Arg-Leu-Tyr-Glu tetrapeptide inhibits tumor progression by suppressing angiogenesis and vascular permeability via VEGF receptor-2 antagonism

Abstract

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE) is known to inhibit vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis in vitro. Herein, we examined its underlying mechanism and antitumor activity associated with vascular remodeling. RLYE inhibited VEGF-A-induced angiogenesis in a mouse model and suppressed VEGF-A-induced angiogenic signal cascades in human endothelial cells. However, RLYE showed no inhibitory effect on VEGF-A-induced proliferation and migration of multiple myeloma cells expressing VEGF receptor (VEGFR)-1, but not VEGFR-2. In addition, RLYE showed no inhibitory effect on angiogenic activities induced by VEGF-B, basic fibroblast growth factor, epithermal growth factor, sphingosine-1-phosphate, and placental growth factor. RLYE bound specifically to VEGFR-2 at the VEGF-A binding site, thereby blocking VEGF-A-VEGFR-2 binding and VEGF-A-induced VEGFR-2 internalization. The RLYE peptide inhibited tumor growth and metastasis via suppression of tumor angiogenesis in tumor-bearing mice. Moreover, RLYE showed a synergistic effect of the cytotoxic agent irinotecan on tumor cell apoptosis and tumor progression via tumor vessel normalization due to stabilization of VE-cadherin-mediated adherens junction, improvement of pericyte coverage, and inhibition of vascular leakage in tumors. Our results suggest that RLYE can be used as an antiangiogenic and tumor blood vessel remodeling agent for inhibition of tumor growth and metastasis by antagonizing VEGFR-2, with the synergistic anti-cancer effect via enhancement of drug delivery and therapeutic efficacy.

N-Terminal Modification of the Tetrapeptide Arg-Leu-Tyr-Glu, a Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) Antagonist, Improves Antitumor Activity by Increasing its Stability against Serum Peptidases

“…RLYE inhibited VEGF-induced angiogenesis in vitro more effectively than KLYD (Baek et al, 2015). Further studies demonstrated that RLYE acts as a VEGFR-2 antagonist and inhibits tumor angiogenesis by binding to VEGFR-2 at the same binding site as VEGF-A (Baek et al, 2015(Baek et al, , 2017. Here, we found that RLYE had a short half-life in serum; therefore, we developed a more stable peptide, Ac-RLYE, by N-terminal acetylation of RLYE.…”

“…HCT116 cells were cultured in RPMI medium supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 100 U/ml penicillin-streptomycin antibiotic solution in a humidified atmosphere of 5% CO 2 at 37°C. HUVECs were maintained and cultured in M199 as previously described (Baek et al, 2017), and only passages 2-6 were used for all experiments.…”

“…In Vitro Angiogenesis Assay. Angiogenic activity was determined by measurements of endothelial cell migration and tube formation as described previously (Baek et al, 2017). Chemotactic migration was analyzed using Boyden chambers.…”

“…After incubation at 37°C for 4 hours, the cells that migrated to the lower side of the filter were fixed and stained with hematoxylin and eosin and counted using a phase-contrast microscope. On the other hand, the formation of tube-like structure was determined after culture of HUVECs on growth factor reduced Matrigel (Baek et al, 2017). Twenty-four well culture plates were coated with Matrigel according to the manufacturer's instructions.…”

“…Tumor size was measured in two dimensions using calipers. The tumor volume (cubic millimeter) was calculated using the ellipsoid formula: width 2  length  0.52 (Baek et al, 2017).…”