Recombinant Snake Venom Cystatin Inhibits Tumor Angiogenesis in vitro and in vivo Associated with Downregulation of VEGF-A165, Flt-1 and bFGF

Qun      Xie; Nanhong      Tang; Rong      Wan; Yuanlin      Qi; Xu      Lin; Jianyin      Lin

Abstract

Previous studies have shown that recombinant snake venom cystatin (sv-cystatin) inhibits the invasion and metastasis of tumor cells in vitro and in vivo. The purpose of this study was to investigate the ability of recombinant sv-cystatin to inhibit tumor angiogenesis in vitro and in vivo, and the mechanisms underlying this effect. Recombinant sv-cystatin inhibited proliferation of human umbilical vein endothelial cells (HUVECs) at 100 and 200 μg/mL after 72, 96 and 120 h. Recombinant sv-cystatin also inhibited tumor–endothelial cell adhesion at 25, 50, 100 and 200 μg/mL. Recombinant sv-cystatin inhibited capillary-like tube formation by HUVECs at 10, 25, 50, 100 and 200 μg/mL following 12, 24 and 36 h incubation. Furthermore, recombinant sv-cystatin significantly suppressed microvessel density (MVD) of lung tumor colonies in C57BL/6 mice inoculated in the lateral tail vein with B16F10 melanoma cells. Administration of recombinant sv-cystatin significantly decreased MVD of primary tumor tissues in nude mice implanted subcutaneously with human hepatocellular carcinoma cells (MHCC97H). Exposure of B16F10 and MHCC97H cells to increasing doses of recombinant sv-cystatin suppressed secretion of vascular endothelial growth factor (VEGF)-A165 and basic fibroblast growth factor (bFGF) into the surrounding medium (P<0.05). The expression of fms-related tyrosine kinase 1 (Flt-1) protein in HUVECs was decreased by 25, 50, 100 and 200 μg/mL recombinant sv-cystatin (P<0.05). This study demonstrates that recombinant sv-cystatin inhibits tumor angiogenesis associated with downregulation of VEGF-A165, Flt-1 and bFGF. This suggests that recombinant sv-cystatin may have potential pharmaceutical applications as an antiangiogenic and antimetastatic therapeutic agent.

Keywords: Angiogenesis, Basic fibroblast growth factor, Cystatin, Endothelial cell, Fms-related tyrosine kinase 1, Snake venom, Vascular endothelial growth factor-A165

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