Impairment of the cell-to-matrix adhesion and cytotoxicity induced by the Mediterranean jellyfish Pelagia noctiluca venom and its fractions in cultured glioblastoma cells

<p>Abstract</p> <p>Background</p> <p>The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited source of new active substances in the field of the development of bioactive products. In our study, we have investigated the efficiency of the venom from the Mediterranean jellyfish, <it>Pelagia noctiluca</it> and its fractions for anti-proliferative and anti-cell adhesion to cell–extracellular matrix activities.</p> <p>Results</p> <p>Our experiments have indicated that the separation of the Mediterranean jellyfish <it>Pelagia noctiluca</it> crude venom extract by sephadex G-75 chromatography led to four fractions (F1, F2, F3, and F4). Among the four fractions F1 and F3 were cytotoxic against U87 cells with IC50 values of 125 and 179 μg/ml respectively. The venom, F1, F2 and F 3 showed significant anti-proliferative activity in time-dependent manner. Our results also suggest that these fractions and the venom are able to inhibit cell adhesion to fibrinogen in dose-dependent manner. This inhibition is reliant on its ability to interact with integrins.</p> <p>Conclusions</p> <p>To conclude, we have demonstrated for the first time that <it>Pelagia noctiluca</it> venom and its fractions especially (F1 and F2) display potent anti-tumoral properties. Separation by sephadex G-75 chromatography give rise to more active fractions than the crude venom extract. The purification and the determination of chemical structures of compounds of these active fractions are under investigation. Overall, <it>Pelagia noctiluca</it> venom may has the potential to serve as a template for future anticancer-drug development.</p

Inhibitory effect of phenolic extract from squirting cucumber (<i>Ecballium elaterium</i> (L.) A. Rich) seed oil on integrin-mediated cell adhesion, migration and angiogenesis

International audienceIntegrin targeted therapies by natural bioactive compounds have attracted attention in the field of oncology and cancer treatment. This study evaluates the potential of phenolic extract from the medicinal herb Ecballium elaterium L. seed oil (PEO) to inhibit the adhesion and migration of the highly invasive human fibrosarcoma cell line HT1080. At safe concentrations (up to 40 mg mL −1), results show that PEO dosedependently inhibits adhesion and migration of HT1080 to fibronectin (IC 50 = 18 mg mL −1) and fibrinogen (IC 50 = 12.86 mg mL −1). These observations were associated with the reduction of cell motility and migration velocity as revealed in the Boyden chamber and random motility using two-dimensional assays, respectively. Additional experiments using integrin blocking antibodies showed that PEO at the highest safe concentration (40 mg mL −1) competitively inhibited the attachment of HT1080 cell to anti-avb3 (>98%), anti-a5b1 (>86%), and to a lesser extent anti-a2 (>50%) immobilized antibodies, suggesting that avb3 and a5b1 integrins were selectively targeted by PEO. Moreover, PEO specifically targeted these integrins in human microvascular endothelial cells (HMEC-1) and dose-dependently blocked the in vitro tubulogenesis. In the CAM model, PEO inhibited the VEGF-induced neoangiogenesis confirming its antiangiogenic effect. Collectively, these results indicate that PEO holds promise for the development of natural integrin-targeted therapies against fibrosarcoma

La famille Kunitz/BPTI des venins de serpents: Structure, classification et potentiel pharmacologique

International audienceSnake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. Generally, these inhibitors are formed by 60 amino acids approximately. Their folding is characterised by a canonical loop that binds in a complementary manner to the active site of serine protease. Some variants from snake venoms show only weak inhibitory activity against proteases while others are neurotoxic. Moreover, proteases inhibitors are involved in various physiological processes, such as blood coagulation, fibrinolysis, and inflammation. Also, these molecules showed an anti-tumoral potent and anti-metastatic effect. Interestingly, Kunitz/BPTI peptides can have exquisite binding specificities and possess high potency for their targets making them excellent therapeutic candidates.Les venins des serpents sont riches en inhibiteurs de sérine protéases qui sont membres de la famille Kunitz / BPTI (inhibiteur de la trypsine pancréatique bovine). Ces inhibiteurs sont en général formés par environ 60 acides aminés. Leur modèle structural est caractérisé par la présence d’une boucle canonique qui se lie d’une manière complémentaire au site actif des sérines protéases. Certains inhibiteurs ne montrent qu'une faible activité inhibitrice contre la protéase tandis que d'autres sont neurotoxiques. De plus, ces peptides sont impliqués dans de divers processus physiologiques, tels que la coagulation sanguine, la fibrinolyse et l'inflammation. En outre, ces molécules ont montré un puissant effet anti-tumoral et anti-métastatique. Ainsi, les inhibiteurs de type Kunitz/BPTI peuvent avoir des spécificités de liaison et possèdent un potentiel élevé envers leurs cibles qui les rend d'excellents candidats thérapeutiques

Snake Venom Proteins Isolated from Tunisian Vipers: Pharmacological and Therapeutic Overview

: The venoms of Tunisian wildlife snakes are complex mixtures containing proteins/ peptides and non-protein molecules. Proteins and peptides are the most abundant compounds responsible for the biological effects of venoms. Snake venoms proteins have enzymatic or nonenzymatic activities, which are grouped into different families, including C-type lectin proteins, disintegrins (long, medium and short disintegrins), Kunitz-type serine protease inhibitors, natriuretic- like peptides, vascular endothelial growth factor-related proteins, L-amino acid oxidases, phospholipases A2 and serine proteinases. With technological advancements, the toxic effects of venoms were turned into potential benefits for clinical diagnosis, basic research and development of new research tools and drugs of potential clinical use. Our research team has shown that Macrovipera lebetina and Cerastes cerastes venom components of Tunisian wildlife snakes had great potential for the development of new drugs for the treatment of cancer, angiogenesis disorders or cardiovascular diseases. This review is an overview of snake venom proteins from Macrovipera lebetina and Cerastes cerastes and their biochemical, pharmacological and molecular characterization and their importance as protein resources with therapeutic potential

Expression of the First Recombinant Anti-Tumoral Snake Venom Kunitz-Type Serine Protease Inhibitor

International audiencePIVL is a Kunitz-type serine protease inhibitor that was previously characterized from Tunisian snake venom, Macrovipera lebetina transmediterranea. It reduced glioblastoma cells’ development and significantly blocked angiogenesis in in-vitro and ex-vivo models. PIVL exerted these effects by interfering with αvβ3 integrin. In order to produce a biological active recombinant, the cDNA cloning and expression of PIVL was performed in Escherichia coli (BL21)-DE3 cells using pET-22b (+) vector. The recombinant PIVL protein (rPIVL) was purified by nickel affinity chromatography and has recognized monoclonal anti-His antibody. Functionally, rPIVL exhibited potent anti-tumor cell effects as well as anti-angiogenesis properties. Interestingly, we found that both native PIVL (nPIVL) and rPIVL modulated PI3/AKT and MAPK signaling pathways. In all, our results showed that we have successfully expressed the first active anti-oncogenic snake venom Kunitz-type protease inhibitor that can be a potential therapeutic drug against glioblastoma, in its native or recombinant form

Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF(165) in E-coli

International audienceVascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF(165) in convenient yield. In this study, recombinant human VEGF(165) was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF(165) was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC)

Investigation of the Renal Protective Effect of Combined Dietary Polyphenols in Streptozotocin-Induced Diabetic Aged Rats

Natural polyphenols are widely reported to have a large range of pharmacological properties, especially antioxidant activities and free radical scavenging capacities. In this study, we investigate the effects of naringin, chlorogenic acid, and quercetin mixtures (NCQ) on renal fibrosis in streptozotocin (STZ)-induced diabetic aged rats and its underlying mechanisms for ten consecutive weeks. The oxidative defense system in the kidneys of treated rats was found to be improved. Several biomarkers were investigated including the blood urea nitrogen, creatinine, and uric acid. Moreover, antioxidant parameters were evaluated and we found that superoxide dismutase, catalase, glutathione peroxidase, Na+-K+-ATPase activities, the nitric oxide production, the protein carbonyl, the advanced oxidation protein products, lipid peroxidation, and reduced glutathione levels were all significantly balanced and close to control values. In addition, NCQ restored renal injuries and fibrosis as assessed by histological method and molecular biology investigation of the matrix metalloproteinase, the transforming growth factor-beta TGF-&beta;, the tumor necrosis factor TNF&alpha;, and p53 expression. Our study proposes the NCQ combination as potential plant-derived bioactive compounds to prevent diabetic nephropathy

Expression of a functional recombinant C-type lectin-like protein lebecetin in the human embryonic kidney (HEK) cells.

International audienceLebecetin is an anticoagulant C-type lectin-like protein (CLPs) that was previously isolated from Macrovipera lebetinavenom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib (GPIb) and to exhibit a broad spectrum of inhibitory activities on various integrin-mediated functions of tumour cells, including adhesion, proliferation, and cell migration. The present study aimed to investigate the structure-function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells (HEK)using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co-localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal-chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represent a prerequisite for functioning

Targeting α1 inserted domain (I) of α1β1 integrin by Lebetin 2 from M. lebetina transmediterranea venom decreased tumorigenesis and angiogenesis.

International audienceThrough the recent development of knowledge in biotechnology and bioinformatics, snake venoms are widely used to develop new drugs to treat diseases such as hypertension and cancer. We have previously reported that Lebetin 2 isolated from Macrovipera lebetina transmediterranea venom displays a potent anti-platelet activity and exerts a cardioprotective effect in ischemia-reperfusion (IR) injury model. Here, we report that Lebetin 2 possess an anti-tumor effect by targeting the integrin receptor function. It was thus able to inhibit both adhesion and migration of pheochromocytoma cells (PC12) and α1β1 integrin-expressing CHO cells (CHO-α1) to type I and IV collagens. Moreover, this peptide affects proliferation of PC12 cells by modulating AKT phosphorylation. Furthermore, Lebetin 2 exhibits a potent anti-angiogenic effect as assessed in vitro and ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Interestingly, the interaction mode of Lebetin 2 with the integrin α1β1, assessed in silico, showed that the peptide represents a steric obstruction preventing the collagen from enforcing the interactions with the integrin

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis.

International audience: Development and homeostasis of the vascular system requires integrin-promoting endothelial cell adhesion, migration and survival. Nowadays, integrins represent potential targets for pharmacological agents and open new avenues for the control of metastatic spread in the treatment of tumor malignancies. We have already reported that PIVL, a serine protease inhibitor isolated from Macrovipera lebetina venom, displays an anti-tumor effect through the interference with integrin receptor function. Here, we report that PIVL inhibits human vascular endothelial cell adhesion and migration onto fibrinogen and fibronectin in a dose-dependent manner without any cytotoxicity. Furthermore, we show that PIVL increases microtubule dynamic instability in HMEC-1 transfected with EGFP-tagged α-tubulin. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrate that PIVL exhibits a strong anti-angiogenic effect both in vitro and in vivo. Interestingly, results herein reveal that the potent anti-angiogenic properties of PIVL are mediated by its RGD-like motif ((41)RGN(43))