ABSTRACT
The cellular transmembrane receptor Neuropilin-1(NRP-1) is overexpressed in tumour tissue and endothelial cells of tumour vessels, whereas it has limited expression in normal tissues. This study aimed to design a novel recombinant protein tTF-EG3287, which consisting of the truncated tissue factor (tTF) and the NRP-1 targeting peptide EG3287. The procoagulant protein selectively activates blood coagulation in tumour vessels once bound to the cell surface of the tumour vasculature by a targeting peptide EG3287. In this study, procoagulant activity of the recombinant protein tTF-EG3287 was evaluated by Spectozyme FXa assay. NRP-1 targeting ability was analysed by fluorescence confocal microscopy and flow cytometry. The living imaging system was used to assess the tumour targeting ability of recombinant proteins tTF-EG3287 in vivo. Tumour growth inhibition showed effective antitumor activity in HepG2 tumour-bearing nude mice. Histological study showed obvious thrombosis and thromboembolism in tumour vessels and cell necrosis of tumour tissue, without any clear side effect such as thrombosis in other organs.
Subject(s)
Neuropilin-1/metabolism , Thromboplastin/pharmacology , Animals , Cell Line , Cell Line, Tumor , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Hep G2 Cells , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Peptide Fragments/pharmacology , Peptides/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , Xenograft Model Antitumor Assays/methodsABSTRACT
Class three semaphorins were originally identified as mediators of axon guidance, which repelled axons and collapsed growth cones. As a member of class three semaphorins, semaphorin3F (Sema3F) has been found to have similar effects on tumor cells and endothelial cells and also is implicated in the signaling of tumor metastasis by forming a complex with neuropilins and plexins. In this study, our laboratory produced a monoclonal antibody against the C-terminal domain of Sema3F (Sema3Fc mAb) using the hybridoma method, expecting to explore the potential role of the antibody and its application in the detection of Sema3F. The capture enzyme-linked immunosorbent assay (ELISA) method indicated that mAb belonged to the IgM subclass and purified Sema3Fc mAb had a titer of 5.12 × 105 against Sema3Fc by indirect ELISA. In addition, results showed that the Sema3Fc mAb could be applied in such experiments as Western blotting, flow cytometry, immunofluorescence, and immunocytochemical staining. It indicates the Sema3Fc mAb is available in the detection of Sema3F with specificity and will help further study the role and mechanism of Sema3F among tumor cells.
