新穎醣胺多醣結合胜肽於腫瘤標靶與抑制之功能分析

Functional Characterization of A Novel Glycosaminoglycan Binding Peptide on Tumor Targeting and Suppression

蔡岡橋(Kang-Chiao Tsai)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2014年)

https://doi.org/10.6843/NTHU.2014.00178

摘要

細胞表面的醣胺多醣(glycosaminoglycans)與趨化素(chemokines)、凝血素(thrombins)及生長因子間交互作用被視為與增殖(proliferation)、黏附(adhesion)、移行(migration)及血管新生(angiogenesis)等癌化過程有關。本實驗室先前於人類嗜酸性白血球陽離子蛋白(human eosinophil cationic protein、hECP)中核心硫酸乙醯肝素結合區域(heparan sulfate binding motif)發展出一段無毒性醣胺多醣結合胜肽(glycosaminoglycan binding peptide、GBP)。本研究利用酵素連結免疫分析(enzyme-linked immunosorbent assay、ELISA)證實帶有綠色螢光重組蛋白的GBP (eGFP-GBP)具有結合至老鼠直腸癌(CT-26)及人類肺癌(A549)上皮細胞表面的能力。進一步利用CT-26皮下同種移植之腫瘤小鼠模式研究eGFP-GBP於活體循環組織特異性，免疫化學染色(immunohistochemistry、IHC)結果顯示eGFP-GBP主要標的至CT-26腫瘤、肝臟以及腎臟；在同一動物模式中，普魯士染色(Prussian Blue staining)結果顯示帶有GBP的磁性奈米粒子(GBP-conjugated magnetic nanoparticle、MNP-GBP)亦有標的至CT-26腫瘤的能力。此外，本研究利用體外移行實驗(in vitro migration assay)發現GBP具有抑制CT-26和A549癌細胞以及人類臍帶靜脈內皮細胞(HUVEC)的移行能力。透過活體動物實驗分析GBP對血管新生及腫瘤生長的影響，結果顯示GBP能有效抑制斑馬魚腸下靜脈血管(subintestinal vessel、SIV)的生長，亦能在A549皮下異種移植之腫瘤小鼠模式中抑制腫瘤生長。本研究展現GBP不但具有標靶表皮癌細胞的能力，亦有抑制癌細胞惡化的潛力。因此，GBP具有潛力應用於臨床轉譯醫學，能用於開發癌症用藥的新配方。

關鍵字

胜肽

並列摘要

Cell surface glycosaminoglycans (GAGs), which play diverse roles in cancer progression to regulate proliferation, adhesion, migration, and angiogenesis, interact with molecules including chemokines, thrombins and growth factors. We have recently identified a non-cytotoxic and sulfated GAG-binding peptide (GBP) derived from core heparan sulfate binding motif of human eosinophil cationic protein (hECP). Recombinant enhanced green fluorescent protein-fused GBP (eGFP-GBP) bound to mouse colon (CT-26) and human lung (A549) epithelial cancer cells in a dose-dependent manner determined by enzyme-linked immunosorbent assay (ELISA). Through in vivo tissue targeting experiment carried out in subcutaneous CT-26 syngeneic tumor mouse model (Balb/c), eGFP-GBP signals were detected in subcutaneous CT-26 tumor site, liver and kidney by immunohistochemistry (IHC) staining. In addition, GBP-conjugated magnetic nanoparticle (MNP-GBP) signals were also observed in subcutaneous CT-26 tumor site using Prussian blue staining. Furthermore, in vitro migration assay indicated that the migration activities of CT-26 and A549 cell and human umbilical vein endothelial cell (HUVEC) were significantly inhibited upon treatment of GBP. Moreover, GBP inhibited the growth of subintestinal vessel (SIV) in zebrafish and suppressed tumor growth in subcutaneous A549 xenogeneic tumor mouse model (SCID). Taken together, this study demonstrated that GBP possessed not only epithelial tumor targeting activity but also anti-migration and anti-angiogenesis activities. Thus, further development of GBP as a reagent for application in translational medicine is feasible.

參考文獻

1. Prydz K, Dalen KT (2000) Synthesis and sorting of proteoglycans. J Cell Sci 113 Pt 2: 193-205.

2. Tumova S, Woods A, Couchman JR (2000) Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. Int J Biochem Cell Biol 32: 269-288.

3. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, et al. (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68: 729-777.

4. Hacker U, Nybakken K, Perrimon N (2005) Heparan sulphate proteoglycans: the sweet side of development. Nat Rev Mol Cell Biol 6: 530-541.

5. Madonna R, De Caterina R (2014) Potential roles of vessel wall heparan sulfate proteoglycans in atherosclerosis. Vascul Pharmacol 60: 49-51.