The pseudopeptide HB-19 binds to cell surface nucleolin and inhibits angiogenesis

Abstract

Background: Nucleolin is a protein over-expressed on the surface of tumor and endothelial cells. Recent studies have underlined the involvement of cell surface nucleolin in tumor growth and angiogenesis. This cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules like integrins, selectins or laminin-1, lipoproteins and viruses (HIV and coxsackie B). HB-19 is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits both tumor growth and angiogenesis.

Methodology/principal findings: In the present work, we further investigated the biological actions of pseudopeptide HB-19 on HUVECs. In a previous work, we have shown that HB-19 inhibits the in vivo angiogenesis on the chicken embryo CAM assay. We now provide evidence that HB-19 inhibits the in vitro adhesion, migration and proliferation of HUVECs without inducing their apoptosis. The above biological actions seem to be regulated by SRC, ERK1/2, AKT and FAK kinases as we found that HB-19 inhibits their activation in HUVECs. Matrix metalloproteinases (MMPs) play crucial roles in tumor growth and angiogenesis, so we investigated the effect of HB-19 on the expression of MMP-2 and we found that HB-19 downregulates MMP-2 in HUVECs. Finally, down regulation of nucleolin using siRNA confirmed the implication of nucleolin in the biological actions of these peptides.

Conclusions/significance: Taken together, these results indicate that HB-19 could constitute an interesting tool for tumor therapy strategy, targeting cell surface nucleolin.

Figure 1

HB-19 inhibits the in vitro adhesion, proliferation, migration and motility of HUVECs. (A) Inhibition of HUVECs adhesion by HB-19. An equal number of HUVECs were incubated with increasing concentrations of HB-19 for 30 min before seeding. After a 45 min incubation period, adherent cells were measured by the crystal violet assay. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments. (B) Inhibition of HUVECs proliferation by HB-19. Cells were cultured for 3 days in presence of increasing concentrations of HB-19. Cell proliferation was quantified by crystal violet staining. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments. (C) Migration of cells through Transwell filters. The lower compartment of Transwell filters (8 μm pores) was filled with growth media containing 0.25% BSA. An equal number of HUVECs was re suspended in growth medium containing 0.25% BSA and increasing concentrations of HB-19, and transferred into Transwell inserts. Cells that successfully migrated through the filter pores, were fixed, stained and quantified by counting the entire area of each filter. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments. (D) Confluent cell monolayers were scratched and cells were left to heal the wound in the presence of increasing concentrations of HB-19. 48 h later the plates were photographed.

Figure 2

HB-19 does not induce apoptosis of HUVECs. (A and C), endothelial cells were incubated with increasing concentrations of HB-19 and 24 h later the number of apoptotic cells was measured by FACS analysis. (B), H₂O₂ was used as positive control.

Figure 3

HB-19 down-regulates MMP2 in HUVECs. (A) Endothelial cells were cultured in a minimal medium with increasing concentrations of HB-19 and 8 h later the supernatants were analyzed for MMP2 activity by zymography. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments. (B) Endothelial cells were incubated with increasing concentrations of HB-19 and 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for MMP2 or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments.

Figure 4

HB-19 signalling down-regulates pSRC, pFAK, pAKT and pERK1/2. Western blot analysis of phosphorylated SRC, FAK, AKT and ERK1/2, in cells stimulated by increasing concentrations of HB-19 for 15 minutes. The blots were stripped and re probed for total SRC, HSC70, HSC70 and total ERK1/2 respectively. Results are expressed as % change relative to the control and are mean values ± SE from at least 3 independent experiments.

Figure 5

Effect of nucleolin (NCL) knockdown on HB-19 biological actions. Down-regulation of nucleolin (NCL) mRNA (A) and protein (B) using specific siRNA targeting nucleolin mRNA. Effect of HB-19 (10μΜ) on proliferation (C) and adhesion (D) of HUVECs. The last two bars of each diagram indicate HUVECs that were transiently transfected with siRNA targeting nucleolin (NCL). Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments.

Figure 6

Effect of nucleolin (NCL) knockdown on HB-19-induced signal transduction. Western blot analysis of phosphorylated SRC and ERK1/2 in HUVECs stimulated with 10 μM HB-19 for 15 minutes. The blots were stripped and re probed for HSC70. The last two bars of each diagram indicate HUVECs that were transiently transfected with siRNA targeting nucleolin. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments.