臺灣博碩士論文加值系統

實驗室先前的研究中發現強力黴素(Doxycycline, DOX)可以抑制口腔鱗狀細胞癌(Oral squamous cell carcinoma, OSCC)基質金屬蛋白酶-9(Matrix metalloproteinase-9, MMP-9)的基因表達，MMP-9可以將細胞外基質的明膠(Gelatin)分解，幫助癌細胞轉移。接著發現DOX是和TGF-β/Smad pathways中的Smad4進行結合來抑制MMP-9的基因表達。最後透過去除強力黴素C-4上的二甲基氨來移除抗生素活性減少抗藥性的生成，並保有抑制MMP-9的活性。本篇研究中要探討強力黴素C-9上官能基是否會影響MMP-9的抑制能力，並加上親水性官能基增加藥物在水中的溶解度。本篇合成出9-Nitro-4-dedimethylamino doxycycline(TMC-2)和9-Amino-4-dedimethylamino doxycycline(TMC-3)並利用HPLC和1H-NMR進行鑑定確認修飾上官能基。利用細胞存活率分析(MTT assay)得知我們合成出來的藥物對正常細胞毒性較小，而對癌細胞毒性較大，表示我們的藥物對人體的副作用較低。並利用明膠蛋白酵素電泳(Gelatin zymography)分析TMC-2和TMC-3抑制MMP-9的能力，發現在C-9修飾上拉電子基會導致抑制MMP-2和MMP-9的活性下降，而修飾上推電子基可以保持抑制MMP-2和MMP-9的能力。

Previous studies in our laboratory have found that doxycycline (DOX) inhibits the expression of matrix metalloproteinase-9 in oral squamous cell carcinoma. MMP-9 can decompose gelatin to help the metastasis of cancer cells. Our lab previously found that DOX binds to Smad4 in TGF-β/Smad pathways to inhibit gene expression of MMP-9. By removing dimethylamine from DOX C-4 position can reduce the DOX’s antibiotic activity, while retains its activity of inhibiting MMP-9. In this study, we first investigate whether the C-9 functional group on DOX affect the inhibitory ability of MMP-9. Moreover, the addition of hydrophilic functional group improves the solubility of the drug for oral administration. 9-Nitro-4-dedimethylamino doxycycline (TMC-2) and 9-Amino-4-dedimethylamino doxycycline (TMC-3) were both synthesized and identified by HPLC and 1H-NMR. Cell viability analysis was used to find that the drugs we synthesized were less toxic to normal cells and more toxic to cancer cells, indicating that our drugs have lower side effects on humans. We also performed gelatin zymography to analyze the ability of TMC-2 and TMC-3 in inhibition of MMP-9. In conclusion, we found that the electron-withdrawing group of C-9 position led to a decrease in the activities of inhibiting MMP-2 and 9; however, the electron-donating group on C-9 can regain it’s the ability of inhibiting MMP-2 and MMP-9 activities.

中文摘要 ..............................................i
英文摘要 ..............................................iii
誌謝 ..............................................xii
壹、 研究背景 ........................................1
一、 口腔鱗狀細胞癌（Oral squamous cell carcinoma,OSCC)3
1. 口腔鱗狀細胞癌轉移作用 ........................3
2. 基質金屬蛋白酶在口腔鱗狀細胞癌的作用................4
二、 基質金屬蛋白酶 (Matrix metalloproteinases, MMPs) 4
1. MMPs的功能 ................................5
2. MMPs的活化及抑制劑 ........................6
三、 四環黴素（Tetracycline） ........................7
1. 抗生素特性 ................................8
2. 非抗生素特性 ................................8
3. 化學修飾四環黴素（Chemically modified tetracyclines, CMTs） ...............................10
四、 乙型轉化生長因子 (Transforming growth factor-β, TGF-β) ...............................................11
1. Smad訊號傳遞路徑 (Smad-dependent pathway).......12
2. 非Smad傳遞路徑 (Smad-independent pathway).......13
貳、 研究目的 .......................................14
參、 材料與方法 ...............................15
一、 材料 .......................................15
1. 化學修飾（Chemical modification） .......15
2. 高壓液相層析儀（High pressure liquid chromatography, HPLC） ...............................16
3. 旋轉濃縮儀 (rotary evaporator) ...............16
4. 冷凍乾燥機 (Freeze dryer) ...............16
5. 細胞培養 (Cell culture) .......................16
6. 明膠蛋白酵素電泳（Gelatin zymography） .......17
二、 方法 .......................................18
1. 化學修飾強力黴素TMC-1、TMC-2、TMC-3的合成 .......18
i. 化學修飾-甲基化（Chemical modification-Methylated） .......................................................18
ii. 化學修飾-去季氨基 （Chemical modification-elimination of the quaternary amino group） .......19
iii. 化學修飾-硝基化 （Chemical modification-Nitration） .......................................................20
iv. 化學修飾-胺基化 （Chemical modification-Amination） .......................................................20
2. 高效能液相層析儀分析強力黴素衍生物 ...............22
3. 核磁共振分析強力黴素衍生物 .......................23
4. 細胞培養 (Cell culture) .......................23
i. 細胞存活率分析 (MTT assay) ...............24
ii. DOX與CMT-8抑制SCC-15基質金屬蛋白酶-9 .......25
iii. 明膠蛋白酵素電泳（Gelatin zymography）分析基質金屬蛋白酶-9含量 .............................................25
iv. 膠片分析 .......................................26
肆、 研究之結果 ...............................27
一、 化學修飾四環黴素 ...............................27
1. 4-去二甲基氨強力黴素（4-Dedimethylamino doxycycline, TMC-1）合成步驟優化 .......................27
2. 9-硝基-4-去二甲基氨強力黴素之合成 ...............28
3. 9-胺基-4-去二甲基氨強力黴素(9-Amino-4-dedimethylamino doxycycline, TMC-3) ...............29
二、 DOX、TMC-1, 2, 3對SCC-15及MRC-5細胞毒性測試......30
三、 用DOX、TMC-1、2和3抑制TGF-β1所誘導的MMP-2和9.....31
伍、 討論 .......................................32
一、 化學修飾DOX ...............................32
二、 DOX、TMC-1、2和3抑制TGF-β1誘導SCC-15的MMP-9表現..34
陸、 結論 .......................................36
柒、 參考資料 .......................................37
圖和表 ...............................................41
附錄 ...............................................70

1.Rowe, R.G. and S.J. Weiss, Breaching the basement membrane: who, when and how? Trends Cell Biol, 2008. 18(11): p. 560-74.
2.Shen, L.C., et al., Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma--in vitro and in vivo studies. Oral Oncol, 2010. 46(3): p. 178-84.
3.林志強, The study of suppression mechanism of MMP-9 expression level in oral squamous carcinoma cells by doxycycline. National Cheng Kung University, 2013.
4.張文馨, Expression and characterization of recombinant human Smad4. National Cheng Kung University, 2015.
5.Golub, L.M., et al., Tetracyclines Inhibit Connective Tissue Breakdown: New Therapeutic Implications for an Old Family of Drugs. Critical Reviews in Oral Biology ＆ Medicine, 1991. 2(3): p. 297-321.
6.廖靜洳, The study of the inhibition of Matrix-metalloproteinase-9 in oral squamous cell carcinoma by tetracycline analogs. National Cheng Kung University, 2017.
7.Warnakulasuriya, S., G. Sutherland, and C. Scully, Tobacco, oral cancer, and treatment of dependence. Oral Oncol, 2005. 41(3): p. 244-60.
8.Lacy, P.D., J. Edward L. Spitznagel, and J.F. Piccirillo, Development of a New Staging System for Recurrent Oral Cavity and Oropharyngeal Squamous Cell Carcinoma. Cancer, 1999.
9.Choi, S. and J. Myers, Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. Journal of dental research, 2008. 87(1): p. 14-32.
10.Clark, A.G. and D.M. Vignjevic, Modes of cancer cell invasion and the role of the microenvironment. Curr Opin Cell Biol, 2015. 36: p. 13-22.
11.Mishev, G., et al., Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma. Biotechnol Biotechnol Equip, 2014. 28(6): p. 1138-1149.
12.Page-McCaw, A., A.J. Ewald, and Z. Werb, Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol, 2007. 8(3): p. 221-33.
13.Mooren, F.C., Encyclopedia of Exercise Medicine in Health and Disease. 2012.
14.Sorsa, T., L. Tjäderhane, and T. Salo, Matrix metalloproteinases (MMPs) in oral diseases. Oral diseases, 2004. 10(6): p. 311-318.
15.Bourboulia, D. and W.G. Stetler-Stevenson. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion. in Seminars in cancer biology. 2010. Elsevier.
16.Stetler-Stevenson, W.G., The tumor microenvironment: regulation by MMP-independent effects of tissue inhibitor of metalloproteinases-2. Cancer and Metastasis Reviews, 2008. 27(1): p. 57-66.
17.Gialeli, C., A.D. Theocharis, and N.K. Karamanos, Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. The FEBS journal, 2011. 278(1): p. 16-27.
18.Chopra, I. and M. Roberts, Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev., 2001. 65(2): p. 232-260.
19.Fuoco, D., Classification framework and chemical biology of tetracycline-structure-based drugs. Antibiotics, 2012. 1(1): p. 1.
20.Pioletti, M., et al., Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. The EMBO journal, 2001. 20(8): p. 1829-1839.
21.Sapadin, A.N. and R. Fleischmajer, Tetracyclines: nonantibiotic properties and their clinical implications. Journal of the American Academy of Dermatology, 2006. 54(2): p. 258-265.
22.Ramamurthy, N., E. Zebrowski, and L. Golub, The effect of alloxan diabetes on gingival collagen metabolism in rats. Archives of oral biology, 1972. 17(11): p. 1551-1560.
23.Golub, L., et al., Minocycline reduces gingival collagenolytic activity during diabetes: preliminary observations and a proposed new mechanism of action. Journal of periodontal research, 1983. 18(5): p. 516-526.
24.Wennström, J.L., et al., 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): p. 753-761.
25.Devulapalli Narasimha Swamy, S.S., S. Moogla, and V. Kapalavai, Chemically modified tetracyclines: The novel host modulating agents. Journal of Indian Society of Periodontology, 2015. 19(4): p. 370.
26.Chang, K., et al., Tetracyclines inhibit Porphyromonas gingivalis‐induced alveolar bone loss in rats by a non‐antimicrobial mechanism. Journal of periodontal research, 1994. 29(4): p. 242-249.
27.Syed, S., et al., A phase I and pharmacokinetic study of Col-3 (Metastat), an oral tetracycline derivative with potent matrix metalloproteinase and antitumor properties. Clinical cancer research, 2004. 10(19): p. 6512-6521.
28.Chu, Q.S., et al., A phase II and pharmacological study of the matrix metalloproteinase inhibitor (MMPI) COL-3 in patients with advanced soft tissue sarcomas. Investigational new drugs, 2007. 25(4): p. 359.
29.Sporn, M.B. and A.B. Roberts, Peptide growth factors and their receptors. Handbook of experimental pharmacology, 1990. 95: p. 3-15.
30.Ebner, R., et al., Determination of type I receptor specificity by the type II receptors for TGF-beta or activin. Science, 1993. 262(5135): p. 900-902.
31.López-Casillas, F., et al., Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites. The Journal of cell biology, 1994. 124(4): p. 557-568.
32.Border, W.A. and N.A. Noble, Transforming growth factor β in tissue fibrosis. New England journal of medicine, 1994. 331(19): p. 1286-1292.
33.Flaumenhaft, R., et al., Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity. The Journal of cell biology, 1992. 118(4): p. 901-909.
34.Sun, L., et al., Transforming growth factor-β1 promotes matrix metalloproteinase-9–mediated oral cancer invasion through snail expression. Molecular Cancer Research, 2008. 6(1): p. 10-20.
35.Xie, H., et al., Infiltrated pre-adipocytes increase prostate cancer metastasis via modulation of the miR-301a/androgen receptor (AR)/TGF-β1/Smad/MMP9 signals. Oncotarget, 2015. 6(14): p. 12326.
36.Villar, V., J. Kocic, and J.F. Santibanez, Skip regulates TGF-β1-induced extracellular matrix degrading proteases expression in human PC-3 prostate cancer cells. Prostate cancer, 2013. 2013.
37.Lei, H., et al., The effects of genistein on transforming growth factor-β1-induced invasion and metastasis in human pancreatic cancer cell line Panc-1in vitro. Chinese medical journal, 2012. 125(11): p. 2032-2040.
38.Wiercinska, E., et al., The TGF-β/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): p. 657-666.
39.Schmierer, B. and C.S. Hill, TGFβ–SMAD signal transduction: molecular specificity and functional flexibility. Nature reviews Molecular cell biology, 2007. 8(12): p. 970.
40.Massagué, J., J. Seoane, and D. Wotton, Smad transcription factors. Genes ＆ development, 2005. 19(23): p. 2783-2810.
41.Derynck, R. and Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature, 2003. 425(6958): p. 577.
42.Zhang, Y.E., Non-Smad pathways in TGF-β signaling. Cell research, 2009. 19(1): p. 128.
43.Li, H.-Y., et al., TGF-β signaling regulates p-Akt levels via PP2A during diapause entry in the cotton bollworm, Helicoverpa armigera. Insect biochemistry and molecular biology, 2017. 87: p. 165-173.