Abstract
TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.
Highlights
Complex containing MMP-2,2 membrane type metalloprotease-1, tissue inhibitor of metalloprotease-2 [8], and the CLC-3 chloride channel at the surface of glioma cells and mediate the internalization and down-regulation of both MMP-2 and CLC-3 [7, 8]
We show that surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2 using siRNA-mediated specific knockdown of annexin A2 levels
We demonstrate that treatment of human umbilical vein endothelial cell (HUVEC) in culture with TM601 inhibits the activity of tissue plasminogen activator present in the cell culture supernatants
Summary
Complex containing MMP-2,2 membrane type metalloprotease-1, tissue inhibitor of metalloprotease-2 [8], and the CLC-3 chloride channel at the surface of glioma cells and mediate the internalization and down-regulation of both MMP-2 and CLC-3 [7, 8]. Using mouse tumor models, a bio-conjugate of chlorotoxin with the nearly infrared dye Cy5.5 (CTX:Cy5.5) was shown to efficiently detect and monitor multiple tumor types, including glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma following intravenous injection [10] These studies demonstrated that binding of CTX:Cy5.5 bio-conjugate to MCF-7 breast carcinoma cells in vitro is facilitated by the expression of exogenous MMP-2. We found that TM601 binds a wide range of tumor cell types but is internalized by proliferating human vascular endothelial cells [11] These studies demonstrated an anti-angiogenic effect of TM601 using both the chicken chorioallantoic membrane assays and the mouse Matrigel plug assays. Consistent with the inhibition of cell surface protease activity, we show that TM601 inhibits trans-well migration of both HUVEC and U373-MG glioma cells in response to PDGF-CC stimulation
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