inhibitors, Chem. Commun. (Camb.) 2006, 14, 629-631.
    49. Chen, J. Y.; Tang, Y. A.; Huang, S. M.; Juan, H. F.; Wu, L. W.; Sun, Y. C.; Wang, S. C.; Wu, K. W.; Balraj, G.; Chang, T. T.; Li, W. S.; Cheng, H. C.; Wang, Y. C. A novel sialyltransferase inhibitor suppresses FAK/paxillin signaling and cancer angiogenesis and metastasis pathways, Cancer. Res. 2011, 71, 473-483.
    50. 傅志偉, 由自然界靈感設計及合成出抗癌症和抗癌症轉移試劑：細胞和動物體內之活性測試評估, 國立中央大學化學學系博士論文, 2015.
    51. Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2019, CA Cancer J Clin 2019, 69, 7-34.
    52. Aysola, K.; Desai, A.; Welch, C.; Xu, J.; Qin, Y.; Reddy, V.; Matthews, R.; Owens, C.; Okoli, J.; Beech, D. J.; Piyathilake, C. J.; Reddy, S. P.; Rao, V. N. Triple negative breast cancer - an overview, Hereditary Genet 2013, Suppl 2, 001.
    53. Ma, X.; Dong, W.; Su, Z.; Zhao, L.; Miao, Y.; Li, N.; Zhou, H.; Jia, L. Functional roles of sialylation in breast cancer progression through miR-26a/26b targeting ST8Sia4, Cell Death Dis. 2016, 7, e2561.
    54. Lu, J.; Isaji, T.; Im, S.; Fukuda, T.; Hashii, N.; Takakura, D.; Kawasaki, N.; Gu, J. Beta-galactoside alpha2,6-sialyltranferase 1 promotes transforming growth factor-beta-mediated epithelial-mesenchymal transition, J. Biol. Chem. 2014, 289, 34627-34641.
    55. Frohlich, T.; Reiter, C.; Saeed, M. E. M.; Hutterer, C.; Hahn, F.; Leidenberger, M.; Friedrich, O.; Kappes, B.; Marschall, M.; Efferth, T.; Tsogoeva, S. B. Synthesis of thymoquinone-artemisinin hybrids: New potent antileukemia, antiviral, and antimalarial agents, ACS Med Chem Lett 2018, 9, 534-539.
    56. Luu, T. H.; Bard, J. M.; Carbonnelle, D.; Chaillou, C.; Huvelin, J. M.; Bobin-Dubigeon, C.; Nazih, H. Lithocholic bile acid inhibits lipogenesis and induces apoptosis in breast cancer cells, Cell Oncol (Dorder) 2018, 41, 13-24.
    57. Fu, C.-W.; Chang, K.-H.; Jen, Y. C.; Chang, T. T.; Li, W.-S. Synthesis of amino acid-comprising sialyltransferase inhibitors and their antimetastatic activities against human breast cancer cells, J. Chin. Chem. Soc. 2016, 63, 171-180.
    58. Legeay, J. C.; Vanden Eynde, J. J.; Bazureau, J. P. Sequential synthesis of a new analogue of amlodipine bearing a short amino polyethyleneglycol chain, Tetrahedron 2007, 63, 12081-12086.
    59. Sofia Svedhem, C.-A. H., Jing Shi, Peter Konradsson, Bo Liedberg, and Stefan C. T. Svensson Synthesis of a series of oligo(ethylene glycol)-terminated alkanethiol amides designed to address structure and stability of biosensing interfaces, J. Org. Chem. 2001, 4494-4503.
    60. Aucagne, V.; Valverde, I. E.; Marceau, P.; Galibert, M.; Dendane, N.; Delmas, A. F. Towards the simplification of protein synthesis: Iterative solid-supported ligations with concomitant purifications, Angew. Chem. Int. Ed. Engl. 2012, 51, 11320-11324.

    下載圖示
    QR CODE