Coupling to a cancer-selective heparan-sulfate-targeted branched peptide can by-pass breast cancer cell resistance to methotrexate

Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications

Decoding the role of HSPGs in tumor onset and progression through use of branched peptide probe

Heparan sulfate Proteoglycans (HSPGs) play a number of signaling and structural roles in tumor progression and metastasis spread. The biological function of HSPGs resides in their ability of interaction with many different types of ligands like growth factors, morphogens, chemokines and proteins of extracellular matrix (ECM). These bindings activate signaling pathways that modulate major transformations of cancer cells, leading to tumor growth, migration, invasion and metastasis. HSPGs are over-expressed on cancer cell membranes. The tetra-branched peptide NT4 binds with high selectivity to different human cancer cells and tissues. Its ability to discriminate between tumor and healthy tissues resides in the high-affinity binding to HSPGs and can be exploited by conjugating NT4 with different functional units, like chemotherapeutical drugs and tracers for cancer cell imaging and therapy. In this work, we test NT4 ability to interfere with processes mediated by HSPGs in tumor cell adhesion, migration and matrix invasiveness. Since HSPGs modulate also neo-angiogenesis, because of their expression by endothelial cells of microvessels that generate new vasculature, we focus also on NT4 role in endothelial cell proliferation, migration and tube formation, in the presence of Fibroblast Growth Factor-2 (FGF-2). Since the internalization and trafficking behaviour of HSPGs seems to reveal a clathrin and caveolin- independent, but dynamin-dependent endocytic pathway, we investigate the pathway used by NT4 to enter cells. Last, but not least, we radiolabel NT4 with 18F in order to measure tumor uptake and whole-body biodistribution in a mouse model of breast cancer, using in vivo PET imaging. NT4-HSPG interactions and consequent modulation of signaling pathways can prove the importance of NT4 peptide as a specific tool to enlighten the role of HSPG in tumor onset and progression

Insights into the role of sulfated glycans in cancer cell adhesion and migration through use of branched peptide probe

9noopenThe tetra-branched peptide NT4 selectively binds to different human cancer cells and tissues. NT4 specifically binds to sulfated glycosaminoglycans on cancer cell membranes. Since sulfated glycosaminoglycans are involved in cancer cell interaction with the extracellular matrix, we evaluated the effect of NT4 on cancer cell adhesion and migration. We demonstrated here that the branched peptide NT4 binds sulfated glycosaminoglycans with high affinity and with preferential binding to heparan sulfate. NT4 inhibits cancer cell adhesion and migration on different proteins, without modifying cancer cell morphology or their ability to produce protrusions, but dramatically affecting the directionality and polarity of cell movement. Results obtained by taking advantage of the selective targeting of glycosaminoglycans chains by NT4, provide insights into the role of heparan sulfate proteoglycans in cancer cell adhesion and migration and suggest a determinant role of sulfated glycosaminoglycans in the control of cancer cell directional migration.openBrunetti, Jlenia; Depau, Lorenzo; Falciani, Chiara; Gentile, Mariangela; Mandarini, Elisabetta; Riolo, Giulia; Lupetti, Pietro; Pini, Alessandro; Bracci, LuisaBrunetti, Jlenia; Depau, Lorenzo; Falciani, Chiara; Gentile, Mariangela; Mandarini, Elisabetta; Riolo, Giulia; Lupetti, Pietro; Pini, Alessandro; Bracci, Luis

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

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

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 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