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系統識別號 U0026-2308201318103400
論文名稱(中文) 探討Calprotectin在口腔癌及基質細胞所扮演的角色
論文名稱(英文) Studying the Role of Calprotectin in Oral Cancer and Stromal Cells
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 陳怡雯
研究生(英文) Yi-Wen Chen
學號 T16004043
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 指導教授-吳梨華
口試委員-蔡森田
口試委員-張文粲
中文關鍵字 S100A8  S100A9  口腔癌  血管新生 
英文關鍵字 S100A8  S100A9  oral cancer  angiogenesis 
學科別分類
中文摘要 在台灣口腔癌是常見的男性癌症之一。Calprotectin是由S100A8及S100A9這兩個蛋白質所形成的異型二聚體。這兩個蛋白質是屬於可與鈣離子結合的S100蛋白質家族成員,主要是表現在一些骨隨細胞。Calprotectin 除了表現在與發炎相關的細胞外,在許多不同類型的癌症中也發現calprotectin的存在。最近也發現,在急性及慢性發炎中,calprotectin可作為發炎前期的指標。因此,calprotectin在與發炎相關的癌症中扮演一個相當重要的角色,其中也包含了口腔癌。雖然目前關於calprotectin的功能已有一些研究,但其在口腔癌中所扮演的角色目前還並不清楚。根據我們實驗室之前針對口腔癌病人檢體做的免疫組織化學染色切片顯示:在腫瘤周邊的基質細胞內,其S100A9的表現量上升,與病人有較差的預後呈正相關。本篇研究主要的目的是要探討calprotectin在口腔癌細胞、內皮細胞及單核球中所扮演的角色。其中,內皮細胞及單核球富含於腫瘤基質中。利用免疫螢光染色,我們發現calprotectin不只表現在腫瘤細胞中,也表現在腫瘤周邊的基質細胞中,其中包含了表現CD15的嗜中性白血球,以及表現CD11b和CD68的單核球或巨噬細胞。這個結果表示calprotectin對於口腔癌可能同時具有自體分泌及旁分泌的作用。雖然改變calprotecctin在細胞內的表現量對於細胞生長及入侵的能力在不同的細胞株而有不同的結果,但對於細胞的爬行能力扮演著促進的角色。除此之外,由口腔癌細胞分泌的calprotectin能抑制抗發炎因子IL-10的產生,並促進血管新生。與in vitro實驗結果相符,S100A9具有促進腫瘤在老鼠體內生長的能力。最後總結,calprotectin,特別是S100A9,在口腔癌形成過程中扮演促癌的角色。了解calprotectin在口腔癌細胞及基質細胞中所扮演的角色對於未來研發新的口腔癌的治療方法會有所幫助。
英文摘要 Oral cancer is one prevalent male cancer type in Taiwan. Calprotectin is a heterodimer consisting of S100A8 and S100A9. Both proteins belong to a large calcium-binding S100 family and are predominantly expressed by myeloid cells. In addition to their expression in inflammatory cells, calprotectin has been detected in many cancer types and has recently emerged as a pro-inflammatory marker for both acute and chronic inflammatory diseases. Taken together, calprotectin may play a prominent role in inflammation-associated cancer including oral cancer. Although a number of putative functions have been proposed for calprotectin, its biological role particularly in oral cancer remains poorly defined. Using immunohistochemistry staining, we were able to find that the increase of S100A9, a subunit of calprotectin, in the stromal tissue adjacent the tumor tissues was significantly associated with poor clinical outcome. The purpose of this study is to investigate the role of calprotectin in oral cancer cells, endothelial cells and monocytes. The latter two types are rich in tumor stroma. Immunofluorescence microscopy showed that calprotectin was not only detected in oral cancer cells but also in their surrounding stromal cells including monocytes or macrophages expressing CD68+ or CD11b+, and neutrophils expressing CD15+ cells, suggesting the possibility of autocrine and paracrine effect of calprotectin on oral tissues. Although altering the expression of calprotectin affected cell proliferation and invasion in cell type-dependent manner, calprotecin functioned as a promoting molecule for cell migration. Moreover, calprotectin secreted from oral cancer cells inhibited anti-inflammatory IL-10 production and acted as positive regulator in angiogenesis. S100A9 manifested a more potent tumor promoting effect in vitro. Consistent with the notion, S100A9 promoted tumorigenesis in vivo. Together, stromal calprotectin especially S100A9 may play an oncogenic role in oral carcinogenesis. Understanding the potential role of calprotectin in oral cancer and stromal cells will facilitate the development of new therapeutic approaches for oral cancer treatment.
論文目次 中文摘要 I
Abstract II
致謝 IV
Content VI
Table, figure and appendix contents IX
Abbreviations XI
I. Introduction 1
1-1 Oral cancer 1
1-2 Cancer cells and their microenvironment 1
1-3 Calprotectin 2
1-4 Calprotectin in stroma and cancer 2
II. Specific aims 5
III. Materials and methods 6
3-1 Materials 6
3-2 Cell culture 7
3-3 Construct pcDNA3.1A-S100A8 or S100A9 plasmids 8
3-4 Establish S100A8 or S100A9 overexpressing stable clones 8
3-5 Establish S100A8 or S100A9 knockdown cells 8
3-6 Doubling time 9
3-7 Wound healing 9
3-8 Cell invasion assay 9
3-9 Prepare conditioned medium (CM) 10
3-10 Semi-quantitative reverse transcription-PCR (RT-PCR) 10
3-11 MTT assay 10
3-12 Migration assay 11
3-13 Tube formation assay 11
3-14 Western blot analysis 11
3-15 Immunofluorescence staining 12
3-16 Xenograft transplantation 12
IV. Results 13
4-1 Calprotectin was expressed in oral cancer cells and certain stromal cell types including monocytes/macrophages and neutrophils 13
4-2 Altered S100A8 or S100A9 expression affected oral cancer cell proliferation 13
4-3 S100A8 or S100A9 induced migration but inhibited invasion in oral cancer cells 14
4-4 S100A8 or S100A9 secreted from cancer cells inhibited the production of anti-inflammatory cytokine IL-10 15
4-5 S100A8 or S100A9 acted as positive regulator in angiogenesis 16
4-6 S100A9 promoted turmorigenesis in vivo 16
V. Discussion 18
References 21
參考文獻 1. Massano J, Regateiro FS, Januario G, Ferreira A. Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics. 2006;102:67-76.
2. Geneva: World Health Organization 2003. pp 6–7
3. Tsai KY, Su CC, Lin YY, Chung JA, Lian Ie B. Quantification of betel quid chewing and cigarette smoking in oral cancer patients. Community dentistry and oral epidemiology. 2009;37:555-61.
4. Lian Ie B, Tseng YT, Su CC, Tsai KY. Progression of precancerous lesions to oral cancer: Results based on the Taiwan National Health Insurance Database. Oral oncology. 2013;49:427-30.
5. Genden EM, Ferlito A, Bradley PJ, Rinaldo A, Scully C. Neck disease and distant metastases. Oral oncology. 2003;39:207-12.
6. Leite IC, Koifman S. Survival analysis in a sample of oral cancer patients at a reference hospital in Rio de Janeiro, Brazil. Oral oncology. 1998;34:347-52.
7. Whiteside TL. The tumor microenvironment and its role in promoting tumor growth. Oncogene. 2008;27:5904-12.
8. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nature reviews Cancer. 2009;9:239-52.
9. Bhowmick NA, Moses HL. Tumor-stroma interactions. Current opinion in genetics & development. 2005;15:97-101.
10. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008;454:436-44.
11. Hunter MJ, Chazin WJ. High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. The Journal of biological chemistry. 1998;273:12427-35.
12. Marenholz I, Heizmann CW, Fritz G. S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochemical and biophysical research communications. 2004;322:1111-22.
13. Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell. 1999;97:889-901.
14. Roth J, Vogl T, Sorg C, Sunderkotter C. Phagocyte-specific S100 proteins: a novel group of proinflammatory molecules. Trends in immunology. 2003;24:155-8.
15. Benedyk M, Sopalla C, Nacken W, Bode G, Melkonyan H, Banfi B, et al. HaCaT keratinocytes overexpressing the S100 proteins S100A8 and S100A9 show increased NADPH oxidase and NF-kappaB activities. The Journal of investigative dermatology. 2007;127:2001-11.
16. Lagasse E, Clerc RG. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation. Molecular and cellular biology. 1988;8:2402-10.
17. Yui S, Nakatani Y, Mikami M. Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity. Biological & pharmaceutical bulletin. 2003;26:753-60.
18. Bode G, Luken A, Kerkhoff C, Roth J, Ludwig S, Nacken W. Interaction between S100A8/A9 and Annexin A6 Is Involved in the Calcium-induced Cell Surface Exposition of S100A8/A9. Journal of Biological Chemistry. 2008;283:31776-84.
19. Gebhardt C, Nemeth J, Angel P, Hess J. S100A8 and S100A9 in inflammation and cancer. Biochemical pharmacology. 2006;72:1622-31.
20. Salama I, Malone PS, Mihaimeed F, Jones JL. A review of the S100 proteins in cancer. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology. 2008;34:357-64.
21. Hermani A, Hess J, De Servi B, Medunjanin S, Grobholz R, Trojan L, et al. Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2005;11:5146-52.
22. Kawai H, Minamiya Y, Takahashi N. Prognostic impact of S100A9 overexpression in non-small cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2011;32:641-6.
23. Ichikawa M, Williams R, Wang L, Vogl T, Srikrishna G. S100A8/A9 activate key genes and pathways in colon tumor progression. Molecular cancer research : MCR. 2011;9:133-48.
24. Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin WJ, Hashemi M, et al. S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway. Journal of leukocyte biology. 2008;83:1484-92.
25. Qin F, Song Y, Li Z, Zhao L, Zhang Y, Geng L. S100A8/A9 induces apoptosis and inhibits metastasis of CasKi human cervical cancer cells. Pathology oncology research : POR. 2010;16:353-60.
26. Ghavami S, Kerkhoff C, Los M, Hashemi M, Sorg C, Karami-Tehrani F. Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions. Journal of leukocyte biology. 2004;76:169-75.
27. Viemann D, Barczyk K, Vogl T, Fischer U, Sunderkotter C, Schulze-Osthoff K, et al. MRP8/MRP14 impairs endothelial integrity and induces a caspase-dependent and -independent cell death program. Blood. 2007;109:2453-60.
28. Eue I, Konig S, Pior J, Sorg C. S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells. International immunology. 2002;14:287-97.
29. Leukert N, Sorg C, Roth J. Molecular basis of the complex formation between the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14). Biological chemistry. 2005;386:429-34.
30. Lemarchand P, Vaglio M, Mauel J, Markert M. Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation. The Journal of biological chemistry. 1992;267:19379-82.
31. van den Bos C, Roth J, Koch HG, Hartmann M, Sorg C. Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton. Journal of immunology (Baltimore, Md : 1950). 1996;156:1247-54.
32. Kostura MJ, Tocci MJ, Limjuco G, Chin J, Cameron P, Hillman AG, et al. Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proceedings of the National Academy of Sciences of the United States of America. 1989;86:5227-31.
33. Moore KW, O'Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annual review of immunology. 1993;11:165-90.
34. Platzer C, Meisel C, Vogt K, Platzer M, Volk HD. Up-regulation of monocytic IL-10 by tumor necrosis factor-alpha and cAMP elevating drugs. International immunology. 1995;7:517-23.
35. Li C, Zhang F, Wang Y. S100A proteins in the pathogenesis of experimental corneal neovascularization. Molecular vision. 2010;16:2225-35.
36. Li C, Li S, Jia C, Yang L, Song Z, Wang Y. Low concentration of S100A8/9 promotes angiogenesis-related activity of vascular endothelial cells: bridges among inflammation, angiogenesis, and tumorigenesis? Mediators of inflammation. 2012;2012:248574.
37. Riva M, He Z, Kallberg E, Ivars F, Leanderson T. Human S100A9 protein is stabilized by inflammatory stimuli via the formation of proteolytically-resistant homodimers. PloS one. 2013;8:e61832.
38. Kim HJ, Kang HJ, Lee H, Lee ST, Yu MH, Kim H, et al. Identification of S100A8 and S100A9 as serological markers for colorectal cancer. Journal of proteome research. 2009;8:1368-79.
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