A Pleiotrophin C-terminus peptide induces anti-cancer effects through RPTPβ/ζ

<p>Abstract</p> <p>Background</p> <p>Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor.</p> <p>Results</p> <p>Our results show that P(122-131) inhibits <it>in vitro </it>adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPβ/ζ. In addition, P(122-131) inhibits angiogenesis <it>in vivo</it>, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk¹/₂. Finally, P(122-131) not only interacts with RPTPβ/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPβ/ζ expression with the RNAi technology.</p> <p>Conclusions</p> <p>In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPβ/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.</p

Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin

BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy

Hemodialysis Removes Uremic Toxins That Alter the Biological Actions of Endothelial Cells

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure

Nucleolin mediates the antiangiogenesis effect of the pseudopeptide N6L

BACKGROUND: Nucleolin is a protein over-expressed on the surface of activated cells. Recent studies have underlined the involvement of cell surface nucleolin in angiogenesis processes. 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. N6L is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits cell proliferation. RESULTS: In the present work, we further investigated the mechanisms of action of pseudopeptide N6L on angiogenesis using HUVECs. We provide evidence that N6L inhibits the in vitro adhesion, proliferation and migration of HUVECs without inducing their apoptosis. In addition, we found that N6L downregulates MMP-2 in HUVECs. The above biological actions are regulated by SRC, ERK1/2, AKT and FAK kinases as we found that N6L inhibits their activation in HUVECs. Finally, down regulation of nucleolin using siRNA demonstrated the implication of nucleolin in the biological actions of these peptides. CONCLUSIONS: Taken together, these results indicate that N6L could constitute an interesting therapeutic tool for treating diseases associated with excessive angiogenesis

Effect of pre- or post-HD serum on the expression of TIMP-1 and TIMP-2.

<p>(A): HUVEC were incubated in culture medium supplemented with 20% pre- or post- HD serum. 4 h later the medium was replaced by minimal medium and 8 h or 20 h later the supernatants were analyzed for TIMP-1 and TIMP-2 proteins by SDS-PAGE. (B): HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for TIMP-1, TIMP-2 or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^* and ^** represent p<0.05 and p<0.01 respectively.</p

Effect of pre- or post-HD serum on the expression of collagen-IV and elastin.

<p>HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for Collagen IV, Elastin or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^*, ^** and ^*** represent p<0.05, p<0.01 and p<0.001 respectively.</p