Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed

Chlorin e6 embedded in phospholipid nanoparticles equipped with specific peptides: Interaction with tumor cells with different aminopeptidase N expression

A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system, either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mechanisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles

Chlorin e6 Phospholipid Delivery System Featuring APN/CD13 Targeting Peptides: Cell Death Pathways, Cell Localization, In Vivo Biodistribution

We have previously designed a phospholipid delivery system for chlorin e6 to increase the efficacy of photodynamic therapy involving a second-generation photosensitizer. Further research into the matter led to double modification of the obtained nanoparticles with ligands exhibiting targeting and cell-penetrating effects: an NGR-containing peptide and heptaarginine (R7), respectively. This study investigated the cell death pathway on HT-1080 tumor cells after treatment with the proposed compositions: the chlorin e6 phospholipid composition and the two-peptide chlorin e6 phospholipid composition. It was demonstrated that most of the cells died by apoptosis. Colocalization analysis of chlorin e6 in the phospholipid composition with two peptides showed mitochondria are one of the targets of the photosensitizer. An HT-1080 tumor-bearing mouse model was used to evaluate the biodistribution of the drug in tumor, liver, and kidney tissues after administration of the study compositions in comparison with free chlorin e6. The photosensitizer mostly accumulated in the tumor tissue of mice administered the phospholipid compositions, and accumulation was increased 2-fold with the peptide-containing composition and approximately 1.5-fold with the unenhanced composition, as compared with free chlorin e6. The enhancement of the chlorin e6 phospholipid composition with targeting and cell-penetrating peptides was found to be effective both in vitro and in vivo

Chondroitin Sulfate and Fucosylated Chondroitin Sulfate as Stimulators of Hematopoiesis in Cyclophosphamide-Induced Mice

The immunosuppression and inhibition of hematopoiesis are considered to be reasons for the development of complications after intensive chemotherapy and allogeneic hematopoietic stem cell transplantation. Chondroitin sulfate (CS), isolated from the fish Salmo salar, and fucosylated chondroitin sulfate (FCS), isolated from the sea cucumber Apostichopus japonicus, were studied for their roles as stimulators of hematopoiesis in a model of cyclophosphamide-induced immunosuppression in mice. The recombinant protein r G-CSF was applied as a reference. The studied polysaccharides were shown to stimulate the release of white and red blood cells, as well as platelets from bone marrow in immunosuppressed mice, while r G-CSF was only responsible for the significant increase in the level of leucocytes. The analysis of different populations of leucocytes in blood indicated that r G-CSF mainly stimulated the production of neutrophils, whereas in the cases of the studied saccharides, increases in the levels of monocytes, lymphocytes and neutrophils were observed. The normalization of the level of the pro-inflammatory cytokine IL-6 in the serum and the recovery of cell populations in the spleen were observed in immunosuppressed mice following treatment with the polysaccharides. An increase in the proliferative activity of hematopoietic cells CD34(+)CD45(+) was observed following ex vivo polysaccharide exposure. Further study on related oligosaccharides regarding their potential as promising drugs in the complex prophylaxis and therapy of hematopoiesis inhibition after intensive chemotherapy and allogeneic hematopoietic stem cell transplantation seems to be warranted

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.Fil: Cumashi, Albana. Universita degli Studi G. D Annunzio; ItaliaFil: Ushakova, Natalia A.. Academia Rusa de Ciencias Médicas; RusiaFil: Preobrazhenskaya, Marina E.. Academia Rusa de Ciencias Médicas; RusiaFil: D'Incecco, Armida. Universita degli Studi G. D Annunzio; ItaliaFil: Piccoli, Antonio. Consorzio Mario Negri Sud; ItaliaFil: Totani, Licia. Consorzio Mario Negri Sud; ItaliaFil: Tinari, Nicola. Universita degli Studi G. D Annunzio; ItaliaFil: Morozevich, Galina E.. Academia Rusa de Ciencias Médicas; RusiaFil: Berman, Albert E.. Academia Rusa de Ciencias Médicas; RusiaFil: Bilan, María. Academia Rusa de Ciencias; RusiaFil: Usov, Anatolii I.. Academia Rusa de Ciencias; RusiaFil: Ustyuzhanina, Nadezhda E.. Academia Rusa de Ciencias; RusiaFil: Grachev, Alexey A.. Academia Rusa de Ciencias; RusiaFil: Sanderson, Craig J.. Scottish Association for Marine Sciences; Reino UnidoFil: Kelly, Maeve. Scottish Association for Marine Sciences; Reino UnidoFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Iacobelli, Stefano. Universita degli Studi G. D Annunzio; ItaliaFil: Nifantiev, Nikolay E.. Academia Rusa de Ciencias; Rusi

Sulfate content, anti-inflammatory and anti-coagulant activities of the polysaccharide preparations from <i>L. saccharina</i> brown seaweed.

a<p>Six animals received 0.9% (wt/vol) NaCl instead of polysaccharides.</p>b<p>The anti-inflammatory activity was determined as the effect on PMN transmigration to the peritoneal cavity of rats (six animals in each group, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017283#s3" target="_blank"><i>Results</i></a>). The polysaccharide preparations were injected intravenously with a dose of 1.0 and 4.0 mg/kg of the rat weight. Data are presented as mean ± SEM. N is the number of rats in the group.</p>c<p>Anticoagulant activity was measured as the APTT related to a heparin standard with an activity of 140 U/mg. Data are shown as mean ± SEM; N = 4.</p

Effect of polysaccharide preparations on tumor growth and angiogenesis <i>in vivo</i>.

<p>C57BL/6 (B6) mice were injected with 500 µl of Matrigel containing 1×10⁵ B16-F10 cells in PBS or 100 µg of a non-fractionated polysaccharide mixture <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. After 6–7 days, tumors were removed and hemoglobin content was evaluated by using the Drabkin colorimetric method. Results are expressed as the amount of hemoglobin (mg)/Matrigel weight (mg) (<b>A</b>) (**<i>P</i><0.01). (<b>B</b>) Flow cytometry analysis of the frequency of CD34⁺ endothelial cells on Matrigel plugs embedded with B16 melanoma cells. (**<i>P</i><0.01) (<b>C</b>) <i>In vitro</i> cell growth of B16 melanoma cells exposed to 100 µg/ml of <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. Data are the mean ± SEM of three independent experiments. (<b>D</b>) B6 mice were injected with 500 µl Matrigel containing 1×10⁵ B16-F10 cells. <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b> were injected <i>i.p.</i> at doses of 50 mg/kg every 3 days and compared to control (PBS). Tumors were removed on day 21 post-implantation, photographed (<b>D</b>) and analyzed for CD31⁺ associated blood vessels (<b>E</b>), microvessel density (<b>F</b>) and weight (<b>G</b>). (*<i>P</i><0.05; **<i>P</i><0.01).</p

Selective inhibitory effects of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on FBS-induced HUVEC tubulogenesis.

<p>(<b>A</b>) Representative photographs of HUVEC cultured on Matrigel in the presence of FBS along with 100 µg/ml of indicated parent fucoidan or purified fractions. (<b>B</b>) Quantitative analysis of tube-like structures <i>in vitro</i> using three different polysaccharide concentrations. Analysis was obtained by counting closed areas (tubes) in at least four different fields. Data are collected from at least three independent experiments. All data were expressed as the percentage of tubes/cm² of treated cells <i>vs</i> control: filled, open and grey bars indicate the effect induced by 100, 10 or 1 µg/ml polysaccharides respectively.</p