Patil Shilpa S, Railkar Reema, Swain Monalisa, Atreya Hanudatta S, Dighe Rajan R, Kondaiah Paturu
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
J Neurooncol. 2015 Jun;123(2):225-35. doi: 10.1007/s11060-015-1800-7. Epub 2015 May 6.
Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface α5β1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 × 10(8)) and Tomlinson J (Library size 1.37 × 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.
胰岛素样生长因子结合蛋白2（IGFBP2）在胶质母细胞瘤（GBM）组织中高度上调，并且一直是预后指标之一。有令人信服的证据表明IGFBP2在胶质瘤细胞增殖、迁移和侵袭中发挥重要作用。细胞外的IGFBP2通过其羧基末端的精氨酸-甘氨酸-天冬氨酸（RGD）基序可以与细胞表面的α5β1整合素结合，并激活整合素信号下游的通路。已知这种IGFBP2激活的整合素信号在IGFBP2介导的胶质瘤细胞侵袭中起关键作用。因此，一种能够抑制IGFBP2与细胞表面相互作用的IGFBP2羧基末端结构域的分子抑制剂具有重要的治疗意义。为了开发IGFBP2的分子抑制剂，我们使用人重组IGFBP2筛选了单链可变片段（scFv）噬菌体展示文库Tomlinson I（文库大小为1.47×10⁸）和Tomlinson J（文库大小为1.37×10⁸）。筛选后，我们获得了三种IGFBP2特异性结合物，其中一种scFv B7J在其羧基末端结构域与IGFBP2表现出更好的结合，阻断了IGFBP2与细胞表面的结合，降低了胶质瘤细胞条件培养基中基质金属蛋白酶2的活性，并抑制了IGFBP2诱导的胶质瘤细胞迁移和侵袭。我们首次证明，使用人scFv体外抑制细胞外IGFBP2活性可显著降低胶质瘤细胞的迁移和侵袭。因此，抑制IGFBP2可作为GBM治疗的潜在策略。
