臺灣博碩士論文加值系統

緊密連接蛋白(claudins)家族共有24個成員，在細胞內負責細胞之間的緊密連接，限制物質的進出使細胞產生極性。有趣的是近年來已有許多研究證實,在癌組織中claudins成員不只單純負責細胞屏障的功能，而會因應不同器官的癌組織，某些特定成員會有異常的表現甚至是參與癌的發展過程；如claudin-1已被證實當鼻咽癌細胞受到抗癌藥物的刺激下，會透過大量表現claudin-1使癌細胞產生抗細胞凋亡的能力，然而其中的分子機制並未清楚，因此在本篇論文中我們透過在鼻咽癌細胞株中大量表現claudin-1後，分析細胞癌化的影響。結果顯示在3-D立體培養條件下，大量表現claudin-1不僅會降低癌細胞形成細胞群落的能力，也會使細胞之間的界線變得明顯，進一步經由皮下注射癌細胞至免疫不全鼠也發現claudin-1的表現會抑制腫瘤的形成。由於發現細胞界線的改變，我們透過西方墨點法觀察分化標誌蛋白cytokeratin 8的表現，結果發現，在大量表現claudin-1的兩組clones中，CK8的表現高於原本的鼻咽癌細胞，因此我們認為，在鼻咽癌中大量表現claudin-1會透過促進細胞分化的方式來抑制腫瘤的生長

Claudin superfamily has been demonstrated an important role in cell to cell communication. Of interest, numerous evidences reveal that abnormal expression of claudins, such as claudin-1 contributed to tumor progression. Previous study demonstrated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma (NPC) cells. However, the mechanism is still not clear. In this study, exogenous expression of claudin-1 in NPC cells was utilized to assess effect of claudin-1 in tumor cells. Via anchorage independent assay and 3-D tumor spheroid formation analysis, intriguingly, overexpressed claudin-1 not only reduced tumor cells colonies size, but repressed the cell cell’s connection. Base on the change of the cell’s connection, we examine the cell differentiation marker Cytokeratin 8(CK8), than found the CK8 upregulated in two Claudin-1 stable expressing clones. Thus, our preliminary results show that claudin-1 reduced tumor cells colonies size of NPC.

Abstract I
中文摘要 II
目錄 III
第一章、緒論 1
1-1鼻咽癌 1
1-1.1致病因子 1
1-1.2臨床病徵與治療 2
1-1.3病理分類與研究發展 3
1-2緊密連接蛋白(Claudins) 4
1-3緊密連接蛋白(Claudins)與疾病 6
1-4研究目的 7
第二章、材料與方法 8
2-1化學試劑與材料 8
2-2溶液 9
2-3實驗方法與步驟 11
2-3.1細胞培養 11
2-3.2蛋白質製備 11
2-3.3西方墨點法 12
2-3.4 MTT Assay 13
2-3.5細胞爬行 13
2-3.6耗氧量及乳酸生成 14
2-3.7 Soft agar assay 14
2-3.8免疫螢光染色 15
第三章、結果 16
3-1建立長期穩定表現Flag-Claudin-1的癌細胞株 16
3-2大量表現claudin-1改變鼻咽癌細胞的型態 17
3-3大量表現claudin-1不會影響細胞生長速率 17
3-4大量表現claudin-1並不會影響細胞遷移的能力 18
3-5大量表現claudin-1不會影響癌細胞的氧氣的消耗 19
3-6大量表現caudin-1會降低細胞在非貼附下形成細胞群落的大小 19
3-73-D細胞培養下大量表現claudin-1會降低細胞形成群落的能力 21
3-8在InVivo的條件下，claudin-1會降低腫瘤形成的能力 21
3-9大量表現claudin-1會改變細胞分化的狀態 22
3-10抗癌藥物刺激，claudin-1的增加也會促進細胞的分化 23
第四章、討論 24
第五章、參考文獻 29
第六章、圖 35

1.Wei, W.I. and J.S. Sham, Nasopharyngeal carcinoma. Lancet, 2005. 365(9476): p. 2041-54.
2.Cheng, Y.J., et al., Cigarette smoking, alcohol consumption and risk of nasopharyngeal carcinoma in Taiwan. Cancer Causes Control, 1999. 10(3): p. 201-7.
3.Hildesheim, A., et al., Association of HLA class I and II alleles and extended haplotypes with nasopharyngeal carcinoma in Taiwan. J Natl Cancer Inst, 2002. 94(23): p. 1780-9.
4.Wei, Y.S., et al., Interleukin-10 gene promoter polymorphisms and the risk of nasopharyngeal carcinoma. Tissue Antigens, 2007. 70(1): p. 12-7.
5.Thorley-Lawson, D.A., Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol, 2001. 1(1): p. 75-82.
6.Dodd, L.E., et al., Genes involved in DNA repair and nitrosamine metabolism and those located on chromosome 14q32 are dysregulated in nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev, 2006. 15(11): p. 2216-25.
7.Shaffer, R., et al., Deriving prostate alpha-beta ratio using carefully matched groups, long follow-up and the phoenix definition of biochemical failure. Int J Radiat Oncol Biol Phys, 2011. 79(4): p. 1029-36.
8.Geara, F.B., et al., Carcinoma of the nasopharynx treated by radiotherapy alone: determinants of distant metastasis and survival. Radiother Oncol, 1997. 43(1): p. 53-61.
9.Huncharek, M. and B. Kupelnick, In regards to Baujat et al.: Chemotherapy in locally advanced nasopharyngeal carcinoma: An individual patient data meta-analysis of eight randomized trials and 1753 patients (Int J Radiat Oncol Biol Phys 2006;64:47-56). Int J Radiat Oncol Biol Phys, 2006. 65(3): p. 958; author reply 958-9.
10.Mundt, A.J., et al., Patterns of failure following high-dose chemotherapy and autologous bone marrow transplantation with involved field radiotherapy for relapsed/refractory Hodgkin's disease. Int J Radiat Oncol Biol Phys, 1995. 33(2): p. 261-70.
11.Hsu, H.C., et al., Pathology of nasopharyngeal carcinoma. Proposal of a new histologic classification correlated with prognosis. Cancer, 1987. 59(5): p. 945-51.
12.Cho, W.C., Nasopharyngeal carcinoma: molecular biomarker discovery and progress. Mol Cancer, 2007. 6: p. 1.
13.Lo, K.W. and D.P. Huang, Genetic and epigenetic changes in nasopharyngeal carcinoma. Semin Cancer Biol, 2002. 12(6): p. 451-62.
14.Ma, B.B., E.P. Hui, and A.T. Chan, Systemic approach to improving treatment outcome in nasopharyngeal carcinoma: current and future directions. Cancer Sci, 2008. 99(7): p. 1311-8.
15.Singh, A.B., A. Sharma, and P. Dhawan, Claudin family of proteins and cancer: an overview. J Oncol, 2010. 2010: p. 541957.
16.Shen, L., C.R. Weber, and J.R. Turner, The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state. J Cell Biol, 2008. 181(4): p. 683-95.
17.Sawada, N., et al., Tight junctions and human diseases. Med Electron Microsc, 2003. 36(3): p. 147-56.
18.Cereijido, M., R.G. Contreras, and L. Shoshani, Cell adhesion, polarity, and epithelia in the dawn of metazoans. Physiol Rev, 2004. 84(4): p. 1229-62.
19.Krause, G., et al., Structure and function of claudins. Biochim Biophys Acta, 2008. 1778(3): p. 631-45.
20.Feigin, M.E. and S.K. Muthuswamy, Polarity proteins regulate mammalian cell-cell junctions and cancer pathogenesis. Curr Opin Cell Biol, 2009. 21(5): p. 694-700.
21.Furuse, M., et al., Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol, 1993. 123(6 Pt 2): p. 1777-88.
22.Morita, K., et al., Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci U S A, 1999. 96(2): p. 511-6.
23.Furuse, M., et al., A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol, 1998. 143(2): p. 391-401.
24.Lal-Nag, M. and P.J. Morin, The claudins. Genome Biol, 2009. 10(8): p. 235.
25.Furuse, M., et al., Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures. J Cell Sci, 1996. 109 ( Pt 2): p. 429-35.
26.Kubota, K., et al., Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions. Curr Biol, 1999. 9(18): p. 1035-8.
27.Stevenson, B.R., et al., Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol, 1986. 103(3): p. 755-66.
28.Hamazaki, Y., et al., Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule. J Biol Chem, 2002. 277(1): p. 455-61.
29.Williams, L.A., et al., Identification and characterisation of human Junctional Adhesion Molecule (JAM). Mol Immunol, 1999. 36(17): p. 1175-88.
30.Yamamoto, Y., et al., Distinct roles of Rab3B and Rab13 in the polarized transport of apical, basolateral, and tight junctional membrane proteins to the plasma membrane. Biochem Biophys Res Commun, 2003. 308(2): p. 270-5.
31.Wu, X., et al., Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc Natl Acad Sci U S A, 2000. 97(8): p. 4233-8.
32.Nakamura, T., et al., huASH1 protein, a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene, localizes to both nuclei and cell-cell tight junctions. Proc Natl Acad Sci U S A, 2000. 97(13): p. 7284-9.
33.Piontek, J., et al., Formation of tight junction: determinants of homophilic interaction between classic claudins. FASEB J, 2008. 22(1): p. 146-58.
34.Wilcox, E.R., et al., Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell, 2001. 104(1): p. 165-72.
35.Al-Haggar, M., et al., Familial hypomagnesemia with hypercalciuria and nephrocalcinosis: unusual clinical associations and novel claudin16 mutation in an Egyptian family. Clin Exp Nephrol, 2009. 13(4): p. 288-94.
36.Martin, T.A. and W.G. Jiang, Loss of tight junction barrier function and its role in cancer metastasis. Biochim Biophys Acta, 2009. 1788(4): p. 872-91.
37.Tan, C., et al., Regulation of tumor angiogenesis by integrin-linked kinase (ILK). Cancer Cell, 2004. 5(1): p. 79-90.
38.Mullin, J.M., et al., Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology. J Cell Physiol, 1992. 152(1): p. 35-47.
39.Schoenenberger, C.A., et al., Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras. J Cell Biol, 1991. 112(5): p. 873-89.
40.Soini, Y., Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology, 2005. 46(5): p. 551-60.
41.Kominsky, S.L., et al., Loss of the tight junction protein claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast. Oncogene, 2003. 22(13): p. 2021-33.
42.Kwon, M.J., et al., Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications. Carcinogenesis, 2010. 31(6): p. 974-83.
43.Michl, P., et al., Claudin-4 expression decreases invasiveness and metastatic potential of pancreatic cancer. Cancer Res, 2003. 63(19): p. 6265-71.
44.Lee, J.W., et al., Increased expressions of claudin-1 and claudin-7 during the progression of cervical neoplasia. Gynecol Oncol, 2005. 97(1): p. 53-9.
45.Reichert, M., T. Muller, and W. Hunziker, The PDZ domains of zonula occludens-1 induce an epithelial to mesenchymal transition of Madin-Darby canine kidney I cells. Evidence for a role of beta-catenin/Tcf/Lef signaling. J Biol Chem, 2000. 275(13): p. 9492-500.
46.Jenkins, E.L., et al., Chronic hypoxia down-regulates tight junction protein ZO-2 expression in children with cyanotic congenital heart defect. ESC Heart Fail, 2016. 3(2): p. 131-137.
47.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.
48.Jin, T., I. George Fantus, and J. Sun, Wnt and beyond Wnt: multiple mechanisms control the transcriptional property of beta-catenin. Cell Signal, 2008. 20(10): p. 1697-704.
49.Lee, J.W., et al., Upregulated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma cells. Int J Cancer, 2010. 126(6): p. 1353-66.
50.Hanahan, D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1): p. 57-70.
51.Reddy, S.P., et al., Prognostic significance of keratinization in nasopharyngeal carcinoma. Am J Otolaryngol, 1995. 16(2): p. 103-8.
52.Brodeur, G.M., Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer, 2003. 3(3): p. 203-16.
53.Huo, Q., et al., Claudin-1 protein is a major factor involved in the tumorigenesis of colorectal cancer. Anticancer Res, 2009. 29(3): p. 851-7.
54.Bhat, A.A., et al., Claudin-1 promotes TNF-alpha-induced epithelial-mesenchymal transition and migration in colorectal adenocarcinoma cells. Exp Cell Res, 2016. 349(1): p. 119-127.
55.Pope, J.L., et al., Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling. Gut, 2014. 63(4): p. 622-34.
56.Lourenco, S.V., et al., Oral squamous cell carcinoma: status of tight junction claudins in the different histopathological patterns and relationship with clinical parameters. A tissue-microarray-based study of 136 cases. J Clin Pathol, 2010. 63(7): p. 609-14.
57.Li, W.J., et al., Expression of claudin-1 and its relationship with lymphatic microvessel generation in hypopharyngeal squamous cell carcinoma. Genet Mol Res, 2015. 14(4): p. 11814-26.
58.Hsueh, C., et al., Expression pattern and prognostic significance of claudins 1, 4, and 7 in nasopharyngeal carcinoma. Hum Pathol, 2010. 41(7): p. 944-50.
59.Dhawan, P., et al., Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer. J Clin Invest, 2005. 115(7): p. 1765-76.
60.Jian, Y., et al., Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells. Biochem Biophys Res Commun, 2015. 466(3): p. 356-61.
61.Lippoldt, A., et al., Organization of choroid plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, -2 and -5 expression by protein kinase C. Neuroreport, 2000. 11(7): p. 1427-31.
62.Leotlela, P.D., et al., Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility. Oncogene, 2007. 26(26): p. 3846-56.
63.Yan, M., et al., IKKalpha restoration via EZH2 suppression induces nasopharyngeal carcinoma differentiation. Nat Commun, 2014. 5: p. 3661.
64.Nowak, D., D. Stewart, and H.P. Koeffler, Differentiation therapy of leukemia: 3 decades of development. Blood, 2009. 113(16): p. 3655-65.
65.Rane, J.K., D. Pellacani, and N.J. Maitland, Advanced prostate cancer[mdash]a case for adjuvant differentiation therapy. Nat Rev Urol, 2012. 9(10): p. 595-602.