Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation.

Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells.

Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. We previously demonstrated that MMR-deficient tumour cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G(2) phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analogue BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumour cells to DNA damage induced by BLM in G(2). The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no additional effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was observed also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient versus MMR-deficient cells to BLM-induced chromatid breaks in the G(2) phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.

Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide.

Vascular endothelial growth factor receptor-1 (VEGFR-1) exists in two isoforms: a membrane-bound isoform (mVEGFR-1) and a soluble one (sVEGFR-1). mVEGFR-1 is involved in endothelial cell migration and survival supported by VEGF-A and placenta growth factor (PlGF), whereas the biologic function of sVEGFR-1 has not been fully elucidated. We previously reported that sVEGFR-1 induces endothelial cell motility and promotes endothelial cell adhesion. In this study, we tested a set of VEGFR-1-derived peptides for their ability to interfere with endothelial cell migration. Peptide B3 was found to specifically inhibit cell migration induced by sVEGFR-1 and by mVEGFR-1-specific ligands. Moreover, peptide B3 markedly hampered angiogenesis in vitro and in vivo and was found to interfere with VEGFR-1 homodimerisation. Altogether, these data demonstrate that peptide B3 might be a useful tool for the specific inhibition of VEGFR-1 function and might represent a basis for the development of new anti-angiogenic compounds.