Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran.
Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.
Mol Biol Rep. 2020 Mar;47(3):2061-2071. doi: 10.1007/s11033-020-05306-9. Epub 2020 Feb 18.
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, β-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.
肿瘤血管生成使肿瘤细胞能够向血流中生长和迁移，并启动转移。血管内皮生长因子 (VEGF) A 和 B 作为血管生长的重要调节因子，与 VEGFR1 和 VEGFR2 的相互作用触发血管生成过程。因此，阻止这些相互作用导致了 VEGF/VEGFRs 信号通路的有效阻断。在这项研究中，使用 MTT 测定法研究了一种 23 个氨基酸线性肽 (VGB4) 的抑制作用，该肽与 VEGFR1 和 VEGFR2 结合，对 VEGF 刺激的人脐静脉内皮细胞 (HUVECs) 和高转移性人乳腺癌细胞 MDA-MB-231 的增殖。为了评估 VGB4 的抗迁移潜力，在 48 和 72 小时进行了 HUVEC 和 MDA-MB-231 细胞划痕愈合测定。此外，通过实时定量 PCR 和 Western blot 定量分析与细胞迁移和侵袭相关的 VEGF 下游信号通路，在 4T1 肿瘤组织和 MDA-MB-231 细胞中检测到 VEGF 下游信号通路。结果表明，VGB4 以剂量和时间依赖的方式显著抑制 HUVEC 和 MDA-MB-231 细胞的增殖，并延长 HUVEC 和 MDA-MB-231 细胞的迁移。我们还观察到，与对照组相比，VGB4 处理的 4T1 肿瘤组织中粘着斑激酶 (FAK)、桩蛋白、基质金属蛋白酶-2 (MMP-2)、RAS 相关 C3 肉毒梭菌底物 1 (Rac1)、P21 激活激酶-2 (PAK-2) 和肌动蛋白的转录本和蛋白水平显著降低。与对照组相比，VGB4 处理的 MDA-MB-231 细胞和 VGB4 处理的 4T1 肿瘤组织中磷酸化 VEGFR1、磷酸化 VEGFR2、波形蛋白、β-连环蛋白和 Snaill 的蛋白水平明显降低，这一点通过 Western blot 得到证实。这些结果以及我们之前的研究证实，由于多种信号介质的失活，双重阻断 VEGFR1 和 VEGFR2 可有效抑制肿瘤生长和转移。
