成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	鄭瑞 Cheng, Jui
論文名稱：	設計針對Galectin-1的aptamer與FTY720連接的奈米藥物在頭頸癌治療效果探討 Design of Gal-1 aptamer conjugated FTY720-nanomedicine as a novel anti-head and neck cancer therapy
指導教授：	謝達斌 Shieh, Dar-Bin
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	42
中文關鍵詞：	芬戈莫德、頭頸癌、適體、半乳糖凝集素、奈米藥物
外文關鍵詞：	FTY720, head and neck cancer, aptamer, galectin, nanoparticle
相關次數：	點閱：117 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

FTY720 是一種用於治療多發性硬化症的雙性分子藥物，它具有形成微胞與微酯體的特性，利於使用在雞尾酒療法並透過高滲透長滯留效應促進藥物吸收、分佈、代謝、排除，延長藥物在體內循環的時間。FTY720的分子機制是透過調節Sphingosine-1-phosphate（S1P）與受體Sphingosine-1-phosphate receptores（S1PR1/3/4/5）信號路徑去調節免疫反應減少中央神經系統的發炎反應。在S1P的信號路徑中，sphingosine會被sphingosine kinases（SPHK1/2 ）磷酸化並傳送到細胞膜外與細胞膜上的五個與G蛋白連接的Sphingosine-1-phosphate receptores（SPHKs）反應並啟動多個下游的信號路徑。先前的研究指出抑制S1PRs的信號路徑可以抑制癌細胞增生、轉移、侵襲周邊組織，但是這條細胞信號路徑在口腔癌上的相關研究資料非常的稀少。在最近的研究發現Galectin-1（Gal-1）會在頭頸癌中大量表現並參與了多條癌症發生的基因調控路徑，而且針對Gal-1標靶的aptamer分子近期也被陳玉玲老師的研究團隊研發出來並可以針對肺癌病灶進行專一性的標靶。再加上最近有其他研究指出Gal-1與S1PR1信號路徑間有相關性，而且目前也沒有利用Gal-1分子標靶與FTY720藥物合併在頭頸癌的治療上。因此，在我的研究中，我發現S1PR1的蛋白質表現量與Gal-1的蛋白質表現量是有負相關性的，而且FTY720可以在IC50介於8到15 ?M 的濃度間對頭頸癌細胞株有毒殺效果，這個結果高於其他文獻實驗的其他種癌細胞。在頭頸癌前期的癌細胞中，我發現他的毒殺濃度是介於8.12與8.15 ?M之間，而在頭頸癌末期的細胞株中，毒殺濃度則是介於10.72到15.64 ?M之間。在FTY720對於頭頸癌轉移實驗中，末期的癌細胞株在受到FTY720藥物濃度高於1.25 ?M時，抑制癌細胞轉移的能力有限，甚至還增進了癌細胞的轉移，而在前期的癌細胞株中，FTY720藥物濃度高於2.5 ?M時，癌細胞轉移會被抑制。在TEM觀察中，我所合成的aptamer連接FTY720藥物分子是圓形的，大小介於180-200奈米之間，而且aptamer連接FTY720藥物分子對於頭頸癌細胞株的毒殺能力是有增進的，特別是在末期的癌細胞株，降低了所需藥物濃度的20.62到34.5％。這些結果顯示，FTY720可以自體租裝成奈米藥物並提升了藥物對頭頸癌細胞株的毒殺效果，特別是在與標靶Gal-1的aptamer分子連接後。但是FTY720藥物對癌細胞轉移的抑制受到癌症階段的影響。針對aptamer連接FTY720藥物分子對於細胞分子機制與體內研究藥物治療效果的研究需要未來做進一步的驗證。

FTY720, an amphiphilic molecule approved for the treatment of multiple sclerosis, could form liposome or micelle nano-structures and benefit from potential co-delivery of cocktail regimen and improvement of ADME through EPR effect and extended circulation time. It modulates Sphingosine-1-phosphate (S1P) signaling pathways through S1PR1/3/4/5 to regulate immune functions to attenuate neuron-inflammation in the central nervous system. In S1P pathway, sphingosine was phosphorylated by sphingosine kinases (SPHK1/2) and transported outside then interacts with five different G-proteins coupled with S1PRs to activate multiple downstream signals. Previous studies showed that S1PRs’ signaling blockage could inhibit tumor proliferation, migration, and invasion. However, this signaling pathway in oral carcinogenesis remained poorly understood. Recent studies showed that Galectin-1 (Gal-1) is over-expressed in head and neck cancer (HNC) and involved in multiple oncogenic signaling. The Gal-1 specific aptamer was developed by Dr. Yuh-Ling Chen and was able to provide selective targeting to lung cancer lesions. Besides, recent study showed a cross-talk between Gal-1 and S1PR1 signaling in gastric cancer model. To date, there is no integrated approach to target Gal-1 with FTY720 to improve therapeutic efficacy in HNC. In my research, I discovered that S1PR1 protein expression were negatively correlated to Gal-1 protein expression. FTY720 could induce cytotoxicity for HNC cells with IC50 at a range of 8 to 15 ?M, higher than other cancer types reported. The cytotoxicity of FTY720 to cancer lines from early-stage disease was between 8.12 and 8.15 µM for IC50, while they were between 10.72 and 15.64 µM for cell lines from late-stage disease. FTY720 had limited inhibition of cell migration in the HNC cell lines derived form late-stage disease at dose higher than 1.25 µM, migration was even promoted at higher dose. For the HNC cell lines derived from early-stage, cell migration was inhibited at doses higher than 2.5 µM. TEM observation showed that aptamer conjugated FTY720 nanoparticle has a round shape of 180-200 nm in diameter. Aptamer-conjugation was able to improve the cytotoxicity to HNC lines, especially for the late-stage disease, to decrease the IC50 to 20.62-34.5% of the non-targeting drugs. This study showed that FTY720 could form self-assembled nanoparticles and dose dependently induce cytotoxicity to HNC lines, especially when conjugated with aptamer targeting to Gal-1. However, their effects to cell migration were dependent on disease stage. Further experiments to dissect molecular mechanisms in the observed efficacy and in vivo validation of the therapeutic nanoparticle conjugation is encouraged.

Introduction 1 
1. Head and neck cancer 1 
2. Head and neck cancer treatment 1 
3. Sphingosine 1-phosphate pathway 2 
4. Fingolimod (FTY720) 3 
5. Galectin-1 protein 3 
6. Targeting Galectin-1 as an anti-cancer treatment 4 
7. The interplay between Galectin-1 and FTY720 and potential synergism in combined targeting anti-cancer treatment 5 
Study basis and specific aims 7 
Material and methods 8 
1. Cell lines and culture condition 8 
2. Cell cryopreservation and thawing 8 
3. Cell counting 9 
4. Aptamer sequence and refolding 9 
5. Cell proliferation assay 9 
6. Wound healing assay 9 
7. Protein extraction and quantification 10 
8. Western blotting 10 
9. Synthesis of AptC7@2,2′- (Ethylenedithio) diacetic acid 11 
10. AptC7@2,2′- (Ethylenedithio) diacetic acid precipitation 11 
11. Synthesis of AptC7@2,2′- (Ethylenedithio) diacetic acid@FTY720 12 
12. Characterization of the nanoparticle size 12 
13. Characterization of the nanoparticle shape and element distribution 12 
14. Analyze the nanoparticle concentration 13 
Results 14 
1. HNSCC S1PR1 and Galectin 1 expressions in HNSCC lines derived from different clinical stages 14 
2. The effect of FTY720 treatment to HNSCC cell migration 14 
3. Cytotoxicity of FTY720 to HNSCC lines 14 
4. Characterization of the anti-Gal-1 aptamer conjugated FTY720 nano medicine 15 
5. The cytotoxicity of FTY720@AptC7 nanoparticles to late-stage HNSCC cell lines 15 
Discussion 16 
Conclusion 21 
Tables 22 
Table 1. The antibodies and the using condition in this study 22 
Table 2. HNSCC lines stages 23 
Figures 24 
Figure 1. S1PR1 and Gal-1 protein expression profile 24 
Figure 2. FTY720 inhibit early-stage HNSCC cancer cell migration 25 
Figure 3. Cytotoxicity test of FTY720 and FTY720@AptC7 on HNSCC 26 
Figure 4. Gal-1 specific aptamer conjugate FTY720 nanoparticle synthesis idea 27 
Figure 5. Gal-1 specific aptamer conjugate FTY720 nanoparticle size and shape 29 
Figure 6. Gal-1 specific aptamer conjugate FTY720 nanoparticle element mapping 31 
Appendix Figures 32 
Appendix Figure 1. Ten leading causes of death in Taiwan 32 
Appendix Figure 2. Sphingosine metabolism pathway 33 
Appendix Figure 3. FTY720 intrinsic/extrinsic pathways 34 
Appendix Figure 4. Gal-1 specific aptamer (AP-74 M-545) 35 
Appendix Figure 5. Interaction between Gal-1 and S1PR1 in gastric cancer model 37 
References 38
                                    

Bernier, Jacques, Christian Domenge, Mahmut Ozsahin, Katarzyna Matuszewska, Jean-Louis Lefèbvre, Richard H. Greiner, Jordi Giralt, Philippe Maingon, Frédéric Rolland, Michel Bolla, Francesco Cognetti, Jean Bourhis, Anne Kirkpatrick, and Martine van Glabbeke. 2004. 'Postoperative Irradiation with or without Concomitant Chemotherapy for Locally Advanced Head and Neck Cancer', New England Journal of Medicine, 350: 1945-52.
Chen, C. L., E. Meng, S. T. Wu, H. F. Lai, Y. S. Lu, M. H. Yang, C. W. Tsao, C. C. Kao, and Y. L. Chiu. 2021. 'Targeting S1PR1 May Result in Enhanced Migration of Cancer Cells in Bladder Carcinoma', Cancers (Basel), 13.
Chiang, W. F., S. Y. Liu, L. Y. Fang, C. N. Lin, M. H. Wu, Y. C. Chen, Y. L. Chen, and Y. T. Jin. 2008. 'Overexpression of galectin-1 at the tumor invasion front is associated with poor prognosis in early-stage oral squamous cell carcinoma', Oral Oncol, 44: 325-34.
Cramer, J. D., B. Burtness, Q. T. Le, and R. L. Ferris. 2019. 'The changing therapeutic landscape of head and neck cancer', Nat Rev Clin Oncol, 16: 669-83.
Ding, Y. M., J. H. Dong, L. L. Chen, and H. D. Zhang. 2009. 'Increased expression of galectin-1 is associated with human oral squamous cell carcinoma development', Oncol Rep, 21: 983-7.
Dings, R. P. M., M. C. Miller, R. J. Griffin, and K. H. Mayo. 2018. 'Galectins as Molecular Targets for Therapeutic Intervention', Int J Mol Sci, 19.
Dings, R. P., M. C. Miller, I. Nesmelova, L. Astorgues-Xerri, N. Kumar, M. Serova, X. Chen, E. Raymond, T. R. Hoye, and K. H. Mayo. 2012. 'Antitumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding', J Med Chem, 55: 5121-9.
Dings, Ruud P. M., Emily S. Van Laar, Melissa Loren, Jeremy Webber, Yan Zhang, Stephen J. Waters, John R. MacDonald, and Kevin H. Mayo. 2010. 'Inhibiting Tumor Growth by Targeting Tumor Vasculature with Galectin-1 Antagonist Anginex Conjugated to the Cytotoxic Acylfulvene, 6-Hydroxylpropylacylfulvene', Bioconjugate Chemistry, 21: 20-27.
Gheysen, L., L. Soumoy, A. Trelcat, L. Verset, F. Journe, and S. Saussez. 2021. 'New Treatment Strategy Targeting Galectin-1 against Thyroid Cancer', Cells, 10.
GRIFFIOEN, Arjan W., Daisy W. J. VAN DER SCHAFT, Annemarie F. BARENDSZ-JANSON, Andrew COX, Harry A. J. STRUIJKER BOUDIER, Harry F. P. HILLEN, and Kevin H. MAYO. 2001. 'Anginex, a designed peptide that inhibits angiogenesis', Biochemical Journal, 354: 233-42.
Ho, A. S., D. H. Kraus, I. Ganly, N. Y. Lee, J. P. Shah, and L. G. Morris. 2014. 'Decision making in the management of recurrent head and neck cancer', Head Neck, 36: 144-51.
Höög, Johanna L., Eva Gluenz, Sue Vaughan, and Keith Gull. 2010. 'Ultrastructural Investigation Methods for Trypanosoma brucei.' in, Electron Microscopy of Model Systems.
Inagaki, Toshiro, Satoshi Matsuwari, Rei Takahashi, Kei-Kichi Shimada, Kimiko Fujie, and Sakan Maeda. 1994. 'Establishment of human oral-cancer cell lines (KOSC-2 and -3) carrying p53 and c-myc abnormalities by geneticin treatment', International Journal of Cancer, 56: 301-08.
Johnson, D. E., B. Burtness, C. R. Leemans, V. W. Y. Lui, J. E. Bauman, and J. R. Grandis. 2020. 'Head and neck squamous cell carcinoma', Nat Rev Dis Primers, 6: 92.
Kalhori, V., M. Magnusson, M. Y. Asghar, I. Pulli, and K. Tornquist. 2016. 'FTY720 (Fingolimod) attenuates basal and sphingosine-1-phosphate-evoked thyroid cancer cell invasion', Endocr Relat Cancer, 23: 457-68.
Koonce, N. A., R. J. Griffin, and R. P. M. Dings. 2017. 'Galectin-1 Inhibitor OTX008 Induces Tumor Vessel Normalization and Tumor Growth Inhibition in Human Head and Neck Squamous Cell Carcinoma Models', Int J Mol Sci, 18.
Lankadasari, M. B., J. S. Aparna, S. Mohammed, S. James, K. Aoki, V. S. Binu, S. Nair, and K. B. Harikumar. 2018. 'Targeting S1PR1/STAT3 loop abrogates desmoplasia and chemosensitizes pancreatic cancer to gemcitabine', Theranostics, 8: 3824-40.
Momose, F., T. Araida, A. Negishi, H. Ichijo, S. Shioda, and S. Sasaki. 1989. 'Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas', J Oral Pathol Med, 18: 391-5.
Nambiar, D. K., T. Aguilera, H. Cao, S. Kwok, C. Kong, J. Bloomstein, Z. Wang, V. S. Rangan, D. Jiang, R. von Eyben, R. Liang, S. Agarwal, A. D. Colevas, A. Korman, C. T. Allen, R. Uppaluri, A. C. Koong, A. Giaccia, and Q. T. Le. 2019. 'Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance', J Clin Invest, 129: 5553-67.
Patmanathan, S. N., S. P. Johnson, S. L. Lai, S. Panja Bernam, V. Lopes, W. Wei, M. H. Ibrahim, F. Torta, P. Narayanaswamy, M. R. Wenk, D. R. Herr, P. G. Murray, L. F. Yap, and I. C. Paterson. 2016. 'Aberrant expression of the S1P regulating enzymes, SPHK1 and SGPL1, contributes to a migratory phenotype in OSCC mediated through S1PR2', Sci Rep, 6: 25650.
Perez Saez, J. M., P. F. Hockl, A. J. Cagnoni, S. P. Mendez Huergo, P. A. Garcia, S. G. Gatto, J. P. Cerliani, D. O. Croci, and G. A. Rabinovich. 2021. 'Characterization of a neutralizing anti-human galectin-1 monoclonal antibody with angioregulatory and immunomodulatory activities', Angiogenesis, 24: 1-5.
Perillo, N. L., K. E. Pace, J. J. Seilhamer, and L. G. Baum. 1995a. 'Apoptosis of T cells mediated by galectin-1', Nature, 378: 736-9.
Perillo, Nancy L., Karen E. Pace, Jeffrey J. Seilhamer, and Linda G. Baum. 1995b. 'Apoptosis of T cells mediated by galectin-1', Nature, 378: 736-39.
Pignon, J. P., A. le Maitre, E. Maillard, J. Bourhis, and Mach-Nc Collaborative Group. 2009. 'Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): an update on 93 randomised trials and 17,346 patients', Radiother Oncol, 92: 4-14.
Rodriguez, Y. I., L. E. Campos, M. G. Castro, A. Aladhami, C. A. Oskeritzian, and S. E. Alvarez. 2016. 'Sphingosine-1 Phosphate: A New Modulator of Immune Plasticity in the Tumor Microenvironment', Front Oncol, 6: 218.
Rodriguez-Remirez, M., L. Del Puerto-Nevado, M. J. Fernandez-Acenero, M. Cruz-Ramos, L. Garcia-Garcia, S. Solanes, E. Molina-Roldan, J. Garcia-Foncillas, and A. Cebrian. 2021. 'Targeting Galectin-1 by Aflibercept Strongly Enhances Its Antitumor Effect in Neuroendocrine Carcinomas', Neuroendocrinology, 111: 146-57.
Rostami, N., A. Nikkhoo, A. Ajjoolabady, G. Azizi, M. Hojjat-Farsangi, G. Ghalamfarsa, B. Yousefi, M. Yousefi, and F. Jadidi-Niaragh. 2019. 'S1PR1 as a Novel Promising Therapeutic Target in Cancer Therapy', Mol Diagn Ther, 23: 467-87.
Strader, C. R., C. J. Pearce, and N. H. Oberlies. 2011. 'Fingolimod (FTY720): a recently approved multiple sclerosis drug based on a fungal secondary metabolite', J Nat Prod, 74: 900-7.
Suwanwela, J., and T. Osathanon. 2017. 'Inflammation related genes are upregulated in surgical margins of advanced stage oral squamous cell carcinoma', J Oral Biol Craniofac Res, 7: 193-97.
Takahashi, Kikuo, Harusachi Kanazawa, Yukihiro Akiyama, Shigeko Tazaki, Masaaki Takahara, Toshitaka Muto, Hideki Tanzawa, and Ken-ichi Sato. 1989. 'Establishment and characterization of a cell line (SAS) from poorly differentiated human squamous cell carcinoma of the tongue', Journal of The Japanese Stomatological Society, 38: 20-28.
Takasaki, T., K. Hagihara, R. Satoh, and R. Sugiura. 2018. 'More than Just an Immunosuppressant: The Emerging Role of FTY720 as a Novel Inducer of ROS and Apoptosis', Oxid Med Cell Longev, 2018: 4397159.
Thijssen, V. L., B. Barkan, H. Shoji, I. M. Aries, V. Mathieu, L. Deltour, T. M. Hackeng, R. Kiss, Y. Kloog, F. Poirier, and A. W. Griffioen. 2010. 'Tumor cells secrete galectin-1 to enhance endothelial cell activity', Cancer Res, 70: 6216-24.
Tsai, H. C., and M. H. Han. 2016. 'Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway: Therapeutic Targets in Autoimmunity and Inflammation', Drugs, 76: 1067-79.
Tsai, Y. T., C. H. Liang, J. H. Yu, K. C. Huang, C. H. Tung, J. E. Wu, Y. Y. Wu, C. H. Chang, T. M. Hong, and Y. L. Chen. 2019. 'A DNA Aptamer Targeting Galectin-1 as a Novel Immunotherapeutic Strategy for Lung Cancer', Mol Ther Nucleic Acids, 18: 991-98.
V.Kalyana Chakravarthy, D.Gowri Sankar. 2011. 'LC determination of fingolimod in bulk and its pharmaceutical formulation', Imperial J. Medicinal & Organic Chemistry.
Warnakulasuriya, S. 2009. 'Global epidemiology of oral and oropharyngeal cancer', Oral Oncol, 45: 309-16.
Wu, M. H., H. C. Hong, T. M. Hong, W. F. Chiang, Y. T. Jin, and Y. L. Chen. 2011. 'Targeting galectin-1 in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis by downregulating MCP-1/CCL2 expression', Clin Cancer Res, 17: 1306-16.
Yang, C. Y., and C. L. Meng. 1994. 'Regulation of PG synthase by EGF and PDGF in human oral, breast, stomach, and fibrosarcoma cancer cell lines', J Dent Res, 73: 1407-15.
You, X., Y. Wang, J. Wu, Q. Liu, D. Chen, D. Tang, and D. Wang. 2018. 'Galectin-1 Promotes Metastasis in Gastric Cancer Through a Sphingosine-1-Phosphate Receptor 1-Dependent Mechanism', Cell Physiol Biochem, 51: 11-30.

校內：2027-09-20公開
校外：2027-09-20公開電子論文尚未授權公開，紙本請查館藏目錄

簡易檢索 / 詳目顯示

相關論文