The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells

<div><p>Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in <i>in vivo</i> models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.</p></div

a) NT4 binding to HUVEC endothelial cells tested by cytofluorimetry: Increasing amounts of heparin displaced NT4 binding; b) Effect of NT4 on proliferation was assessed in the presence of FGF2 and heparin.

<p>a) NT4 binding to HUVEC endothelial cells tested by cytofluorimetry: Increasing amounts of heparin displaced NT4 binding; b) Effect of NT4 on proliferation was assessed in the presence of FGF2 and heparin.</p

Effect of NT4 on HUVEC tube formation; a, b, c) representative images of tubes after 3 h of incubation at 37°C in the presence of FGF2 and NT4; d) variation in number of branching nodes with respect to untreated cells after 2, 3 and 6 h.

<p>Images of the wells were analyzed for the number of nodes.</p

The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells - Fig 2

<p>a) Effect of NT4 on migration of HUVEC on different coatings in a cell layer wound healing assay, measured as % of gap closure. HUVEC were plated on wells coated with collagen IV, fibronectin or on uncoated wells where a silicon spacer had been placed immediately before cell plating. Once cells had reached confluence, the silicon spacer was removed and cells were treated with NT4 peptide (10 μM) for 24 hours; b) Complete gap closure on fibronectin, untreated cells and C) impairment of HUVEC gap closure by NT4 on fibronectin.</p

The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells - Fig 4

<p>a) and b) Collagen I degradation measured after 72 h of incubation. 100% indicates intact collagen. Experiments were performed in quadruplicate; c) Collagen I invasion assay; d) MDA-MB-231 crossed a porous membrane coated with collagen (upper panel), but invasion was impaired by NT4 (10 μM) (lower panel).</p

Relaxin prevents TGF-β1 induced down regulation of MMP-2 and MMP-9 expression and up-regulation of TIMP-2 in NIH/3T3 and primary neonatal cardiac fibroblasts.

<p>A–C, F–H). Representative confocal immunofluorescence images of NIH/3T3 cells (A–C) and primary neonatal cardiac fibroblasts (F–H) cultured in the indicated experimental conditions and immunostained with antibodies against MMP-2 (A,F; cyan), MMP-9 (B,G; green) or TIMP-2 (C,H, green). In B and G the nuclei are labeled in red with propidium iodide. The histograms show the corresponding densitometric analyses of the intensity of MMP-2, MMP-9 and TIMP-2 fluorescence signals. D–E) Western blotting analyses of the expression of (D) MMP-2, and (E) MMP-9 proteins in neonatal cardiac fibroblasts. In the histograms the densitometric analyses of the bands normalized to GAPDH are reported. Data are representative of at least three independent experiments with similar results. Significance of differences: *p<0.05 <i>vs</i> control, °p<0.05 <i>vs</i> TGF-β1.</p

Relaxin prevents the TGF-β1-induced down-regulation of Notch-1 pathway in primary neonatal cardiac fibroblasts.

<p>A) RT-PCR of Notch-1 expression. B,C) Western blotting analysis of activated intracellular form of Notch-1 (Notch-ICD, B) ) and of Notch-1 ligand, Jagged-1(C). The densitometric analyses of the bands normalized to GAPDH are reported in the histograms. D) Confocal immunofluorescence analysis of Notch-1 (green) and Hes-1 (cyan) expression. For the analysis of Notch-1, the cells were stained with a specific antibody recognizing both the membrane Notch-1 receptor and its activated intracellular form, Notch-ICD. Densitometric analyses of Notch-ICD and Hes-1 fluorescent signals are reported in the corresponding histogram. Significance of differences: *p<0.05 <i>vs</i> control, °p<0.05 <i>vs</i> TGF-β1.</p

Characterization and expression of RLX receptor on primary neonatal cardiac fibroblasts.

<p>A) Representative contrast phase microscopy images of first passage neonatal murine neonatal cardiac fibroblasts. B) Representative superimposed differential interference contrast (DIC) and confocal immunofluorescence images of neonatal cardiac fibroblasts immunostained with antibodies against vimentin (green). Nuclei are counterstained in red with propidium iodide. C) Expression of Relaxin family peptide receptor 1 (RXFP1) in neonatal cardiac fibroblasts and NIH/3T3 at mRNA level determined by RT-PCR, and protein level evaluated by Western blotting analysis. The images are representative of at least three independent experiments with similar results.</p

RLX and Notch-1 negatively regulates TGF-β1-induced fibroblast-myofibroblast transition in cardiac fibroblasts.

<p>Neonatal cardiac fibroblasts were cultured for 48 h and treated as indicated. A) Western blotting analysis of NICD expression in the absence (control) or presence of DAPT (5 µM) a pharmacological γ-secretase inhibitor, used to block the generation of NICD. B) Western Blotting analysis of α–sma and MMP-2 expression in the cells treated with DAPT. C) Representative confocal immunofluorescence images cardiac fibroblasts treated with DAPT, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. D) Western blotting analysis of Jagged-1 expression in control cells, cells transfected with non specific scrambled-siRNA (SCR-siRNA) or silenced for the expression of Notch-1 ligand, Jagged-1, by specific Jagged-1 siRNA (Jagged-1 siRNA). E) Western Blotting analysis of α–sma in Jagged-1 silenced cells. F) Representative confocal immunofluorescence images cardiac fibroblasts silenced for Jagged-1 expression, fixed and stained with antibodies against α–sma (green). Nuclei are marked in red with propidium iodide. The densitometric analyses of the bands normalized to GAPDH are reported in histograms in A–E; the densitometric analyses α–sma fluorescent signal are shown in the histograms in and C, F. Significance of differences: *p<0.05 <i>vs</i> control, ^δp<0.05 vs SCR-siRNA, °p<0.05 <i>vs</i> TGF-β1, ^#p<0.05 vs TGF-β1+ RLX.</p

Cancer Selectivity of Tetrabranched Neurotensin Peptides Is Generated by Simultaneous Binding to Sulfated Glycosaminoglycans and Protein Receptors

In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity