Bedeutung der Familien der Src Tyrosinkinasen in der Lymphangiogenese

Abstract

Introduction: The close association of lymphatic and blood vessels and their coordinated development in vivo suggest that some molecules may control both hemangiogenesis and lymphangiogenesis. We have previously shown that the inhibition of Src tyrosine kinase by AZM475271 (AstraZeneca) inhibited hemangiogenesis and suppressed L3.6pl pancreatic tumor growth via induction of tumor endothelial cell apoptosis in mice bearing L3.6pl human pancreatic cancer, but so far there is no evidence about the involvement of Src kinase in lymphangiogenesis and metastatic spread via the lymphatics. We hypothesized that Src tyrosine kinase inhibitors may also generate inhibitory effects on lymphatics, thus, may act as anti-lymphangiogenic agents as well. Material and Methods: Lymphatic endothelial cells (LECs) were isolated by magnetic bead sorting from human dermal microvascular endothelial cells and the purity of isolated LECs exceeded 95 %. Using Western Blot we investigated the ability of the well known Src kinase inhibitor, PP2 (Calbiochem), to block Src kinase activity in the LECs stimulated with VEGF-C. The effect of PP2 on VEGF-C driven proliferation of the LECs was tested in the MTT Proliferation Assay. To investigate the complex process of lymphangiogenesis in vitro, we used a spheroid angiogenesis model. The level of VEGF-C in the supernatant of L3.6pl pancreatic carcinoma cell line was measured by ELISA. We also investigated the migratory activity of isolated LECs following treatment with Src inhibitor using a modified Boyden Chamber Assay. To selectively investigate lymphangiogenesis in vivo, we used the Matrigel Plug Assay and the ingrowth of lymphatic vessels was visualized by LYFE-1 immunostaining. Results: VEGF-C exhibited an increased activity of Src tyrosine kinase in isolated LECs, however the activity was abrogated by treatment with Src kinase inhibitor PP2 in a dose dependent manner. Src kinase inhibitor PP2 also reduced VEGF-C-driven proliferation of isolated LECs in a concentration dependent manner in the MTT Proliferation Assay. PP2 inhibited sprouting of VEGF-C-stimulated LECs in the spheroid assay at even lower concentrations than suggested by the MTT Assay. Src kinase inhibition by PP2 markedly reduced the VEGF-C levels in the supernatant of the highly metastatic pancreatic carcinoma cell line L3.6pl. Inhibition of Src kinase by PP2 also resulted in a significant reduction of the migratory activity of isolated LECs. Lymphatic neovascularization was markedly inhibited in PP2-treated compared to untreated animals in the Matrigel Plug Assay. Conclusion: The Src kinase inhibitor PP2 shows strong anti-lymphangiogenic activity in vitro and in vivo. The combined anti-angiogenic and antilymphangiogenic effects of Src kinase inhibition may provide therapeutic value for the treatment of angiogenesis-dependent diseases and tumor metastasis.