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研究生:洪曉貞
研究生(外文):Shiao-Chen Hung
論文名稱:探討L1細胞黏附分子於舌鱗狀細胞癌中所扮演之角色
論文名稱(外文):Role of L1 Cell Adhesion Molecule in Tongue Squamous Cell Carcinoma
指導教授:謝嘉玲謝嘉玲引用關係
指導教授(外文):Chia-Ling Hsieh
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:癌症生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:49
中文關鍵詞:口腔癌L1細胞黏附分子
外文關鍵詞:Oral cancerL1-CAM
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摘要:
根據行政院衛生署的統計,目前口腔癌排行國人癌症前十大死因中的第六位。其原因不外乎口腔內所發生的癌前病變並不顯著,所以經常被忽略,隨著病程進展,口腔癌有可能經淋巴轉移而導致頸部淋巴結的腫大。目前導致口腔癌化的機制仍有待了解。另一方面,L1細胞黏附分子為大腦和神經組織在發育過程中,一個不可或缺的分子。目前越來越多的研究報告指出,L1細胞黏附分子會在許多的癌細胞中有過度表現的情況,例如大腸直腸癌或是乳癌等。而L1細胞黏附分子過度表現的情況和癌細胞癌化的進程具有相關性且也代表著病患有較差的預後。我們的研究目標是去探討L1細胞黏附分子在舌鱗狀細胞癌的癌化過程中所扮演的角色,並且希望藉此瞭解其中的調控機制。首先,在方法上,我們利用西方墨點法,流式細胞儀以及酵素連結免疫吸附分析發現L1細胞黏附分子在較為惡性的舌鱗狀細胞癌的細胞株中有過度表現的情況。接下來,我們利用建立基因改造模型(Genetically manipulation)的舌鱗狀細胞癌,來達到L1細胞黏附分子在舌鱗狀細胞癌表現量的差異。我們主要利用反轉錄病毒及慢病毒為載體,攜帶L1細胞黏附分子的基因和L1 siRNA來達到過度表達或干擾此蛋白質的表現。體外實驗結果的部份,我們發現L1細胞黏附分子的表現和舌鱗狀細胞癌的細胞黏附能力以及細胞的能動行具有極大的關聯性。除此之外,藉由上皮─間質轉化(epithelial-mesenchymal transition)的標誌的表現與否,包括E-cadherin,Vimentin,Fibronectin的改變我們發現,在舌鱗狀細胞癌中,L1細胞黏附分子會藉由上皮─間質轉化的方式去調控細胞能動性和貼附的能力。另一方面,在動物模式發現當降低L1細胞黏附分子的表現時,腫瘤的生長及轉移能力則明顯地被抑制。同時也發現降低L1細胞黏附分子的表現時,老鼠的存活率明顯地增加。由我們的研究結果可以發現,L1細胞黏附分子可以藉由上皮─間質轉化來調控舌鱗狀細胞癌的癌化過程。除此之外,針對口腔癌的治療,L1細胞黏附分子也許可以當作一個新的分子標靶治療。
Abstract:
Oral cancer is one of the top 10 leading causes of death from cancer in Taiwan. This disease is characterized by poor prognosis and low survival rate. The molecular mechanisms of oral cancer progression are not well understood. Overexpression of L1 cell adhesion molecular (L1-CAM) has been found in many human carcinomas and is associated with tumor progression and poor prognosis. The aim of this study is to investigate the relationship between L1-CAM expression and tumor progression in tongue squamous cell carcinoma (TSCC) with a hope of understanding the molecular mechanisms of TSCC. The expression of L1-CAM in TSCC cell lines was determined by Western blotting, FACS, and ELISA analyses. Genetically manipulated TSCC stable cell lines with either over-expressed or down-regulated L1-CAM were generated by retroviral vector carrying L1-CAM cDNA transfection or lentiviral vector-mediated L1-CAM siRNA delivery, respectively. The resultant cell lines were determined and compared behavior changes including growth rate, migration and invasion in vitro using MTS and Transwell migration assay. We found that L1-CAM was detected in all testing TSCC cell lines (SAS, SCC4, SCC9, SCC25) with a variable expression level and negligible in human normal oral fibroblast cells. Over-expressed L1-CAM in L1-CAM low-expressing SCC25 cells enhanced cell growth, migration, invasion and attachment on fibronectin. Conversely, knockdown L1-CAM in L1-CAM highly-expressing SCC4 cells resulted in decreased cell ability in adhesion and motility. Moreover, L1-CAM mediated cell motility was in accordance with increased E-cadherin and decreased Vimentin and Fibronectin expression, suggesting that L1-CAM is involved in epithelial-mesenchymal transition (EMT) of TSCC. Our results demonstrated the first time that L1-CAM conferred TSCC tumor progression through the pathway of EMT. These findings also suggested that L1-CAM is a promising molecular target for the treatment of oral cancer.
CONTENTS
Contents…………………………………………………………………Ⅰ
Figure contents………………………………………………………….Ⅲ
致謝……………………………………………………………………..Ⅴ
中文摘要………………………………………………………………..Ⅶ
Abstract…………………………………………………………………Ⅷ
1. INTRODUCTION……………………………………………………1
1.1. Oral Cancer.............................................................................................................1
1.2. The role of cell adhesion molecules in human oral cancer……………………….3
1.3. L1 Cell Adhesion Molecule.....................................................................................3
1.4. Epithelial-Mesenchymal Transition……………………………............................5
1.5. Aim of this study……………..…………………………………...........................5
2. MATERIALS AND METHODS…………………………………….7
2.1. Cell lines and cell culture........................................................................................7
2.2.Antibodies .........................................................................................7
2.3. RNA isolation and Real-Time Polymerase Chain Reaction……………………....8
2.4. Western Blotting......................................................................................................8
2.5. ELISA .....................................................................................................................9
2.6. Immunohistochemistry............................................................................................9
2.7. L1CAM surface staining and flow cytometry.......................................................10
2.8. Lentiviral vector Preparation.................................................................................10
2.9.Lentivirus Infection..........................................................................................................11
2.10. Adhesion assay....................................................................................................11
2.11. Transwell- migration assay..................................................................................12
2.12. Matrigel invasion assay.......................................................................................12
2.13. Gelatin zymography............................................................................................13
2.14. Propidium iodide Nucleic acid stain...................................................................13
2.15. MTS assay……………………………………………………………………...14
2.16. In vivo tumor formation assay.............................................................................14
2.17. Statistical analysis………………………………………………………...……14
3. RESULTS……………………………………………………………15
3.1. The expression of L1-CAM mRNA in TSCC cell lines…………….......................15
3.2. L1-CAM was overexpressed in TSCC cell lines…............................................................15
3.3. The correlation between L1-CAM expression level and function of TSCC ……16
3.4. The correlation of L1-CAM and clinicpathologic features……………………...17
3.5. The effect of L1-CAM on adhesion ability and motility of genetically manipulated TSCC stable cell lines in vitro……………………………….……17
3.6. L1-CAM mediated cell migration and invasion were relatived with the gelatinase activity…………………………………………………………………………..19
3.7. L1-CAM was involved in epithelial-mesenchymal transition (EMT) of TSCC...19
3.8. The effect of L1-CAM on cell growth in vitro…..................................................20
3.9. L1-CAM was involved in cell proliferation by affecting cell cycle progression..21
3.10. In vivo tumor formation of genetically manipulated TSCC stable cell lines…..21
4. DISSCUSION......................................................................................22
5. REFERENCES...................................................................................24



Figure Contents
Figure 1. L1-CAM mRNA expression in TSCC cell lines…......………………….29
Figure 2. The expression of L1-CAM in oral cell lines…………………………...30
Figure 3. Adhesion ability of TSCC cell lines………….........................................31
Figure 4. Migratory ability of TSCC cell lines in vitro…………………………32
Figure 5. Invasiveness of TSCC cell lines in vitro………………………………...33
Figure 6. Detection of L1-CAM expression in clinical oral cancer tissue……….34
Figure 7. Down-regulation of L1-CAM significantly decreased cell adhesion ability of SCC4 cells………………..…………………………………...35
Figure 8. Down-regulation of L1-CAM significantly decreased transwell-migration of SCC4 cells in vitro……..…………...................36
Figure 9. Down-regulation of L1-CAM significantly decreased cell invasion of SCC4 cells in vitro…..…………………………………………………37
Figure 10. Overexpression of L1-CAM significantly increased cell adhesion ability of SCC9 and SCC25 cells………………………………………38
Figure 11. Overexpression of L1-CAM significantly increased transwell-migration of SCC25 cells in vitro……………………………………………………39
Figure 12. Overexpression of L1-CAM significantly increased cell
invasion of SCC25 cells in vitro…………............................................40
Figure 13. Coordinate expression of L1-CAM and MMP-2 in TSCC cell lines...41
Figure 14. Down-regulation of L1-CAM reversed EMT in SCC4 cells………….42
Figure 15. Up-regulate L1-CAM expression in SCC25 cell promote EMT……..43
Figure 16. The effect of L1-CAM on cell growth in vitro.….…………………….44
Figure 17. L1-CAM was involved in cell proliferation by affecting cell cycle progression……………………………………………………………...45
Figure 18. Down-regulation of L1-CAM suppressed growth of SCC4 orthotopic tumor……..……………….………………………………………….....46
7. TABLE……………………………………………………………….47
Table 1. The primers of real-time PCR……………………………………………47
Table 2. ELISA of soluble form L1-CAM in C.M of TSCC cell lines……………47
8. SUPPLEMENT……………………………………………………...48
Supplementary 1. The death rate of oral cancer between 1986 to 2008………....48
Supplementary 2. The age-standard mortality rate of oral cancer in Taiwan….48
Supplementary 3. Schematic structure of the L1-CAM protein …………………49
References :
1.Parkin, D. M., Pisani, P., and Ferlay, J. Estimates of the worldwide incidence of eighteen major cancers in 1985. Int J Cancer, 54: 594-606, 1993.
2.Su, C. C., Chung, J. A., Hsu, Y. Y., Huang, S. J., and Lian, I. B. Age at diagnosis and prognosis of oral cancer in relation to the patient''s residential area: Experience from a medical center in Taiwan. Oral Oncol, 2008.
3.Silverman, S., Jr. Demographics and occurrence of oral and pharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc, 132 Suppl: 7S-11S, 2001.
4.Boffetta, P., Hecht, S., Gray, N., Gupta, P., and Straif, K. Smokeless tobacco and cancer. Lancet Oncol, 9: 667-675, 2008.
5.Riedel, F., Goessler, U. R., and Hormann, K. Alcohol-related diseases of the mouth and throat. Dig Dis, 23: 195-203, 2005.
6.Chen, Y. J., Chang, J. T., Liao, C. T., Wang, H. M., Yen, T. C., Chiu, C. C., Lu, Y. C., Li, H. F., and Cheng, A. J. Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis. Cancer Sci, 99: 1507-1514, 2008.
7.Novakova, V. and Laco, J. [Role of human papillomavirus in carcinogenesis of head and neck cancer]. Klin Onkol, 21: 141-148, 2008.
8.Awang, M. N. Betel quid and oral carcinogenesis. Singapore Med J, 29: 589-593, 1988.
9.Yang, Y. H., Chen, C. H., Chang, J. S., Lin, C. C., Cheng, T. C., and Shieh, T. Y. Incidence rates of oral cancer and oral pre-cancerous lesions in a 6-year follow-up study of a Taiwanese aboriginal community. J Oral Pathol Med, 34: 596-601, 2005.
10.Yang, Y. H., Lee, H. Y., Tung, S., and Shieh, T. Y. Epidemiological survey of oral submucous fibrosis and leukoplakia in aborigines of Taiwan. J Oral Pathol Med, 30: 213-219, 2001.
11.Murti, P. R., Gupta, P. C., Bhonsle, R. B., Daftary, D. K., Mehta, F. S., and Pindborg, J. J. Effect on the incidence of oral submucous fibrosis of intervention in the areca nut chewing habit. J Oral Pathol Med, 19: 99-100, 1990.
12.Chen, Y. K., Huang, H. C., Lin, L. M., and Lin, C. C. Primary oral squamous cell carcinoma: an analysis of 703 cases in southern Taiwan. Oral Oncol, 35: 173-179, 1999.
13.Liao, C. T., Kang, C. J., Chang, J. T., Wang, H. M., Ng, S. H., Hsueh, C., Lee, L. Y., Lin, C. H., Cheng, A. J., Chen, I. H., Huang, S. F., and Yen, T. C. Survival of second and multiple primary tumors in patients with oral cavity squamous cell carcinoma in the betel quid chewing area. Oral Oncol, 43: 811-819, 2007.
14.Liao, C. T., Chang, J. T., Wang, H. M., Ng, S. H., Hsueh, C., Lee, L. Y., Lin, C. H., Chen, I. H., Huang, S. F., Cheng, A. J., and Yen, T. C. Analysis of risk factors of predictive local tumor control in oral cavity cancer. Ann Surg Oncol, 15: 915-922, 2008.
15.Hennessey, P. T., Westra, W. H., and Califano, J. A. Human papillomavirus and head and neck squamous cell carcinoma: recent evidence and clinical implications. J Dent Res, 88: 300-306, 2009.
16.Tachezy, R., Klozar, J., Rubenstein, L., Smith, E., Salakova, M., Smahelova, J., Ludvikova, V., Rotnaglova, E., Kodet, R., and Hamsikova, E. Demographic and risk factors in patients with head and neck tumors. J Med Virol, 81: 878-887, 2009.
17.Kalnins, I. K., Leonard, A. G., Sako, K., Razack, M. S., and Shedd, D. P. Correlation between prognosis and degree of lymph node involvement in carcinoma of the oral cavity. Am J Surg, 134: 450-454, 1977.
18.Schuller, D. E., McGuirt, W. F., McCabe, B. F., and Young, D. The prognostic significance of metastatic cervical lymph nodes. Laryngoscope, 90: 557-570, 1980.
19.Ho, C. M., Lam, K. H., Wei, W. I., Lau, S. K., and Lam, L. K. Occult lymph node metastasis in small oral tongue cancers. Head Neck, 14: 359-363, 1992.
20.Ziober, B. L., Silverman, S. S., Jr., and Kramer, R. H. Adhesive mechanisms regulating invasion and metastasis in oral cancer. Crit Rev Oral Biol Med, 12: 499-510, 2001.
21.Georgolios, A., Batistatou, A., Charalabopoulos, A., Manolopoulos, L., and Charalabopoulos, K. The role of CD44 adhesion molecule in oral cavity cancer. Exp Oncol, 28: 94-98, 2006.
22.Yanamoto, S., Kawasaki, G., Yoshitomi, I., Iwamoto, T., Hirata, K., and Mizuno, A. Clinicopathologic significance of EpCAM expression in squamous cell carcinoma of the tongue and its possibility as a potential target for tongue cancer gene therapy. Oral Oncol, 43: 869-877, 2007.
23.Rathjen, F. G. and Schachner, M. Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. Embo J, 3: 1-10, 1984.
24.Moos, M., Tacke, R., Scherer, H., Teplow, D., Fruh, K., and Schachner, M. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature, 334: 701-703, 1988.
25.Brummendorf, T., Kenwrick, S., and Rathjen, F. G. Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations. Curr Opin Neurobiol, 8: 87-97, 1998.
26.Demyanenko, G. P. and Maness, P. F. The L1 cell adhesion molecule is essential for topographic mapping of retinal axons. J Neurosci, 23: 530-538, 2003.
27.Oleszewski, M., Beer, S., Katich, S., Geiger, C., Zeller, Y., Rauch, U., and Altevogt, P. Integrin and neurocan binding to L1 involves distinct Ig domains. J Biol Chem, 274: 24602-24610, 1999.
28.Ruppert, M., Aigner, S., Hubbe, M., Yagita, H., and Altevogt, P. The L1 adhesion molecule is a cellular ligand for VLA-5. J Cell Biol, 131: 1881-1891, 1995.
29.Montgomery, A. M., Becker, J. C., Siu, C. H., Lemmon, V. P., Cheresh, D. A., Pancook, J. D., Zhao, X., and Reisfeld, R. A. Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3. J Cell Biol, 132: 475-485, 1996.
30.Gavert, N., Sheffer, M., Raveh, S., Spaderna, S., Shtutman, M., Brabletz, T., Barany, F., Paty, P., Notterman, D., Domany, E., and Ben-Ze''ev, A. Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis. Cancer Res, 67: 7703-7712, 2007.
31.Nayeem, N., Silletti, S., Yang, X., Lemmon, V. P., Reisfeld, R. A., Stallcup, W. B., and Montgomery, A. M. A potential role for the plasmin(ogen) system in the posttranslational cleavage of the neural cell adhesion molecule L1. J Cell Sci, 112 ( Pt 24): 4739-4749, 1999.
32.Mechtersheimer, S., Gutwein, P., Agmon-Levin, N., Stoeck, A., Oleszewski, M., Riedle, S., Postina, R., Fahrenholz, F., Fogel, M., Lemmon, V., and Altevogt, P. Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins. J Cell Biol, 155: 661-673, 2001.
33.Voura, E. B., Ramjeesingh, R. A., Montgomery, A. M., and Siu, C. H. Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells. Mol Biol Cell, 12: 2699-2710, 2001.
34.Senner, V., Kismann, E., Puttmann, S., Hoess, N., Baur, I., and Paulus, W. L1 expressed by glioma cells promotes adhesion but not migration. Glia, 38: 146-154, 2002.
35.Thies, A., Schachner, M., Moll, I., Berger, J., Schulze, H. J., Brunner, G., and Schumacher, U. Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma. Eur J Cancer, 38: 1708-1716, 2002.
36.Gavert, N., Conacci-Sorrell, M., Gast, D., Schneider, A., Altevogt, P., Brabletz, T., and Ben-Ze''ev, A. L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers. J Cell Biol, 168: 633-642, 2005.
37.Gast, D., Riedle, S., Riedle, S., Schabath, H., Schlich, S., Schneider, A., Issa, Y., Stoeck, A., Fogel, M., Joumaa, S., Wenger, T., Herr, I., Gutwein, P., and Altevogt, P. L1 augments cell migration and tumor growth but not beta3 integrin expression in ovarian carcinomas. Int J Cancer, 115: 658-665, 2005.
38.Primiano, T., Baig, M., Maliyekkel, A., Chang, B. D., Fellars, S., Sadhu, J., Axenovich, S. A., Holzmayer, T. A., and Roninson, I. B. Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements. Cancer Cell, 4: 41-53, 2003.
39.Silletti, S., Yebra, M., Perez, B., Cirulli, V., McMahon, M., and Montgomery, A. M. Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to L1 cell adhesion molecule-dependent motility and invasion. J Biol Chem, 279: 28880-28888, 2004.
40.Meier, F., Busch, S., Gast, D., Goppert, A., Altevogt, P., Maczey, E., Riedle, S., Garbe, C., and Schittek, B. The adhesion molecule L1 (CD171) promotes melanoma progression. Int J Cancer, 119: 549-555, 2006.
41.Gast, D., Riedle, S., Issa, Y., Pfeifer, M., Beckhove, P., Sanderson, M. P., Arlt, M., Moldenhauer, G., Fogel, M., Kruger, A., and Altevogt, P. The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies. Oncogene, 27: 1281-1289, 2008.
42.Savagner, P. Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays, 23: 912-923, 2001.
43.Voulgari, A. and Pintzas, A. Epithelial-mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta, 2009.
44.Thiery, J. P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2: 442-454, 2002.
45.Zavadil, J., Haley, J., Kalluri, R., Muthuswamy, S. K., and Thompson, E. Epithelial-mesenchymal transition. Cancer Res, 68: 9574-9577, 2008.
46.Yanjia, H. and Xinchun, J. The role of epithelial-mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis. Clin Chim Acta, 383: 51-56, 2007.
47.Franz, M., Spiegel, K., Umbreit, C., Richter, P., Codina-Canet, C., Berndt, A., Altendorf-Hofmann, A., Koscielny, S., Hyckel, P., Kosmehl, H., Virtanen, I., and Berndt, A. Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma. Histochem Cell Biol, 131: 651-660, 2009.
48.Yokoyama, K., Kamata, N., Fujimoto, R., Tsutsumi, S., Tomonari, M., Taki, M., Hosokawa, H., and Nagayama, M. Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas. Int J Oncol, 22: 891-898, 2003.
49.Rheinwald, J. G. and Beckett, M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res, 41: 1657-1663, 1981.
50.Ruoslahti, E. Fibronectin and its integrin receptors in cancer. Adv Cancer Res, 76: 1-20, 1999.
51.Shtutman, M., Levina, E., Ohouo, P., Baig, M., and Roninson, I. B. Cell adhesion molecule L1 disrupts E-cadherin-containing adherens junctions and increases scattering and motility of MCF7 breast carcinoma cells. Cancer Res, 66: 11370-11380, 2006.
52.Needham, L. K., Thelen, K., and Maness, P. F. Cytoplasmic domain mutations of the L1 cell adhesion molecule reduce L1-ankyrin interactions. J Neurosci, 21: 1490-1500, 2001.
53.Kujat, R., Miragall, F., Krause, D., Dermietzel, R., and Wrobel, K. H. Immunolocalization of the neural cell adhesion molecule L1 in non-proliferating epithelial cells of the male urogenital tract. Histochem Cell Biol, 103: 311-321, 1995.
54.Kowitz, A., Kadmon, G., Eckert, M., Schirrmacher, V., Schachner, M., and Altevogt, P. Expression and function of the neural cell adhesion molecule L1 in mouse leukocytes. Eur J Immunol, 22: 1199-1205, 1992.
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