O-sulfated bacterial polysaccharides with low anticoagulant activity inhibit metastasis

Heparin-like polysaccharides possess the capacity to inhibit cancer cell proliferation, angiogenesis, heparanase-mediated cancer cell invasion, and cancer cell adhesion to vascular endothelia via adhesion receptors, such as selectins. The clinical applicability of the antitumor effect of such polysaccharides, however, is compromised by their anticoagulant activity. We have compared the potential of chemically O-sulfated and N,O-sulfated bacterial polysaccharide (capsular polysaccharide from E. coli K5 [K5PS]) species to inhibit metastasis of mouse B16-BL6 melanoma cells and human MDA-MB-231 breast cancer cells in two in vivo models. We demonstrate that in both settings, O-sulfated K5PS was a potent inhibitor of metastasis. Reducing the molecular weight of the polysaccharide, however, resulted in lower antimetastatic capacity. Furthermore, we show that O-sulfated K5PS efficiently inhibited the invasion of B16-BL6 cells through Matrigel and also inhibited the in vitro activity of heparanase. Moreover, treatment with O-sulfated K5PS lowered the ability of B16-BL6 cells to adhere to endothelial cells, intercellular adhesion molecule-1, and P-selectin, but not to E-selectin. Importantly, O-sulfated K5PSs were largely devoid of anticoagulant activity. These findings indicate that O-sulfated K5PS polysaccharide should be considered as a potential antimetastatic agent.</p

Heparanase Levels Are Elevated in the Urine and Plasma of Type 2 Diabetes Patients and Associate with Blood Glucose Levels

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans. Utilizing an ELISA method capable of detection and quantification of heparanase, we examined heparanase levels in the plasma and urine of a cohort of 29 patients diagnosed with type 2 diabetes mellitus (T2DM), 14 T2DM patients who underwent kidney transplantation, and 47 healthy volunteers. We provide evidence that heparanase levels in the urine of T2DM patients are markedly elevated compared to healthy controls (1162±181 vs. 156±29.6 pg/ml for T2DM and healthy controls, respectively), increase that is statistically highly significant (P<0.0001). Notably, heparanase levels were appreciably decreased in the urine of T2DM patients who underwent kidney transplantation, albeit remained still higher than healthy individuals (P<0.0001). Increased heparanase levels were also found in the plasma of T2DM patients. Importantly, urine heparanase was associated with elevated blood glucose levels, implying that glucose mediates heparanase upregulation and secretion into the urine and blood. Utilizing an in vitro system, we show that insulin stimulates heparanase secretion by kidney 293 cells, and even higher secretion is observed when insulin is added to cells maintained under high glucose conditions. These results provide evidence for a significant involvement of heparanase in diabetic complications

BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues

Background: The bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. Methods: BMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. Results: N-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. Conclusions: In BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. General significance: This study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders

Water diffusion in chemical and physical polysaccharidic gels

Diffusion of water is a major interest in many food and biotechnological applications. In this work we determined the self-diffusion coefficient of water and sugars in chemical and physical polysaccharidic gels, by low field nuclear magnetic resonance. The physical gels were obtained from agar powder (gelagar type R, B&V, Parma Italy) with a final polymer concentration ranged between 0.2 and 1% (m/v). The chemical gels were synthetized from carboxymethylcellulose crosslinking with epichlorhydrin and PEG. All the materials were characterized by their rheological properties both by means of small amplitude oscillatory measurements and large deformation measurements with puncture test and uniaxial compression. Structural informations about the matrix, like the average pore size, can be obtained from self-diffusion coefficient of water

Characterization of Cyclodextrin Cross-linked Polymers Used in Environmental Applications by Solid-state NMR Spectroscopy: a Historical Review

Cross-linked cyclodextrin polymers are attracting increasing interest not only from the scientific community but also from industry. These commercial polymers, discovered in the late 1960s, have applications in many fields, from pharmaceuticals to food processing, chromatography, cosmetics, membrane materials, and the environment. More recently, this class of functional polymers have been proposed as biosorbents to treat wastewater contaminated by conventional pollutants, such as metals, polycyclic aromatic hydrocarbons, phenols, and dyes, or emerging pollutants such as hormones, antibiotics, alkylphenols, and fluorine derivatives. However, although many results have been published, many questions remain not only on the chemical structure of the macromolecular networks of these polymers but also on the biosorption mechanisms involved in their use as biosorbents to treat pollutants. Solid-state NMR spectroscopy can help to answer these questions. This chapter aims to summarize and discuss the role of solid-state NMR spectroscopy in characterizing the structure of cross-linked cyclodextrin polymers and the interactions involved in biosorption using the state of the art and our own research results obtained over 25 years