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International audience; Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE “modulator” capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

International audience; Heparanase is an endo-β-D-glucuronidase that plays an important role in cancer progression, in particular during tumor angiogenesis and metastasis. Inhibiting this enzyme is considered as one of the most promising approaches in cancer therapy. Heparin is a complex glycoaminoglycan known as a strong inhibitor of heparanase. It is primarily used in clinical practice for its anticoagulant activities, which may not be compatible with its use as anti-angiogenic agent. In this study, we described the production of ultra-low-molecular-weight heparins (ULMWH) by a physicochemical method that consists in a hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. We assessed the structural characteristics, anticoagulant and anti-heparanase activities of the obtained heparin derivatives and compared them with three commercial low-molecular-weight heparins (LMWH), glycol-split non-anticoagulant heparins and heparins produced by enzymatic methods. ULMWH generated by the physicochemical method were characterized by high anti-heparanase and moderate anticoagulant activities. These heparin derivatives might be potential candidates for cancer therapy when a compromise is needed between anti-heparanase and anticoagulant activities.

The performance of microwaves irradiation (MAE and VMAE) to extract pigments from two marine microalgae was compared to conventional processes (cold and hot soaking and ultrasound-assisted extraction). Pigments were quantified by RP-HPLC and extraction performance was assessed regarding rapidity, reproducibility and extraction yields. Scanning electron microscopy was used at all extraction steps to assess the impact of the process on microalgal cell integrity. Freeze-drying and pigments extraction preserved microalgae cell integrity (except sonication) and evoked agglutination in superposed cells layers. All processes performed on Dunaliella tertiolecta (chlorophyte) lead to rapid pigments extraction, and equivalent pigments extraction yields, the absence of frustule allowing immediate solvent penetration in microalgae cells. In contrast, presence of the frustule in the diatom Cylindrotheca closterium (bacillariophyte) constituted a mechanical barrier to pigment extraction. MAE was identified as the best extraction process for CC pigments as it combined rapidity, reproducibility, homogeneous heating and high extraction yields. (C) 2010 Elsevier Ltd. All rights reserved.

International audience; Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 µg · mL −1 (0.17 µM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 µg· mL −1 (13.3 µM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 820 40 µg· mL −1 (66.7 µM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained.

Objective— In human abdominal aortic aneurysm, the accumulation of blood-derived cells and proteases within the mural thrombus plays a pivotal role in the evolution toward vessel wall rupture. We sought to identify peptides released from abdominal aortic aneurysm specimens, characterized by an intraluminal thrombus. Methods and Results— Intraluminal thrombus samples were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus. Conclusion— Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.

Low circulating VVH7-like immunoreactivity (VVH7 i.r) level was amazingly observed in human diabetic sera. Here, we examined the impact of diabetes type, clinico-biological features and metabolic control on circulating VVH7 i.r level in this disease. ELISA test was used to measure VVH7 i.r in sera of 120 diabetic patients (type 1 diabetes in 64, type 2 diabetes in 56). Three enzymatic tests were also applied to determine serum cathepsin D (CD), dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE) activities. A subgroup of 24 type 1 diabetic patients negative for microalbuminuria and hypertension were submitted to an ambulatory blood pressure monitoring to evaluate the relationship between VVH7 i.r level and blood pressure parameters. The mean serum concentration of VVH7 i.r was drastically reduced in diabetic patients (0.91+/-0.93 micromol/l versus 5.63+/-1.11 micromol/l in controls) (p0.001). A negative correlation between VVH7 i.r level and daytime diastolic blood pressure existed in type 1 diabetic patients. There was no association of low VVH7 i.r with either type of diabetes or HbA1c level. An increase of cathepsin D activity was found in serum of diabetic patients compared to controls (0.47 U/ml versus 0.15 U/ml, respectively) whereas DPPIV activity was significantly decreased in diabetic sera (50.81 U/ml versus 282.10 U/l respectively). Diminution of VVH7 i.r in sera of diabetic patients was confirmed but still remained unexplained. Relationships between higher systolic blood pressure and decrease of VVH7 i.r reinforce the need to investigate this pathway in this disease to elucidate its role in macro- and micro-angiopathy.

Unsupplemented caprine whey was fermented by 25 cheese microflora in order to produce peptides from α -lactalbumin ( α -la) and/or β -lactoglobulin ( β -lg) hydrolysis. Fourteen hydrolysates enriched in peptides mainly released from α -la were obtained. Angiotensin-I-converting enzyme (ACE) inhibitory activity of each hydrolysate was investigated. Six of them had high ACE inhibitory activities ranging from 31% to 56%. The highest ACE inhibitory activity was obtained after whey fermentation by the microflora from 18 months ripened Comte cheese. The microflora was identified as a co-culture of Candida parapsilosis and Lactobacillus paracasei . Hydrolysate activity remained stable after pepsin, trypsin and chymotrypsin treatments simulating an in vitro gastrointestinal digestion. This hydrolysate was further fractionated by RP-HPLC. The peptide exhibiting the highest ACE inhibitory activity was characterised as WLAHK ( α -la f (104–108): Trp–Leu–Ala–His–Lys). WLAHK was resistant toward pepsin and trypsin treatments but was digested by chymotrypsin.

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (

International audience; The microwave-assisted synthesis of a family of 2,8-substituted thiazoloquinazolinones is described. The preliminary evaluation of the antiproliferative activity and the capacity of these molecules to inhibit CDKs and GSK-3 are reported. A lead compound was identified, constituting a scaffold from which more potent inhibitors could be designed.

This paper describes an enzyme linked immunosorbent assay (ELISA) for the quantification of bioactive peptides: hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) using an anti-rabbit secondary antibody conjugated to peroxidase for sandwich antibody assay. Bovine serum albumin (BSA) conjugated to VV-hemorphin-7 was readily coated on a polystyrene microplate. Data acquisition on microtiter wells is performed by an ELISA reader. The standard curve was produced for 1 × 10 −13 to 2 × 10 −5 M VV-hemorphin-7. The minimum detectable amount of VV-hemorphin-7 is 5 × 10 −13 mol and the relative standard deviation was 8.8% for a 1 × 10 −6 M sample (n = 8). The ELISA procedure was selective with respect to structurally similar compounds. This method was applied to quantify hemorphin-7 peptides in bovine plasma. © 2002 Elsevier Science B.V. All rights reserved.

Hemorphin peptides, issued from hemoglobin, are emerging as endogenous bioactive peptides derived from in vivo tissular degradation of hemoglobin. In order to find the enzymes which could be implicated in the in vivo release of these peptides, the major lysosomal enzyme cathepsin D was selected, and a study of its activity towards hemoglobin and hemorphins was performed. In this paper, it is shown that according to the primary specificity of cathepsin D towards hemoglobin, this enzyme could constitute a good candidate for the in vivo release of two hemorphins: LVV-hemorphin-7 and VV-hemorphin-7. Moreover, these products, especially VV-hemorphin-7, are resistant to an extended cleavage by the enzyme. Although LVV-hemorphin-7 exhibits a lower resistance, an extended incubation with cathepsin D led to the release of the stable peptide VV-hemorphin-7.

[ 125 I]-Ang IV binding to rabbit collecting duct cell membranes was inhibited by hemorphins (H), a class of endogenous peptides obtained by hydrolysis of the β chain of hemoglobin. The most potent competitors were those with a valine in their N-terminal part such as LVV-H7 and VV-H7 (IC 50 = 1.3 n M ) followed by VV-H8 and K 6 VV-H7 (5.1 n M ). The same H, like Ang IV, interacted with aminopeptidase N (APN) as shown by their inhibitory effect (28–36%) on APN activity. HPLC analysis showed that only H with a N-terminal valine or leucine were hydrolyzed. Since H are detected in the body fluids, they are likely to act as endogenous competitors of Ang IV.