Apoptosis induction in human lung and colon cancer cells via impeding VEGF signaling pathways.

There is ample evidence to suggest that vascular endothelial growth factor (VEGF) is a potent mitogen factor in vasculogenesis and angiogenesis and that blockade of VEGF-mediated signals can also prevent tumor growth via enforcing cell apoptosis. In the current study, we assessed the suppressing effect of VGB4, a VEGF antagonist peptide with the binding ability to both VEGF receptor1 and VEGF receptor2, on VEGF-induced proliferation and migration of the human lung adenocarcinoma cell line A549 and the human colon adenocarcinoma cell line HT29 using MTT assay, colony formation assay, and Scratch-wound assay. To evaluate the apoptotic inductive effect of VGB4 on A549 and HT29 cells, apoptosis analysis was carried out by flow cytometry and TUNEL assay. Likewise, p53 and PTEN expression level was examined by immunofluorescence microscopy. In addition, the level of proteins involved in VEGF signaling pathways related to apoptosis was investigated using western blot analysis. Our results indicated that VGB4 markedly inhibited VEGF-induced proliferation and migration, and induced apoptosis of A549 and HT29 cells dose dependently. Encouragingly, significant downregulation of B-cell lymphoma 2 (Bcl2), X-linked inhibitor of apoptosis, Procaspase9, and procaspase3, as well as upregulation of PTEN and P53 tumor suppressors, BCL2 associated X, Cytochrome c, cleaved caspase9, and cleaved caspase3 in VGB4-treated A549 and HT29 cells, further confirmed the profound inductive influence of VGB4 on apoptotic pathways. These findings along with the results from our previous studies show that VGB4 may be considerable for cancer therapy.

Targeting signaling pathways of VEGFR1 and VEGFR2 as a potential target in the treatment of breast cancer.

Tumor angiogenesis allows tumor cells to grow and migrate toward the bloodstream and initiate metastasis. The interactions of vascular endothelial growth factors (VEGF) A and B, as the important regulating factors for blood vessel growth, with VEGFR1 and VEGFR2 trigger angiogenesis process. Thus, preventing these interactions led to the effective blockade of VEGF/VEGFRs signaling pathways. In this study, the inhibitory effect of a 23-mer linear peptide (VGB4), which binds to both VEGFR1 and VEGFR2, on VEGF-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) and highly metastatic human breast cancer cell MDA-MB-231 proliferation was examined using MTT assay. To assess the anti-migratory potential of VGB4, HUVECs and also MDA-MB-231 cells wound healing assay was carried out at 48 and 72 h. In addition, downstream signaling pathways of VEGF associated with cell migration and invasion were investigated by quantification of mRNA and protein expression using real-time quantitative PCR and western blot in 4T1 tumor tissues and MDA-MB-231 cells. The results revealed that VGB4 significantly impeded proliferation of HUVECs and MDA-MB-231 cells, in a dose- and time-dependent manner, and migration of HUVECs and MDA-MB-231 cells for a prolonged time. We also observed statistically significant reduction of the transcripts and protein levels of focal adhesion kinase (FAK), Paxillin, matrix metalloproteinase-2 (MMP-2), RAS-related C3 botulinum substrate 1 (Rac1), P21-activated kinase-2 (PAK-2) and Cofilin-1 in VGB4-treated 4T1 tumor tissues compared to controls. The protein levels of phospho-VEGFR1, phospho-VEGFR2, Vimentin, ß-catenin and Snail were markedly decreased in both VGB4-treated MDA-MB-231 cells and VGB4-treated 4T1 tumor tissues compared to controls as evidenced by western blotting. These results, in addition to our previous studies, confirm that dual blockage of VEGFR1 and VEGFR2, due to the inactivation of diverse signaling mediators, effectively suppresses tumor growth and metastasis.