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研究生：	林依璇 Lin, Yi-Shiuan
論文名稱：	上皮細胞生長因子受體的路易斯Y醣化促進口腔癌細胞的爬行能力 Lewis Y Glycosylation of Epidermal Growth Factor Receptor Promotes Cell Migration in Oral Cancer Cells
指導教授：	吳華林 Wu, Hua-Lin
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	71
中文關鍵詞：	上皮細胞生長因子受體、路易斯Y 、口腔癌
外文關鍵詞：	EGFR, Lewis y, oral cancer
相關次數：	點閱：66 下載：0
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路易斯Y (Lewis y，Ley)是由含有兩個岩藻醣(fucose)的四醣鏈(Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-R)組成，屬於血型抗原中的路易斯(Lewis) 抗原家族成員之一。Ley被發現會修飾在脂質上而形成醣脂質(glycolipid)或是連結在細胞膜表面的蛋白質上如上皮細胞生長因子受體(epidermal growth factor receptor，EGFR)。在過去已經知道Ley在上皮組織來源的腫瘤常有過度表達的情形，此外，在先前的研究指出Ley的過度表達與臨床上口腔癌病患的預後有密切的關聯性。然而，Ley的表現是否會造成口腔癌細胞的惡性以及其關聯性仍然是不清楚的。在本篇研究中，我們探討了Ley在人類口腔癌細胞的爬行可能造成的影響以及其中的機制。我們分析了Ley在四株不同口腔癌細胞的表現，結果顯示高侵襲性的細胞Ley的表現程度高於低侵襲性的細胞。此外，我們發現在OC2口腔鱗狀細胞癌細胞株的EGFR有大量的Ley修飾。為了評估Ley修飾在EGFR的功能上可能造成的影響，我們抑制了OC2細胞中參與在Ley合成途徑裡的岩藻醣轉移酶(fucosyltransferase I)的表現。結果顯示相較於未處理的OC2細胞，在Ley醣化受到抑制的OC2細胞中，發現由上皮細胞生長因子(EGF)所誘導的細胞爬行有減少的現象，但是細胞增生並沒有變化。此外，當Ley醣化受到抑制時，由EGF所誘導的EGFR磷酸化與下游的Akt及ERK磷酸化都減弱了。抑制Ley的醣化造成也抑制EGFR的雙聚體化並加速EGFR的分解，但對於EGF結合EGFR和EGFR入核的情形沒有顯著的變化。總結以上，我們的研究顯示OC2細胞上EGFR的Ley醣化能增加EGFR的雙聚體化和避免EGFR被分解，進一步能夠幫助EGFR下游訊號的傳導以及由EGF所誘導的細胞爬行。

Lewis y (Ley, Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-R) is a difucosylated oligosaccharide which belongs to Lewis antigen of the blood group system. Ley can be found as a glycolipid or linked to surface proteins such as epidermal growth factor receptor (EGFR). It is well known that Ley overexpression is frequently found in epithelium-derived cancers. In addition, previous studies have indicated that high level of Ley expression significantly correlates with poor prognosis in oral cancer patients. However, the correlation of Ley to the malignancy of oral cancer has not been completely understood. In this study, we investigated the effect and the possible mechanism of Ley on the migration of human oral cancer cells. We examined the expression of Ley in four oral cell lines (HSC-3, OC2, OEC-M1, and SG), and found that the expression of Ley was higher in highly invasive cells (HSC-3 and OC2) than in lowly invasive cells (OEC-M1 and SG). Besides, EGFR was highly glycosylated with Ley only in oral squamous cell carcinoma-derived OC2 cells. To evaluate the influence of Ley glycosylation on the function of EGFR, we knocked down fucosyltransferase I, the key enzyme for Ley synthesis in OC2 cells. The data showed that EGF-induced cell migration was reduced in FUT-1 knockdown OC2 cells compared with wild-type OC2 cells, but cell proliferation was unchanged. Furthermore, OC2 cells with suppression of Ley glycosylation had attenuated EGF-induced phosphorylation of EGFR, Akt, and ERK, two downstream molecules of EGFR signaling. Knockdown of FUT-1 inhibited EGFR dimerization and accelerated its degradation, but had no signiﬁcant changes in the binding of EGF to EGFR and nuclear localization of EGFR. In conclusion, our studies suggest that Ley glycosylation of EGFR in OC2 cells increases EGFR dimerization and prevents its degradation, consequently promoting downstream signal transduction and EGF-induced cell migration.

中文摘要....................................................1
Abstract...................................................2
Acknowledgement............................................3
Contents...................................................4
Contents List of Figures...................................6
Contents List of Appendixes................................7
Abbreviation...............................................8
Introduction..............................................10
Specific Aims.............................................13
Material and Methods
1. Cell Culture...........................................14
1.1 Cell lines 
1.2 Cell culture media 
1.3 Subcultivation 
1.4 Cell counting
1.5 Cell freezing 
1.6 Cell thawing 
2. RNA extraction and reverse transcription...............17
2.1 RNA extraction 
2.2 Reverse transcription 
2.3 Real-time PCR 
3. General Protein Analysis...............................20
3.1 Protein sample preparation 
3.2 Protein quantification
3.3 Electrophoresis 
4. Flow-cytometry assay...................................25
5. Immunoprecipitation assay..............................26
6. Cell proliferation assay...............................27
7. Assay of EGFR, Akt, and ERK1/2 phosphorylation.........28
8. Scratch wound healing assay............................28
9. Cell invasion assay....................................29
10. EGF binding assay.....................................30
10.1 Immunofluorescence stain
10.2 Flow-cytometry
11. EGFR dimerization assay...............................31
12. Nuclear EGFR detection................................32
12.1 Immunofluorescence stain
12.2 Nuclear and cytoplasmic fractionation
13. Statistical Analysis..................................34
Result 
Ⅰ. The expression level of Ley is higher in high-invasive cells than in low-invasive cells..........................35
Ⅱ. EGFR is highly glycosylated with Ley only in oral squamous cell carcinoma-derived OC2 cells.................35
Ⅲ. EGF treatment significantly increases cell migration, but cell proliferation is unchanged in OC2 cells..........36
Ⅳ. Cell proliferation has no significant change in FUT-1 knockdown cells..............................36
Ⅴ. The OC2 cells with suppression of Ley glycosylation has reduced cell migration...................................37
Ⅵ. The OC2 cells with suppression of Ley glycosylation has reduced level of EGF-induced phosphorylation of EGFR, Akt, and ERK..................................................37
Ⅶ. EGF binding affinity is not affected by knockdown of FUT-1....................................................38
Ⅷ. Ligand-induced receptor dimerization is inhibited by suppression of Ley expression............................38
Ⅸ. FUT-1 knockdown accelerates ligand-induced degradation of the EGFR in OC2 cells.................................38
Ⅹ.Suppression of Ley expression has no effect on EGFR nuclear localization in OC2 cells........................39 Ⅺ. Decreased Ley expression in OC2 cells influences EGFR dimerization and degradation, and consequently reducing downstream signal transduction and EGF-induced cell migration................................................39
Discussions..............................................41
References...............................................44
Figures..................................................48
Appendixes...............................................61
Instruments..............................................64
Reagents and Chemicals...................................66
Author’s Resume..........................................71
                                    

1.Su, C.-C. et al. Incidence of oral cancer in relation to nickel and arsenic concentrations in farm soils of patients' residential areas in Taiwan. BMC Public Health 10, 67, (2010).
2.Dabelsteen, E. & Gao, S. ABO Blood-group Antigens in Oral Cancer. Journal of Dental Research 84, 21-28, (2005).
3.Xie, X., Boysen, M., Clausen, O. P. F. & Bryne, M. A. Prognostic Value of Ley and H Antigens in Oral Tongue Carcinomas. The Laryngoscope 109, 1474-1480, (1999).
4.Varki, A. et al. Essentials of Glycobiology. 2nd edn, (Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, 2009).
5.De, V. T., Knegtel, R. M., Holmes, E. H. & Macher, B. A. Fucosyltransferases:structure/function studies. Glycobiology 11, 119R-128R, (2001).
6.Ma, B., Simala-Grant, J. L. & Taylor, D. E. Fucosylation in prokaryotes and eukaryotes. Glycobiology 16, 158R-184R, (2006).
7.Aubert, M. et al. Restoration of a (1,2) Fucosyltransferase Activity Decreases Adhesive and Metastatic Properties of Human Pancreatic Cancer Cells. Cancer Research 60, 1449 –1456, (2000).
8.Zhang, Z. et al. Suppression of FUT1/FUT4 expression by siRNA inhibits tumor growth. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1783, 287-296, (2008).
9.Dettke, M., lﬁ, G. P. & Loibner, H. Activation-dependent expression of the blood group-related Lewis Y antigen on peripheral blood granulocytes. Journal of Leukocyte Biology 68, 511-514, (2000).
10.Klinger, M. Antibodies Directed against Lewis-Y Antigen Inhibit Signaling of Lewis-Y Modified ErbB Receptors. Cancer Research 64, 1087-1093, (2004).
11.Basu, A. et al. Presence of Tumor-associated Antigens in Epidermal Growth Factor Receptors from Different Human Carcinomas. Cancer Research 47, 2531-2536, (1987).
12.Hellstrm, I., Garrigues, J., Garrigues, U. & Hellstrm, K. E. Highly Tumor-reactive, Internalizing, Mouse Monoclonal Antibodies to Ley-related Cell Surface Antigens. Cancer Research 50, 2183-2190, (1990).
13.Kim, Y. S. et al. Expression of LeY and Extended LeY Blood Group-related Antigens in Human Malignant, Premalignant, and Nonmalignant Colonie Tissues. Cancer Research 46, 5985-5992, (1986).
14.Madjd, Z. et al. High expression of Lewis y/b antigens is associated with decreased survival in lymph node negative breast carcinomas. Breast Cancer Research 7, R780, (2005).
15.Farhan, H. et al. Inhibition of Xenograft Tumor Growth and Down-Regulation of ErbB Receptors by an Antibody Directed against Lewis Y Antigen. Journal of Pharmacology and Experimental Therapeutics 319, 1459-1466, (2006).
16.LIU, J.-J. et al. Lewis(y) antigen stimulates the growth of ovarian cancer cells via regulation of the epidermal growth factor receptor pathway. Oncology Reports 23, 833-841, (2010).
17.Huang, S.-F. et al. EGFR protein overexpression and mutation in areca quid-associated oral cavity squamous cell carcinoma in Taiwan. Head & Neck 31, 1068-1077, (2009).
18.Mendelsohn, J. & Baselga, J. The EGF receptor family as targets for cancer therapy. Oncogene 19, 6550 – 6565, (2000).
19.P, B. & J, E. Epidermal growth factor receptor structure, regulation, mitogenic signalling and effects of activation. Anticancer Research 21, 2769–2775, (2001).
20.Lin, S.-Y. et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nature Cell Biology 3, 802-808, (2001).
21.Adachi, S. et al. (-)-Epigallocatechin gallate downregulates EGF receptor via phosphorylation at Ser1046/1047 by p38 MAPK in colon cancer cells. Carcinogenesis 30, 1544-1552, (2009).
22.Chen, I. H. et al. Prognostic significance of EGFR and Her-2 in oral cavity cancer in betel quid prevalent area cancer prognosis. British Journal of Cancer 89, 681-686, (2003).
23.Wu, M. H. et al. Galectin-1-Mediated Tumor Invasion and Metastasis, Up-Regulated Matrix Metalloproteinase Expression, and Reorganized Actin Cytoskeletons. Molecular Cancer Research 7, 311-318, (2009).
24.YONG-KIEvWONG, D., CHANG, K.-W., CHEN, C.-F. & CHANG, R. C.-S. Characterization of Two New Cell Lines Derived From Oral Cavity Human Squamous Cell Carcinomas OC1 and OC2 Journal of Oral and Maxillofacial Surgery 48, 395-390, (1990).
25.Yang, C. & Meng, C. L. Regulation of PGSynthase by EGFand PDGF in HumanOral, Breast, Stomach, and Fibrosarcoma Cancer Cell Lines. Journal of Dental Research 73, 1407-1415, (1994).
26.KASTEN, F. H., PINEDA, L. F. R., SCHNEIDER, P. E., RAWLS, H. R. & FOSTER, T. A. Biocompatibility Testing of An Experimental Fluoride Releasing Resin Using Human Gingival Epithelial Cells in Vitro. In Vitro Cellular a Developmental Biology 25, 57-61, (1989).
27.Smith, P. K. et al. Measurement of protein using bicinchoninic acid Analytical Biochemistry 150, 76-85 (1985).
28.Lampugnani, M. G. Cell Migration into a Wounded Area In Vitro Methods in Molecular Biology 96, 177-182, (1999).
29.Yarden, Y., X., M. & Sliwkowski. Untangling the ErbB signalling network. Nature Reviews Molecular Cell Biology 2, 127-137, (2001).
30.Alwan, H. A. J. Ligand-induced Lysosomal Epidermal Growth Factor Receptor (EGFR) Degradation Is Preceded by Proteasome-dependent EGFR De-ubiquitination. Journal of Biological Chemistry 278, 35781-35790, (2003).
31.Larrain, M. et al. Breast cancer humoral immune response: involvement of Lewis y through the detection of circulating immune complexes and association with Mucin 1 (MUC1). Journal of Experimental & Clinical Cancer Research 28, 121, (2009).
32.van Gisbergen, K. P. J. M. Dendritic Cells Recognize Tumor-Specific Glycosylation of Carcinoembryonic Antigen on Colorectal Cancer Cells through Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin. Cancer Research 65, 5935-5944, (2005).
33.BW, Y. et al. Serological and immunochemical analysis of Lewis y (Ley) blood group antigen expression in epithelial ovarian cancer. International Journal of Cancer 65, 406-412, (1996).
34.Guo, H. B., Randolph, M. & Pierce, M. Inhibition of a Specific N-Glycosylation Activity Results in Attenuation of Breast Carcinoma Cell Invasiveness-related Phenotypes. Journal of Biological Chemistry 282, 22150-22162, (2007).
35.Nonaka, M. et al. Glycosylation-Dependent Interactions of C-Type Lectin DC-SIGN with Colorectal Tumor-Associated Lewis Glycans Impair the Function and Differentiation of Monocyte-Derived Dendritic Cells. The Journal of Immunology 180, 3347-3356, (2008).
36.Zhang, Z. et al. Suppression of FUT1/FUT4 expression by siRNA inhibits tumor growth. Biochimica et Biophysica Acta 1783, 287 – 296, (2008).
37.Azuma, Y. Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis. Biochimica et Biophysica Acta (BBA) - General Subjects 1672, 157-163, (2004).
38.Inufusa, H. et al. Ley glycolipid-recognizing monoclonal antibody inhibits procoagulant activity and metastasis of human adenocarcinoma. International Journal of Oncology 19, 941-946, (2001).
39.Harandi, A., Zaidi, A. S., Stocker, A. M. & Laber, D. A. Clinical Efficacy and Toxicity of Anti-EGFR Therapy in Common Cancers. Journal of Oncology 2009, 1-14, (2009).
40.Saleh, M. N. et al. Phase I Trial of the Anti-Lewis Y Drug Immunoconjugate BR96 Doxorubicin in Patients With Lewis Y-Expressing Epithelial Tumors. Journal of Clinical Oncology 18, 2282-2292, (2000).
41.Schuster, M. et al. Improved Effector Functions of a Therapeutic Monoclonal Lewis Y-Specific Antibody by Glycoform Engineering. Cancer Research 65, 7934-7941, (2005).

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