口腔癌是指包含嘴唇、舌頭、臉頰、嘴巴及口咽部其他部位的癌症總稱，是一種與抽煙、喝酒和嚼食檳榔有關的侵犯性癌症，病患通常在晚期才會表現出徵狀，且在治療之後，復發的機率很高。蛋白質體學是近年來發展的分析技術，對尋找癌化相關的腫瘤標記以作為人類疾病的診斷、治療與預防等研究助益很大。Q. Y. He等人以蛋白質體分析口腔舌癌相關蛋白的表現，發現galectin-1在舌癌的表現與周圍其他正常黏膜組織有明顯的差異。但目前對於galectin-1在口腔內的致癌機制仍不清楚。
　　本實驗乃利用噬菌體呈現技術來鑑定與galectin-1結合之胜肽，期能對galectin-1的致癌機制的解析有所助益。經過四次的噬菌體呈現技術篩選，我們從一隨機表現12個胜肽序列的噬菌體庫中，找到許多能辨識galectin-1蛋白的噬菌體。我們比較這些胜肽的親和性，DNA序列分析結果，發現其中兩段序列在篩選中重覆出現，分析比對人類的基因庫之後，發現Met oncogene相關的tyrosine kinase, RON(recepteur d'origine nantais)具有此兩段序列，我們進而利用免疫共沉澱的方式評量galectin-1與RON(及其相關蛋白Met)間之交互作用，不過無法看到galectin-1與RON或Met免疫共沉澱的現象。然而，以共軛焦顯微鏡觀察galectin-1與RON在HSC-3細胞中表現的情形，可以發現galectin-1與RON具有co-localization的現象，且RON免疫組織染色的結果，亦可看到其在口腔腫瘤組織中的表現情形與galectin-1相似。此外，我們也以哺乳動物雙雜交(Mammalian two- hybrid system)來進一步證明噬菌體呈現所篩選出來之胜肽與galectin-1間的交互作用，結果顯示luciferase活性並沒有明顯的改變。總結來說，利用噬菌體呈現技術我們找到一些galectin-1結合胜肽並分析其氨基酸序列及結合強度，其中兩個出現多次的galectin-1結合胜肽的氨基酸序列與RON的氨基酸序列具高度相似性，雖然我們無法以免疫共沉澱法證明galectin-1與RON間的直接交互作用，但是從免疫染色中，包括腫瘤細胞及組織確實有許多徵兆顯示RON與galectin-1間有相關性，galectin-1在口腔癌癌化的作用機制是否透過RON或其他分子的交互作用還需要進一步實驗加以評估，這些galectin-1結合胜肽或許可作為口腔癌研究的有效工具及當作診斷或治療口腔癌策略之參考。

　Oral cancer is defined to include cancers of the lip, tongue, cheek, other sites of the month, and the oropharynx. Oral carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel quid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. Proteomics is a powerful analytical technology newly developed to identify the molecular makers that are associated with malignant transformation, and also to enhance our study of the diagnosis, treatment and prevention of human diseases. Q. Y. He et al. have reported the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. A number of tumor-associated proteins including galectin-1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosa. But the mechanism of galectin-1 protein on the oral carcinogenesis is still poorly understood.
　The aim of this study is to identify the binding peptides of galectin-1 using phage display technology. After four rounds of panning, several phage plaques that recognized galectin-1 protein were obtained from the 12-mer phage-displayed peptide libraries. The binding affinities of these selected phages to galectin-1 have been compared. DNA sequencing indicated that two peptides sequence displayed on some of the selected phages with high frequency. BLAST analysis against human gene database showed that these two displayed peptide sequences have homology to the sequence of RON, a c-met-related tyrosine kinase protein. The interaction of galectin-1 and RON (and also its related protein, Met) was evaluated by co-immunoprecipitation, but galectin-1 couldn’t co-immunoprecipitate with these two proteins in our results. Co-localization of RON proteins and galectin-1 proteins had been observed in HSC-3 cells by confocal immunofluorescence staining. We also observed that the expression pattern of RON in oral cancer was similar to that of galectin-1 by immunohistochemistry. Mammalian two-hybrid system was used to further demonstrate the interaction between galectin-1 and the selected peptides in cell culture system. Our data showed that there were no significant changed in the luciferase activity of cells transfected with pCMX-VP16-galectin-1. In summary, several galectin-1 binding peptides have been obtained by phage display screening and characterized their binding affinity. Two frequently present peptides bear the sequences with highly homology to that of RON. Although we failed to demonstrate the direct binding of RON and galectin-1 by co-immunoprecipitation, but there were many indications that showed the interaction between RON and galectin-1. The roles of the galectin-1 and RON in oral carcinogenesis need more experiments to explore and understand. The Galectin-1-binding peptides might be a powerful tool for the study of oral carcinogenesis and might open way for discovery of small molecules with diagnostic and therapeutic value on oral squamous carcinoma.

1. L. Adams, G. K. Scott, C. S. Weinberg, Biochim Biophys Acta 1312, 137 (Jun 13, 1996).
2. J. Almkvist, C. Dahlgren, H. Leffler, A. Karlsson, J Immunol 168, 4034 (Apr 15, 2002).
3. S. H. Barondes et al., Cell 76, 597 (Feb 25, 1994).
4. S. H. Barondes, D. N. Cooper, M. A. Gitt, H. Leffler, J Biol Chem 269, 20807 (Aug 19, 1994).
5. C. Birchmeier, W. Birchmeier, E. Gherardi, G. F. Vande Woude, Nat Rev Mol Cell Biol 4, 915 (Dec, 2003).
6. C. Blaser et al., Eur J Immunol 28, 2311 (Aug, 1998).
7. E. W. Brunskill, D. P. Witte, A. B. Shreiner, S. S. Potter, Mech Dev 88, 237 (Nov, 1999).
8. I. Camby et al., J Neuropathol Exp Neurol 61, 585 (Jul, 2002).
9. I. Camby et al., Brain Pathol 11, 12 (Jan, 2001).
10. M. Caron, D. Bladier, R. Joubert, Int J Biochem 22, 1379 (1990).
11. M. Caron, R. Joubert, D. Bladier, Biochim Biophys Acta 925, 290 (Sep 11, 1987).
12. Y. Q. Chen, Y. Q. Zhou, J. H. Fisher, M. H. Wang, Oncogene 21, 6382 (Sep 12, 2002).
13. L. Chiariotti et al., Int J Cancer 64, 171 (Jun 22, 1995).
14. C. T. Chien, P. L. Bartel, R. Sternglanz, S. Fields, Proc Natl Acad Sci U S A 88, 9578 (Nov 1, 1991).
15. J. Y. Choi et al., J Cell Sci 111 (Pt 20), 3035 (Oct, 1998).
16. C. D. Chung, V. P. Patel, M. Moran, L. A. Lewis, M. C. Miceli, J Immunol 165, 3722 (Oct 1, 2000).
17. N. Clausse, F. van den Brule, D. Waltregny, F. Garnier, V. Castronovo, Angiogenesis 3, 317 (1999).
18. D. N. Cooper, S. H. Barondes, J Cell Biol 110, 1681 (May, 1990).
19. D. N. Cooper, S. M. Massa, S. H. Barondes, J Cell Biol 115, 1437 (Dec, 1991).
20. J. D. Cornillot et al., Glycobiology 8, 425 (May, 1998).
21. C. V. Dang et al., Mol Cell Biol 11, 954 (Feb, 1991).
22. A. Danguy, I. Camby, R. Kiss, Biochim Biophys Acta 1572, 285 (Sep 19, 2002).
23. R. J. Davis, W. Shen, Y. I. Sandler, T. A. Heanue, G. Mardon, Mech Dev 102, 169 (Apr, 2001).
24. J. Dodd, T. M. Jessell, J Exp Biol 124, 225 (Sep, 1986).
25. A. Dybwad, O. Forre, J. Kjeldsen-Kragh, J. B. Natvig, M. Sioud, Eur J Immunol 23, 3189 (Dec, 1993).
26. J. Ellerhorst, T. Nguyen, D. N. Cooper, D. Lotan, R. Lotan, Int J Oncol 14, 217 (Feb, 1999).
27. E. R. Fearon et al., Proc Natl Acad Sci U S A 89, 7958 (Sep 1, 1992).
28. S. Fields, O. Song, Nature 340, 245 (Jul 20, 1989).
29. G. Gaudino et al., Oncogene 11, 2627 (Dec 21, 1995).
30. S. M. Hanash, J. Madoz-Gurpide, D. E. Misek, Leukemia 16, 478 (Apr, 2002).
31. F. L. Harrison, T. J. Wilson, J Cell Sci 101 (Pt 3), 635 (Mar, 1992).
32. Q. Y. He, J. Chen, H. F. Kung, A. P. Yuen, J. F. Chiu, Proteomics 4, 271 (Jan, 2004).
33. Q. Y. He, J. F. Chiu, J Cell Biochem 89, 868 (Aug 1, 2003).
34. J. M. Healy et al., Biochemistry 34, 3948 (Mar 28, 1995).
35. L. Hetian et al., J Biol Chem 277, 43137 (Nov 8, 2002).
36. H. Horie, T. Kadoya, Glycoconj J 19, 479 (2004).
37. C. L. Hsu et al., J Biol Chem 278, 23691 (Jun 27, 2003).
38. D. F. Hunt, J Proteome Res 1, 15 (Jan-Feb, 2002).
39. R. Hyde-DeRuyscher et al., Chem Biol 7, 17 (Jan, 2000).
40. M. A. Hynes et al., Ciba Found Symp 145, 189 (1989).
41. P. R. Jungblut et al., Electrophoresis 20, 2100 (Jul, 1999).
42. T. Kidd et al., Cell 92, 205 (Jan 23, 1998).
43. H. W. King, P. R. Tempest, K. R. Merrifield, A. J. Rance, Oncogene 2, 617 (Jun, 1988).
44. J. Kopitz et al., J Biol Chem 276, 35917 (Sep 21, 2001).
45. W. Y. Lee et al., Clin Cancer Res 11, 2222 (Mar 15, 2005).
46. H. Leffler, S. Carlsson, M. Hedlund, Y. Qian, F. Poirier, Glycoconj J 19, 433 (2004).
47. B. Levitan, J Mol Biol 277, 893 (Apr 10, 1998).
48. P. Maggiora et al., Exp Cell Res 288, 382 (Aug 15, 2003).
49. P. Maggiora et al., Oncogene 16, 2927 (Jun 4, 1998).
50. P. J. Mintz et al., Nat Biotechnol 21, 57 (Jan, 2003).
51. D. Pacholsky et al., J Cell Sci 117, 5257 (Oct 15, 2004).
52. A. Paz, R. Haklai, G. Elad-Sfadia, E. Ballan, Y. Kloog, Oncogene 20, 7486 (Nov 8, 2001).
53. N. L. Perillo, K. E. Pace, J. J. Seilhamer, L. G. Baum, Nature 378, 736 (Dec 14, 1995).
54. T. C. Poon, P. J. Johnson, Clin Chim Acta 313, 231 (Nov, 2001).
55. A. C. Puche, F. Poirier, M. Hair, P. F. Bartlett, B. Key, Dev Biol 179, 274 (Oct 10, 1996).
56. G. A. Rabinovich, Br J Cancer 92, 1188 (Apr 11, 2005).
57. G. A. Rabinovich et al., Trends Immunol 23, 313 (Jun, 2002).
58. G. A. Rabinovich et al., J Immunol 160, 4831 (May 15, 1998).
59. G. A. Rabinovich, C. E. Sotomayor, C. M. Riera, I. Bianco, S. G. Correa, Eur J Immunol 30, 1331 (May, 2000).
60. U. B. Rasmussen, V. Schreiber, H. Schultz, F. Mischler, K. Schughart, Cancer Gene Ther 9, 606 (Jul, 2002).
61. C. Ronsin, F. Muscatelli, M. G. Mattei, R. Breathnach, Oncogene 8, 1195 (May, 1993).
62. S. Rorive et al., Glia 33, 241 (Mar 1, 2001).
63. O. Sakamoto et al., J Clin Invest 99, 701 (Feb 15, 1997).
64. G. L. Sanford, S. Harris-Hooker, Faseb J 4, 2912 (Aug, 1990).
65. X. Sanjuan et al., Gastroenterology 113, 1906 (Dec, 1997).
66. R. Schmitz, G. Baumann, H. Gram, J Mol Biol 260, 664 (Aug 2, 1996).
67. C. Serra-Pages et al., Embo J 14, 2827 (Jun 15, 1995).
68. G. P. Smith, Science 228, 1315 (Jun 14, 1985).
69. G. P. Smith, V. A. Petrenko, Chem Rev 97, 391 (Apr 1, 1997).
70. P. R. Srinivas, S. Srivastava, S. Hanash, G. L. Wright, Jr., Clin Chem 47, 1901 (Oct, 2001).
71. Z. Z. Su et al., Proc Natl Acad Sci U S A 93, 7252 (Jul 9, 1996).
72. N. Tinari et al., Int J Cancer 91, 167 (Jan 15, 2001).
73. F. A. van den Brule et al., Eur J Cancer 30A, 1096 (1994).
74. F. A. van den Brule et al., Biochem Biophys Res Commun 209, 760 (Apr 17, 1995).
75. F. A. van den Brule, D. Waltregny, V. Castronovo, J Pathol 193, 80 (Jan, 2001).
76. M. H. Wang et al., Exp Cell Res 226, 39 (Jul 10, 1996).
77. K. A. Wehner, S. J. Baserga, Mol Cell 9, 329 (Feb, 2002).
78. V. Wells, D. Davies, L. Mallucci, Eur J Cancer 35, 978 (Jun, 1999).
79. V. Wells, L. Mallucci, Cell 64, 91 (Jan 11, 1991).
80. W. G. Willats, Plant Mol Biol 50, 837 (Dec, 2002).
81. K. Yamaoka et al., J Neurosci Res 59, 722 (Mar 15, 2000).
82. Y. Q. Zhou, C. He, Y. Q. Chen, D. Wang, M. H. Wang, Oncogene 22, 186 (Jan 16, 2003).