臺灣博碩士論文加值系統

化學療法和放射療法是兩種有效治療鼻咽癌的方法，然而，癌細胞的遠距離轉移卻往往造成治療上的失敗。　我們從鼻咽癌患者分離出轉移到骨髓的鼻咽癌上皮細胞株 (NPC-BM1) 中進一步再分離出不轉移的NPC-BM00，以及高轉移率的NPC-BM29 二種細胞株。　其中NPC-BM29 受到上皮間質轉化 (EMT) 的影響表現出侵入性，由上皮類型轉分化為間質顯型。　NPC-BM00 則表現出典型的多邊形細胞型態，而 NPC-BM29 則可能以各種型態表現，舉凡單極的折射紡錘狀，以及雙極的細胞質突起，或是細長的紡錘狀等。　在 2% 的血清中，NPC-BM29 細胞分裂一次的平均時間比 NPC-BM00 要來得快，分別是 26.76 小時和 30.67 小時。　從創傷－癒合之細胞移動 (Scrape-wound Migration) 實驗中發現不管腫瘤促進因子 (TPA)，甘草酸 (GL)，和抗腫瘤藥物 (HPMPC) 的有無，NPC-BM29 的能動性都比 NPC-BM00 還要高。　轉移的潛在性可以由 92-kDa typeIV collagenases/gelatinase B (MMP-2 和 MMP-9) 的差異性基因表現得知；兩種酵素都和腫瘤細胞的入侵和轉移有關。　NPC-BM29細胞表現MMP-9比NPC-BM00細胞大約高10倍。 與 NPC-BM00 相比較，NPC-BM29 的侵入表現型伴隨著抑制 E-cadherin 以及 β-catenin 的表現，以及提高 Ets1 轉錄因子的表現率。　由流式細胞分析對細胞表面標誌和腫瘤相關分子的 immunophenotypic characterization 可以看出，在 NPC-BM29 中AE3，MAK6 (細胞角質蛋白 8，14，15，16，18和19 的混合)，E-cadherin以及 sialyl Tn 的產生非常明顯，而在 NPC-BM00 中則是 CEA 和 CD54 產生率有顯著的提高。　在 cytokine array ELISA 中，我們發現在第一天 NPC-BM29 相對於 NPC-BM00 來說釋放極高量的 IL-6，IL-8，IL-10 和 IL-13，而兩者在 IL-2 的釋放率中並沒有什麼差別。　值得注意的是，到了第三天，NPC-BM00 製造的 IL-6 比 NPC-BM29 還要多出三倍，然而 IL-8，IL-10和 IL-13 的量與第一天相去無異。　到了第五天，兩者皆保持著差不多高的 IL-6 產生率。　儘管 NPC-BM29 在 IL-6 的產生率並不像 NPC-BM00 一樣，整個試驗中卻持續著產生較高量的 IL-8 和 IL-10。　總括來說，在這項研究中，由上皮形變為間質的特徵以及與這些改變相關的潛在分子，提供了針對研發抗腫瘤細胞轉移的藥物一個舉足輕重的線索。

Concurrent chemotherapy and radiotherapy are effective in treatment of nasopharyngeal carcinoma (NPC). Distant metastasis, however, is the major cause of failure. NPC-BM1, an epithelial cell line established from a bone marrow biopsy of NPC patient, was further separated into non-metastasizing and highly metastasizing cell clones, designated NPC-BM00 and NPC-BM29, respectively. The metastasizing clone NPC-BM29 acquired the invasive phenotype marked by epithelial mesenchymal transition (EMT), transdifferentiation from epithelial type to mesenchymal phenotype. NPC-BM00 cells exhibited typical well-attached polygonal epithelial cell morphology. In contrast, NPC-BM29 was heterogeneous, from refractile, spindle-shaped cells with monopolar and occasional bipolar cytoplasmic processes, to elongated fibroblast-like shape cells. NPC-BM29 cells grew better in 2% serum than the non-metastasizing NPC-BM00 cells, with population doubling time of 26.76 h and 30.67 h, respectively. The scrape-wound migration assay revealed that NPC-BM29 cells exhibit higher motility than NPC-BM00, both in the presence and absence of 12-O-tetradecanyol-phorbol-13-acetate (TPA), glycyrrhizic acid (GL) and HPMPC (Cidofovir). The metastasis potential was confirmed by differential expression of 92-kDa type IV collagenases/gelatinase B (MMP-2 and MMP-9); both enzymes are involved in tumor cell invasion and metastasis. NPC-BM29 cells express approximately more than 10-fold of MMP-9 than NPC-BM00 cells. Compared to NPC-BM00, the acquisition of the invasive phenotype in NPC-BM29 was accompanied by downregulation of E-cadherin and its associated protein�n��-catenin, and with concomitant increased the expression of the transcription factor Ets1. Immunophenotypic characterization of cell surface markers and tumor-associated molecules by Flow cytometric analysis indicated that AE3, MAK6 (a mixture of cytokeratins 8, 14, 15, 16, 18, and 19), E-cadherin and sialyl Tn were significantly upregulated in NPC-BM29 cells, whereas the expression of CEA and CD54 were significantly increased in NPC-BM00 cells. In cytokine array ELISA, we found that
NPC-BM29 consistently released higher levels of IL-6, IL-8, IL-10, and IL-13 than that released by NPC-BM00 cells on day 1. There was no difference in the expression level of IL-2a in both cell lines. Interestingly, on day 3 the production of IL-6 by NPC-BM00 was markedly increased to approximately 3-fold higher than that of released by NPC-BM29. However, the production of IL-8, IL-10, and IL-13 on day 3 remained at the similar level as on day 1. On day 5, both cell lines maintained similar high levels of IL-6 production. Despite the fluctuation of expression level of IL-6, NPC-BM29 consistently produced higher levels of IL-8 and IL-10 throughout the entire period of assays. Taken together, the characterization of morphologic changes from epithelial to mesenchymal transition and the identification of the underlying molecules associated with these changes described in this study provide a lead for the design of specific anti-invasive drugs.

Contents…………………………………………………1
致謝……………………………………………………..2
Abstract ………………………………………………...3-4
中文摘要.….……………………………………………5-6
Introduction …………………………………………….7-10
Material & Methods………………………………...…..11-16
Results…………………………………………….…….17-21
Discussion……………………………………………....22-27
Acknowledges………..…………………………………28
References………………………………………………29-33
Tables …………………………………………………..34
Legends to Figures….………………………………..…35-37
Figures …………………………………………………38-49
Appendix…………………………………………….…50-51

1.Belperio, J. A., M. P. Keane, D. A. Arenberg, C. L. Addison, J. E. Ehlert, M. D. Burdick, and R. M. Strieter. 2000. CXC chemokines in angiogenesis. J. Leukoc Biol. 68:1-8.
2.Bonnet, M., J. M. Guinebretiere, E. Kremmer, V. Grunewald, E. Benhamou, G. Contesso, and I. Joab. 1999. Detection of Epstein-Barr virus in invasive breast cancers. J. Natl. Cancer Inst. 16:1376-1381.
3.Bracke, M. E., F. M. Van Roy, and M. M. Mareel. 1996. The E-cadherin/catenin complex in invasion and metastasis. Curr. Top Microbiol. Immunol. 213:123-161.
4.Carmeliet, P., and R. K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature 407:249-257.
5.Cooper, D. A., P. O. Pehrson, C. Pedersen, M. Moroni, E. Oksenhendler, W. Rozenbaum, N. Clumeck, V. Faber, W. Stille, B. Hirschel, C. Farthing, R. Doherty, and J. M. Yeo. 1993. The efficacy and safety of zidovudine alone or as cotherapy with acyclovir for the treatment of patients with AIDS and AIDS –related complex: a double-blind, randomized trial. AIDS 7:197-207.
6.Delannoy-Courdent, A., V. Mattot, V. Fafeur, W. Fauquette, I. Pollet, T. Calmels, C. Vercamer, B. Boilly, B. Vandenbunder, and X. Desbiens. 1998. The expression of an Ets-1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells. J. Cell Sci. 111:1521-1534.
7.Dittmer, D., and A. Nordheim. 1998. Ets transcription factors and human disease. Biochimica et Biophysica Acta 1377:1-11.
8.Ellis, L. M., and I. J. Fidler. 1996. Angiogenesis and metastasis. Euro. J. Cancer 32:2451-2460.
9.Fandi, A., M. Altun, N. Azli, J. P. Armand, and E. Cvitkovic. 1994. Nasopharyngeal cancers: epidemiology, staging, and treatment. Sem. Oncol. 21:382-397.
10.Folkman, J. 1971. Tumor angiogenesis: therapeutic implications. J. Engl. J. Med. 285:1182-1186.
11.Fukayama, M., J. Chong, and Y. Kaizaki. 1998. Epstein-Barr virus and gastric carcinoma. Gastric Cancer 1:104-114.
12.Fukayama, M., Y. Hayashi, Y. Iwasaki, J. Chong, T. Ooba, T. Takizawa, M. Koike, S. Mizutani, M. Miyaki, and K. Hirai. 1994. Epstein-Barr virus-associated gastric carcinoma and Epstein Barr virus infection of the stomach. Lab. Invest. 71: 73-81.
13.Gilles, F., M. -B. Raes, D. Stehelin, B. Vandenbunder, and V. Fafeur. 1996. The c-Ets-1 proto-oncogene is a new early-response gene differentially regulated by cytokines and growth factors in human fibroblasts. Exp. Cell Res. 222:370-378.
14.Gotzmann, J., M. Mikula, A. Eger, R. Schulte-Hermann, R. Foisner, H. Beug, and W. Mikulits. 2004. Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metrastasis. Mutat. Res. 556:9-20.
15.Ho, J.H. 1978. An epidemiologic and clinical study of nasopharyngeal carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 4:182-198.
16.Kim, K. -R., T. Yoshizaki, H. Miyamori, K. Hasegawa, T. Horikawa, M. Furukawa, S. Harada, M. Seiki, and H. Sato. 2000. Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets-1 and invasive growth. Oncogene 19:1764-1771.
17.Liao, S. -K., Y. –P. Perng, Y. –C. Shen, P. –J. Chung, Y. –S. Chang, and C. –H. Wang. 1998. Chromosomal abnormalities of a new nasopharyngel carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet Cytogenet 103:52-58.
18.Lin, H.-J. and J. –C Lin. 2004. Pathogenesis and therapy for Epstein-Barr virus-associated nasopharyngeal carcinoma. J. Chinese Oncol. Soc. 20:1-17.
19.Lin, J.-C. 2006. Pathogenesis and therapy of Epstein-Barr virus infection: Novel therapeutic approaches, p. 1-35. In New Developments in Epstein-Barr Virus, Constantine S. Umar, ed., Nova Science Publishers, Inc., New York, NY.
20.Liotta, L. A. 1986. Tumor invasion and metastases- Role of the extracellular matrix: rhoads memorial award lecture. Cancer Res. 46:1-7.
21.Nagase, H., and J. F. Woessner. 1999. Matrix Metalloproteinases. J. Biol. Chem. 274:21491-21494.
22.Nakayama, T., M. Ito, A. Ohtsuru, S. Naito, M. Nakashima, J. A. Fagin, S. Yamashita, and I. Sekine. 1996. Expression of the Ets-1 proto-oncogene in human gastric carcinoma: correlation with tumor invasion. Am. J. Pathol. 149:1931-1939.
23.Rickinson, A.B., and E. Kieff. 1996. Epstein-Barr virus, p. 2391-2246. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lippincott-Raven, Philadelphia, Pa.
24.Sato, H., Y. Kida, M. Mai, Y. Endo, T. Sasaki, J. Tanaka, and M. Seiki. 1992. Expression of genes encoding type IV collagen-degrading metalloproteinases and tissue inhibitors of metalloproteinases in various human tumor cells. Oncogene 7:77-83.
25.Shanmugaratnam, K. 1978. Histological typing of nasopharyngeal carcinoma. IARC Sci. Publ. 20:3-12.
26.Shapiro, S. D. 1998. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol. 10:602-608.
27.Takeichi, M. 1993. Cadherins in cancer: implications for invasion and metastasis. Curr. Opin. Cell Biol. 5:806-811.
28.Thiery, J. P. 2002. Epithelial-mesenchymal transitions in tumor progression. Nat. Rev. Cancer 2:442-454.
29.Uraguchi, M., M. Morikawa, M. Shirakawa, K. Sanada, and K. Imai. 2004. Activation of WNT family expression and signaling in squamous cell carcinomas of the oral cavity. J. Dent. Res. 83:327-332.
30.Vihinen, P., and V. -M. Kahari. 2002. Matrix metalloproteinases in cancer: prognostic markers and therapeutic targets. Int. J. Cancer 99:157-166.
31.Weidner, N., J. P. Semple, W. R. Welch, and J. Folkman. 1991. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. J. Engl. J. Med. 324:1-8.
32.Westermarck, J., and V. -M. Kahari. 1999. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 13:781-792.
33.Yang, J., S. A. Mani, J. L. Donaher, S. Ramaswamy, R. A. Itzykson, C. Come, P. Savagner, I. Gitelman, A. Richardson, and R. A. Weinberg. 2004. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117:927-939.
34.Yoshizaki, T., H. Sato, M. Furukawa, and J. S. Pagano. 1998. The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1. Proc. Natl. Acad. Sci. USA 95:3621-3626.
35.Zeng, Q., L. K. McCauley, and C. -Y. Wang. 2002. Hepatocyte growth factor inhibits anoikis by induction of activator protein 1-dependent cyclooxygenase-2. J. Biol. Chem. 227:50137-50142.
36.Ziegler, J. L., R. C. Miner, E. Rosenbaum, E. T. Lennette, E. Shillitoe, C. Casavant, W. L. Drew, L. Mintz, J. Gershow, J. Greenspan, J. Beckstead and K. Yamamoto. 1982. Outbreak of Burkitt’s-like lymphoma in homosexual man. Lancet 2: 631-633.