Fucans, but not fucomannoglucuronans, determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed

Abstract

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.

Figure 1. Putative structures of the main components of the fractions L.s.-1.0 (A) and L.s.-1.25 (B) obtained from the total sulfated polysaccharide preparation L.s.-P extracted from L. saccharina.

Figure 2. Effect of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on PMN adhesion to platelet-coated surface under flow conditions.

Polysaccharide fractions L.s.-1.0 and L.s.-1.25 or a parent mixture L.s.-P were added at a final concentration of 100 µg/ml to a platelet-coated surface and incubated for 10 min at RT. The same concentration of each compound was also added to non-treated (filled bars) or IB4-pretreated (grey bars) PMN suspensions before addition to platelets. Under the flow conditions, migration of PMN was monitored and photographed. Analysis was performed by counting the number of attached PMN per field in at least 4 different fields. The mean percentage ± SEM with respect to control of at least three independent experiments is represented. *P<0.05; **P<0.01.

Figure 3. Selective inhibitory effects of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on FBS-induced HUVEC tubulogenesis.

(A) 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) Quantitative analysis of tube-like structures in vitro 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 vs control: filled, open and grey bars indicate the effect induced by 100, 10 or 1 µg/ml polysaccharides respectively.

Figure 4. (A-B) Specific effect of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on the inhibition of bFGF-induced HUVEC tubulogenesis.

(A) Representative photographs of HUVEC cultured on Matrigel in the presence of bFGF along with 100 µg/ml of the indicated polysaccharide preparations. (B) Quantitative analysis of tube-like structures. All data were expressed as the percentage of tubes/cm² vs control (bFGF). (C,D) Specific effect of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on the inhibition of Balb/c 3T3 adhesion to bFGF. (C) Effects of L.s.-P and L.s.-1.25 on fibroblast cell adhesion to bFGF. Representative images of fibroblast cell adhesion to purified bFGF are shown. The images are representative of three independent experiments. Quantification was performed by counting adhered cells of at least three different fields. Results are expressed as percentage of the treated sample with respect to control (D). ***P<0.001.

Figure 5. Effect of polysaccharide preparations on tumor growth and angiogenesis in vivo.

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 L.s.-P or its fractions L.s.-1.0 and L.s.-1.25. 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) (A) (**P<0.01). (B) Flow cytometry analysis of the frequency of CD34⁺ endothelial cells on Matrigel plugs embedded with B16 melanoma cells. (**P<0.01) (C) In vitro cell growth of B16 melanoma cells exposed to 100 µg/ml of L.s.-P or its fractions L.s.-1.0 and L.s.-1.25. Data are the mean ± SEM of three independent experiments. (D) B6 mice were injected with 500 µl Matrigel containing 1×10⁵ B16-F10 cells. L.s.-P or its fractions L.s.-1.0 and L.s.-1.25 were injected i.p. at doses of 50 mg/kg every 3 days and compared to control (PBS). Tumors were removed on day 21 post-implantation, photographed (D) and analyzed for CD31⁺ associated blood vessels (E), microvessel density (F) and weight (G). (*P<0.05; **P<0.01).

Figure 6. Selective effects of L.s.-P and its fractions L.s.-1.0 and L.s.-1.25 on breast cancer cell adhesion to human platelets.

(A) MDA-MB-231 breast cancer cells were pre-incubated with selected polysaccharide preparations prior to exposure to human platelet-coated plates. The images are representative of three independent experiments. (B) Quantitative analysis of cell adhesion was performed by counting the number of tumor cells adhered to at least three different fields. Results are expressed as mean percentage ± SEM of the treated samples versus control. *P<0.05; **P<0.01.