本研究室先前研究發現口腔鱗狀細胞癌（oral squamous cell carcinoma, OSCC）的轉移與金屬基質蛋白酶-9（Matrix metalloproteinase-9, MMP-9）息息相關。隨後又發現強力黴素（Doxycycline, DOX）可干擾轉化生長因子TGF-β信號傳遞路徑而達到抑制MMP-9的基因表現。本研究利用化學合成方法將四環黴素加以修飾以製造出衍生化合物TMC-1（Tetracycline Modified Compound-1），並用HPLC、ESI-LC-MS及1H-NMR鑑定確認成功修飾，再以TMC-1與DOX來抑制SCC-15口腔癌細胞的MMP-9表達及侵襲能力研究。用明膠蛋白酵素電泳（Gelatin zymography）與UVP照膠系統定量，證明TMC-1在5µg /ml即有明顯的抑制MMP-9的表現，而DOX在10 µg/ml才有明顯的抑制效果。另外我們利用細胞侵襲分析，結果也顯示出TMC-1比DOX有較好的抑制SCC-15細胞侵襲的能力。

The previous studies have shown that the migration of oral squamous cell carcinoma is closely related to the matrix-metalloproteinase-9 (MMP-9) expression. We also found that doxycycline (DOX) is through interfering the TGF-β pathway to inhibit MMP-9 expression. In this study, we synthesised a tetracycline analog, TMC-1 and by using HPLC, ESI-LC-MS and 1H-NMR we confirmed its structure. We use gelatin zymography and UVP system to analyze the expression level of MMP-9 in SCC-15 cell line. According to the results, we found out that MMP-9 expression was inhibited by both TMC-1 and DOX, whereas TMC-1 had a significant effect at 5μg/ml but DOX required 10μg/ml. In addition, we used cell invasion assay, also proved that TMC-1 inhibited SCC-15 cancer cells invasive ability better than DOX.

1. Shen, L. C.; Chen, Y. K.; Lin, L. M.; Shaw, S. Y., Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma--in vitro and in vivo studies. Oral oncology 2010, 46 (3), 178-84.

2. 林志強。四環素抑制口腔鱗狀癌細胞內基質金屬蛋白酶-9表現量之抑制機制的研究，碩士論文。國立成功大學生物科技研究所碩士班。2013。

3. 張文馨。基因重組人類Smad4蛋白之表現與功能分析，碩士論文。國立成功大學化學所碩士班。2015。

4. Golub, L. M.; Ramamurthy, N. S.; McNamara, T. F.; Greenwald, R. A.; Rifkin, B. R., Tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists 1991, 2 (3), 297-321.

5. Mashberg, A.; Barsa, P.; Grossman, M. L., A study of the relationship between mouthwash use and oral and pharyngeal cancer. Journal of the American Dental Association 1985, 110 (5), 731-4.

6. Rodriguez, T.; Altieri, A.; Chatenoud, L.; Gallus, S.; Bosetti, C.; Negri, E.; Franceschi, S.; Levi, F.; Talamini, R.; La Vecchia, C., Risk factors for oral and pharyngeal cancer in young adults. Oral oncology 2004, 40 (2), 207-13.

7. Warnakulasuriya, S., Global epidemiology of oral and oropharyngeal cancer. Oral oncology 2009, 45 (4-5), 309-16.

8. Scully, C.; Bagan, J., Oral squamous cell carcinoma overview. Oral oncology 2009, 45 (4-5), 301-8.

9. Yoon, S. O.; Park, S. J.; Yun, C. H.; Chung, A. S., Roles of matrix metalloproteinases in tumor metastasis and angiogenesis. Journal of biochemistry and molecular biology 2003, 36 (1), 128-37.

10. Egeblad, M.; Werb, Z., New functions for the matrix metalloproteinases in cancer progression. Nature reviews. Cancer 2002, 2 (3), 161-74.
11. Snoek-van Beurden, P. A.; Von den Hoff, J. W., Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. BioTechniques 2005, 38 (1), 73-83.

12. Vu, T. H.; Werb, Z., Matrix metalloproteinases: effectors of development and normal physiology. Genes & development 2000, 14 (17), 2123-33.

13. Fanjul-Fernandez, M.; Folgueras, A. R.; Cabrera, S.; Lopez-Otin, C., Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. Biochimica et biophysica acta 2010, 1803 (1), 3-19.

14. Brew, K.; Dinakarpandian, D.; Nagase, H., Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et biophysica acta 2000, 1477 (1-2), 267-83.

15. Gialeli, C.; Theocharis, A. D.; Karamanos, N. K., Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. The FEBS journal 2011, 278 (1), 16-27.

16. Chopra, I.; Roberts, M., Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and molecular biology reviews : MMBR 2001, 65 (2), 232-60 ; second page, table of contents.

17. Ramamurthy, N. S.; Zebrowski, E. J.; Golub, L. M., The effect of alloxan diabetes on gingival collagen metabolism in rats. Archives of oral biology 1972, 17 (11), 1551-60.

18. Golub, L. M.; Lee, H. M.; Lehrer, G.; Nemiroff, A.; McNamara, T. F.; Kaplan, R.; Ramamurthy, N. S., Minocycline reduces gingival collagenolytic activity during diabetes. Preliminary observations and a proposed new mechanism of action. Journal of periodontal research 1983, 18 (5), 516-26.

19. 陳和儒。四環黴素類藥物抑制基質金屬蛋白酶表現和癌細胞生長研究，碩士論文。國立功大學化學所碩士班。2011。

20. Wennstrom, J. L.; Newman, H. N.; MacNeill, S. R.; Killoy, W. J.; Griffiths, G. S.; Gillam, D. G.; Krok, L.; Needleman, I. G.; Weiss, G.; Garrett, S., Utilisation of locally delivered doxycycline in non-surgical treatment of chronic periodontitis. A comparative multi-centre trial of 2 treatment approaches. Journal of clinical periodontology 2001, 28 (8), 753-61.

21. Swamy, D. N.; Sanivarapu, S.; Moogla, S.; Kapalavai, V., Chemically modified tetracyclines: The novel host modulating agents. Journal of Indian Society of Periodontology 2015, 19 (4), 370-4.

22. Seftor, R. E.; Seftor, E. A.; De Larco, J. E.; Kleiner, D. E.; Leferson, J.; Stetler-Stevenson, W. G.; McNamara, T. F.; Golub, L. M.; Hendrix, M. J., Chemically modified tetracyclines inhibit human melanoma cell invasion and metastasis. Clinical & experimental metastasis 1998, 16 (3), 217-25.

23. Chang, K. M.; Ramamurthy, N. S.; McNamara, T. F.; Evans, R. T.; Klausen, B.; Murray, P. A.; Golub, L. M., Tetracyclines inhibit Porphyromonas gingivalis-induced alveolar bone loss in rats by a non-antimicrobial mechanism. Journal of periodontal research 1994, 29 (4), 242-9.

24. Lokeshwar, B. L.; Escatel, E.; Zhu, B., Cytotoxic activity and inhibition of tumor cell invasion by derivatives of a chemically modified tetracycline CMT-3 (COL-3). Current medicinal chemistry 2001, 8 (3), 271-9.

25. Lee, H. M.; Golub, L. M.; Cao, J.; Teronen, O.; Laitinen, M.; Salo, T.; Zucker, S.; Sorsa, T., CMT-3, a non-antimicrobial tetracycline (TC), inhibits MT1-MMP activity: relevance to cancer. Current medicinal chemistry 2001, 8 (3), 257-60.

26. Tolomeo, M.; Grimaudo, S.; Milano, S.; La Rosa, M.; Ferlazzo, V.; Di Bella, G.; Barbera, C.; Simoni, D.; D'Agostino, P.; Cillari, E., Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines. British journal of pharmacology 2001, 133 (2), 306-14.

27. Shen, L. L.; Zhao, L.; Ma, L.; Yang, J. T.; Jiao, Y.; Xu, J. I.; Fan, S. J., Anticancer activity and radiosensitization of TMC-1 in human cervical cancer HeLa cells. J. Radiat. Res. Radiat. Process 2012.

28. de Larco, J. E.; Todaro, G. J., Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation. Journal of cellular physiology 1978, 94 (3), 335-42.

29. Roberts, A. B.; Anzano, M. A.; Lamb, L. C.; Smith, J. M.; Sporn, M. B., New class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proceedings of the National Academy of Sciences of the United States of America 1981, 78 (9), 5339-43.

30. Rosa, S., Transforming Growth Factor Beta: Role in Cell Growth and Differentiation. Copyright © John Wiley & Sons, Ltd. All rights reserved.2014.

31. Derynck, R., TGF-beta-receptor-mediated signaling. Trends in biochemical sciences 1994, 19 (12), 548-53.

32. Massague, J.; Weis-Garcia, F., Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer surveys 1996, 27, 41-64.

33. Xie, H.; Li, L.; Zhu, G.; Dang, Q.; Ma, Z.; He, D.; Chang, L.; Song, W.; Chang, H. C.; Krolewski, J. J.; Nastiuk, K. L.; Yeh, S.; Chang, C., Infiltrated pre-adipocytes increase prostate cancer metastasis via modulation of the miR-301a/androgen receptor (AR)/TGF-beta1/Smad/MMP9 signals. Oncotarget 2015, 6 (14), 12326-39.

34. Villar, V.; Kocic, J.; Santibanez, J. F., Skip Regulates TGF- beta 1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells. Prostate cancer 2013, 2013, 398253.

35. Han, L.; Zhang, H. W.; Zhou, W. P.; Chen, G. M.; Guo, K. J., The effects of genistein on transforming growth factor-beta1-induced invasion and metastasis in human pancreatic cancer cell line Panc-1 in vitro. Chinese medical journal 2012, 125 (11), 2032-40.

36. Sun, L.; Diamond, M. E.; Ottaviano, A. J.; Joseph, M. J.; Ananthanarayan, V.; Munshi, H. G., Transforming growth factor-beta 1 promotes matrix metalloproteinase-9-mediated oral cancer invasion through snail expression. Molecular cancer research : MCR 2008, 6 (1), 10-20.

37. Wiercinska, E.; Naber, H. P.; Pardali, E.; van der Pluijm, G.; van Dam, H.; ten Dijke, P., The TGF-beta/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system. Breast cancer research and treatment 2011, 128 (3), 657-66.

38. Massague, J.; Seoane, J.; Wotton, D., Smad transcription factors. Genes & development 2005, 19 (23), 2783-810.
39. Schmierer, B.; Hill, C. S., TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nature reviews. Molecular cell biology 2007, 8 (12), 970-82.

40. Horbelt, D.; Denkis, A.; Knaus, P., A portrait of Transforming Growth Factor beta superfamily signalling: Background matters. The international journal of biochemistry & cell biology 2012, 44 (3), 469-74.

41. Kitisin, K.; Saha, T.; Blake, T.; Golestaneh, N.; Deng, M.; Kim, C.; Tang, Y.; Shetty, K.; Mishra, B.; Mishra, L., Tgf-Beta signaling in development. Science's STKE : signal transduction knowledge environment 2007, 2007 (399), cm1.

42. Zhang, L.; Zhou, F.; Garcia de Vinuesa, A.; de Kruijf, E. M.; Mesker, W. E.; Hui, L.; Drabsch, Y.; Li, Y.; Bauer, A.; Rousseau, A.; Sheppard, K. A.; Mickanin, C.; Kuppen, P. J.; Lu, C. X.; Ten Dijke, P., TRAF4 promotes TGF-beta receptor signaling and drives breast cancer metastasis. Molecular cell 2013, 51 (5), 559-72.

43. Sekelsky, J. J.; Newfeld, S. J.; Raftery, L. A.; Chartoff, E. H.; Gelbart, W. M., Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 1995, 139 (3), 1347-58.

44. Savage, C.; Das, P.; Finelli, A. L.; Townsend, S. R.; Sun, C. Y.; Baird, S. E.; Padgett, R. W., Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proceedings of the National Academy of Sciences of the United States of America 1996, 93 (2), 790-4.

45. Raftery, L. A.; Sutherland, D. J., TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Developmental biology 1999, 210 (2), 251-68.

46. Singh, P.; Wig, J. D.; Srinivasan, R., The Smad family and its role in pancreatic cancer. Indian journal of cancer 2011, 48 (3), 351-60.

47. Cook, T.; Urrutia, R., TIEG proteins join the Smads as TGF-beta-regulated transcription factors that control pancreatic cell growth. American journal of physiology. Gastrointestinal and liver physiology 2000, 278 (4), G513-21.

48. Shi, Y.; Massague, J., Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003, 113 (6), 685-700.
49. Shi, Y.; Hata, A.; Lo, R. S.; Massague, J.; Pavletich, N. P., A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 1997, 388 (6637), 87-93.

50. Heldin, C. H.; Miyazono, K.; ten Dijke, P., TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 1997, 390 (6659), 465-71.

51. Huse, M.; Chen, Y. G.; Massague, J.; Kuriyan, J., Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell 1999, 96 (3), 425-36.

52. van Heek, T.; Rader, A. E.; Offerhaus, G. J.; McCarthy, D. M.; Goggins, M.; Hruban, R. H.; Wilentz, R. E., K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis. American journal of clinical pathology 2002, 117 (5), 755-65.

53. Schutte, M.; Hruban, R. H.; Hedrick, L.; Cho, K. R.; Nadasdy, G. M.; Weinstein, C. L.; Bova, G. S.; Isaacs, W. B.; Cairns, P.; Nawroz, H.; Sidransky, D.; Casero, R. A., Jr.; Meltzer, P. S.; Hahn, S. A.; Kern, S. E., DPC4 gene in various tumor types. Cancer research 1996, 56 (11), 2527-30.

54. Hruban, R. H.; Wilentz, R. E.; Goggins, M.; Offerhaus, G. J.; Yeo, C. J.; Kern, S. E., Pathology of incipient pancreatic cancer. Annals of oncology : official journal of the European Society for Medical Oncology 1999, 10 Suppl 4, 9-11.

55. Ali, S.; Cohen, C.; Little, J. V.; Sequeira, J. H.; Mosunjac, M. B.; Siddiqui, M. T., The utility of SMAD4 as a diagnostic immunohistochemical marker for pancreatic adenocarcinoma, and its expression in other solid tumors. Diagnostic cytopathology 2007, 35 (10), 644-8.

56. Yasutome, M.; Gunn, J.; Korc, M., Restoration of Smad4 in BxPC3 pancreatic cancer cells attenuates proliferation without altering angiogenesis. Clinical & experimental metastasis 2005, 22 (6), 461-73.

57. Fink, S. P.; Mikkola, D.; Willson, J. K.; Markowitz, S., TGF-beta-induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines. Oncogene 2003, 22 (9), 1317-23.

58. Myszka, D. G.; Rich, R. L., Implementing surface plasmon resonance biosensors in drug discovery. Pharmaceutical science & technology today 2000, 3 (9), 310-317.
59. John Davies, Surface Analytical Techniques for Probing Biomaterial Processes., 1996.

60. James, H. B.; Guido E. B.; Coy W. W.; Joseph P. P.; Raymond W. W.; Robert B. B., Chemistry of the Tetracycline Antibiotics.1 I. Quaternary Derivatives. J. Am. Chem. Soc., 1958, 80 (7), 1654-1657.

61. Vidal, A.; Sabatini, M.; Rolland-Valognes, G.; Renard, P.; Madelmont, J. C.; Mounetou, E., Synthesis and in vitro evaluation of targeted tetracycline derivatives: effects on inhibition of matrix metalloproteinases. Bioorganic & medicinal chemistry 2007, 15 (6), 2368-74.

62. Lokeshwar, B. L.; Selzer, M. G.; Block, N. L.; Gunja-Smith, Z., Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase secretion by malignant tissues. Cancer research 1993, 53 (19), 4493-8.

63. Smith, C. J.; Sayles, H.; Mikuls, T. R.; Michaud, K., Minocycline and doxycycline therapy in community patients with rheumatoid arthritis: prescribing patterns, patient-level determinants of use, and patient-reported side effects. Arthritis research & therapy 2011, 13 (5), R168.