Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis

Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of... Abstract Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell–platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet–tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure–function relationship or even for future treatments. antimetastatic activity, p-selectin, sulfated fucan, sulfated galactan Introduction Sulfated fucans and sulfated galactans are high molecular weight polyanionic molecules. They are mainly isolated from marine invertebrates and algae. They consist of repeating O-sulfated α-l-fucopyranose (Fucp) or α-l-, α-d-, β-d-galactopyranose (Galp) units, respectively (Pomin 2012), with well-defined sulfation patterns, especially those isolated from sea urchins (Pomin 2015). This structural feature is rare among other well-described sulfated polysaccharides, which permits diverse structure–function studies. In fact, many applications in medicine and biomedical research have been reported for these compounds, mainly in coagulation and fertilization (Pereira et al. 2002; Vilela-silva et al. 2008). Recently, studies on similar classes of polysaccharides from marine sources have also revealed effects on hemostasis, immune modulation and tumor biology (Fitton et al. 2015; Mourão 2015; Fernando et al. 2016), suggesting more extensive medical applications for these molecules. Tumor progression is an integrative process, involving not only cancer cells but also the tumor microenvironment and stromal cells. The capacity of tumor cells to overcome immunological barrier and reach other sites is essential for metastasis (Zeeshan and Mutahir 2017). Hematogenous metastasis is one of the most important pathways for cancer progression and its efficiency is associated with more than 90% of cancer-related deaths (Valastyan and Weinberg 2011). However, shear forces and the presence of immune cells in the bloodstream provide an adverse environment that results in attenuated metastasis. Tumor cells can activate platelets and coagulation, leading to the formation of a tumor cell–platelets–fibrin clot that protects the tumor cells from physical stress and immune surveillance (Borsig et al. 2001). These tumor microemboli is dependent on the platelets’ P-selectin expression, and the absence of tumor-derived glycans or the inhibition of P-selectin has been shown to attenuate metastasis (Kim et al. 1998; Ludwig et al. 2007). Selectins are a family of transmembrane glycoproteins involved in carbohydrate-mediated cell adhesion and are expressed by many cell types. P-selectin is mainly stored within the Weibel-Palade bodies of endothelial cells and in platelet α-granules, thus allowing a rapid presentation upon activation (Kansas 1996). All members of the selectin family recognize the core tetrasaccharide sialyl-Lewisx (sLex) and its isomer sLea, whereas the protein and the carbohydrate backbone to which this ligand is conjugated dictates the specific affinity to the selectins (Kansas 1996). Abnormal glycosylation of cancer cells is considered to be one of the most significant changes in the enhancement of the efficiency of tumor progression. The sialylation of membrane-bound mucins, which raises the affinity to P-selectin, is a striking example (Kim and Varki 1997). Platelets directly interact with tumor cells via P-selectin during hematogenous dissemination, which increases the potential for the tumor cells to reach a distant site (Leblanc and Peyruchaud 2016). Furthermore, since platelets can interact with the endothelium (Ruggeri and Mendolicchio 2007), platelet–tumor cell emboli formation is also important to ensure the effective arrest in capillaries at metastatic sites and to facilitate the extravasation of the tumor cells (Schumacher et al. 2013). Heparin is a glycosaminoglycan (GAG) known to inhibit P-selectin and, consequently, metastasis. However, due to its high anticoagulant activity, it can potentially cause hemorrhage, which greatly limits its use as an antimetastatic drug (Borsig 2010). Therefore, the search for other sulfated polysaccharides with high anti P-selectin activity and an inhibitory effect on metastasis is an interesting area of investigation. Marine invertebrates are a rich source of heparin-like molecules and sulfated polysaccharides have high therapeutic potential (Pavão 2014). Although several studies have been carried out on the pharmacological activity of heparin analogs from marine invertebrates, less is known about the anti P-selectin activity of sulfated fucans and sulfated galactans from sea urchins. Most sulfated fucans and sulfated galactans isolated from the egg jelly of sea urchins have large, simple linear structures, but they can vary in the pattern of sulfation and the position of glycosidic linkage. As shown in Figure 1, the very similar sulfated polysaccharides isolated from Echinometra lucunter, Strongylocentrotus droebachiensis and Strongylocentrotus franciscanus are composed of 2-O-sulfated monosaccharide units that vary both in their glycosidic linkages and their constituent monosaccharides. Whereas E. lucunter contains exclusively 3-linked α-L-Galp (Alves et al. 1997), S. droebachiensis and S. franciscanus contain exclusively 4-linked and 3-linked α-L-Fucp molecules, respectively (Vilela-Silva et al. 1999, 2002). The average molecular mass of the S. droebachiensis fucan is 80 kDa, while the other two are 100 kDa (Pereira et al. 2002). Despite their similarities, these molecules can ensure species-specificity in sea urchin fertilization, and they also have different anticoagulant effects (Pomin and Mourão 2008). This information is summarized in Table I. Fig. 1. View largeDownload slide Structures of the 2-O-sulfated α-l-fucans and α-l-galactan from different species of sea urchins. The three fully characterized structures of the sulfated polysaccharides isolated from the egg jelly coats of sea urchins. They show the same sulfation pattern but differ with respect to the glycosidic linkages and the constituent monosaccharides. Fig. 1. View largeDownload slide Structures of the 2-O-sulfated α-l-fucans and α-l-galactan from different species of sea urchins. The three fully characterized structures of the sulfated polysaccharides isolated from the egg jelly coats of sea urchins. They show the same sulfation pattern but differ with respect to the glycosidic linkages and the constituent monosaccharides. Table I. Structure, average molecular mass and effects on the coagulation of the 2-O-sulfated polysaccharides from the sea urchin species Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 aDetermined by polyacrylamide gel electrophoresis. bThe activity is expressed as international units/mg using a parallel standard curve based on the International Heparin Standard (193 U/mg). a,bData from Pereira et al. (2002). View Large Table I. Structure, average molecular mass and effects on the coagulation of the 2-O-sulfated polysaccharides from the sea urchin species Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 aDetermined by polyacrylamide gel electrophoresis. bThe activity is expressed as international units/mg using a parallel standard curve based on the International Heparin Standard (193 U/mg). a,bData from Pereira et al. (2002). View Large This work aimed to investigate whether specific features of the chemical structure of sulfated fucans and galactans from sea urchins could affect their ability to prevent selectin-mediated metastasis. For this purpose, the very similar sulfated fucans from S. droebachiensis and S. franciscanus and the galactan from E. lucunter were tested in mouse models of P-selectin-dependent tumor progression and inflammation. Results Sulfated polysaccharides inhibit tumor cell binding to P-selectin Recent reports have shown the ability of sulfated polysaccharides to inhibit P-selectin binding to its ligands (Kozlowski et al. 2011; Gomes et al. 2015). This is crucial for the antimetastatic activity of these compounds, which not only prevents colonization of distant sites but also extends the patient’s lifespan in many types of cancer (Borsig, Stevenson, et al. 2007). Due to the similar structures of the three sea urchin sulfated polysaccharides, we assessed their potential to inhibit the adhesion of a human colon adenocarcinoma cell line (LS180) to immobilized P-selectin in vitro. Figure 2 shows the representative curves of P-selectin inhibition with increasing concentrations of the sea urchin sulfated polysaccharides and unfractioned heparin (UFH), which was used as a positive control. All glycans inhibited the binding of tumor cells to the immobilized P-selectin in a dose-dependent manner. In particular, the inhibitory activities of the fucan from S. droebachiensis and the galactan from E. lucunter were more potent (IC50 = 11 and 9,6 μg/mL, respectively) than that for UFH (IC50 = 24,5 μg/mL). On the other hand, the fucan from S. franciscanus only inhibited the binding at significantly higher concentrations (IC50 = 170 μg/mL). Fig. 2. View largeDownload slide In vitro tumor cell binding to immobilized P-selectin is inhibited by sea urchin sulfated polysaccharides. The LS180 cell adhesion to immobilized P-selectin chimeras was measured in the presence of increasing concentrations of unfractionated heparin; E. lucunter 1→3 linked galactan; S. franciscanus 1→3 linked fucan; and S. droebachiensis 1→4 linked fucan. Each curve represents 3 independent experiments. The IC50 values, expressed in μg/mL, were 24,5 for UFH; 9,6 for the E. lucunter galactan; 170 for the S. franciscanus fucan and 11 for the S. droebachiensis fucan. Fig. 2. View largeDownload slide In vitro tumor cell binding to immobilized P-selectin is inhibited by sea urchin sulfated polysaccharides. The LS180 cell adhesion to immobilized P-selectin chimeras was measured in the presence of increasing concentrations of unfractionated heparin; E. lucunter 1→3 linked galactan; S. franciscanus 1→3 linked fucan; and S. droebachiensis 1→4 linked fucan. Each curve represents 3 independent experiments. The IC50 values, expressed in μg/mL, were 24,5 for UFH; 9,6 for the E. lucunter galactan; 170 for the S. franciscanus fucan and 11 for the S. droebachiensis fucan. Sea urchin sulfated polysaccharides prevent tumor cell association with platelets in vivo P-selectin-mediated binding of platelets to the surface of the tumor cells is one of the most important steps for successful hematogenous metastasis. Without the formation of tumor cell–platelet complex, the tumor cells become exposed to physical stress and are susceptible to the immune cells in the bloodstream, which results in reduced metastasis. To determine whether sea urchin compounds can prevent the formation of the tumor cell–platelet complex in vivo, calcein-labeled mouse Lewis lung adenocarcinoma cells (LLC) were injected into C57BL/6 mice via the tail vein after injection of the polysaccharides. The interaction of the tumor cells with platelets was quantified via anti-CD41 immunofluorescence in lung sections excised 30 min after this procedure. As shown in Figure 3, in animals injected with the E. lucunter galactan, 24% of the tumor cells in the pulmonary capillaries were associated with platelets, whereas in those injected with S. droebachiensis fucan, the association was 27%. On the other hand, in the animals that had been injected with phosphate-buffered saline (PBS), 69% of the tumor cells were present in the platelet complexes. These numbers are consistent with the results obtained in vitro. As expected from the previous result, the S. franciscanus fucan did not significantly reduce this association. Fig. 3. View largeDownload slide Inhibition of LLC cells binding to platelets in vivo by the sulfated polysaccharides. (A) Representative images of platelet (red)-tumor cell (green) association in lung sections (DNA in blue) 30 min after injection of the tumor cells into mice that had been pre-treated with the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. (B) 3 × 105 LLC cells were injected into C57BL/6 mice after previous injection of 100 μg per animal of the polysaccharides or injection of PBS as a control group. ***P < 0.001. **P < 0.01. Fig. 3. View largeDownload slide Inhibition of LLC cells binding to platelets in vivo by the sulfated polysaccharides. (A) Representative images of platelet (red)-tumor cell (green) association in lung sections (DNA in blue) 30 min after injection of the tumor cells into mice that had been pre-treated with the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. (B) 3 × 105 LLC cells were injected into C57BL/6 mice after previous injection of 100 μg per animal of the polysaccharides or injection of PBS as a control group. ***P < 0.001. **P < 0.01. Fucan from S. franciscanus does not present antimetastatic activity Since these polysaccharides demonstrated potential to inhibit P-selectin and platelet–tumor cell aggregation and given the major importance of these two factors in metastasis, we decided to determine whether the sulfated polysaccharides had any effect on tumor cell metastatic potential. To investigate this, C57BL/6 mice were injected with the polysaccharides, followed by injection of LLC cells. After 21 days, the lungs were harvested and the macrometastasis quantified. Figure 4 shows that treatment with the E. lucunter galactan or the S. droebachiensis fucan completely prevented metastasis in our model. As expected, the S. franciscanus fucan had no significant antimetastatic activity, despite slightly decreasing the formation of metastasis, with an average of 3,5 metastatic foci per mouse vs. 5.6 in the control group. Fig. 4. View largeDownload slide Sea urchin sulfated polysaccharides attenuate experimental metastasis of LLC adenocarcinoma cells. A total of 106 LLC cells were injected into C57BL/6 mice that had been pre-treated with 100 μg per mouse of the following compounds: PBS (Control); the S. franciscanus fucan; the S. droebachiensis fucan; or the E. lucunter galactan. After 21 days, the mice were euthanized and perfused with PBS, and the macrometastatic foci were quantified. ***P < 0.001. Fig. 4. View largeDownload slide Sea urchin sulfated polysaccharides attenuate experimental metastasis of LLC adenocarcinoma cells. A total of 106 LLC cells were injected into C57BL/6 mice that had been pre-treated with 100 μg per mouse of the following compounds: PBS (Control); the S. franciscanus fucan; the S. droebachiensis fucan; or the E. lucunter galactan. After 21 days, the mice were euthanized and perfused with PBS, and the macrometastatic foci were quantified. ***P < 0.001. Sea urchin polysaccharides prevented P-selectin-dependent inflammatory cell recruitment Activated endothelial cells express surface P-selectin, which binds to leukocytes and is essential for cellular recruitment to the inflamed sites (Mayadas et al. 1993). Since our compounds demonstrated a P-selectin dependent antimetastatic activity, we decided to evaluate whether they have effects on P-selectin dependent mediated leukocyte recruitment in an inflammatory model, using a thioglycolate-induced acute peritonitis model in mice. After 3 h of inflammatory stimulus, differential counting of leukocytes was performed in the peritoneal wash of mice and the polymorphonuclear leukocytes (PMN) were quantified. Additionally, the total cell recruitment to the peritoneal cavity was also counted. Figure 5 shows that, in accordance with the previous results (Figures 2–4), the S. franciscanus fucan was not able to significantly prevent PMN recruitment or reduce total cell migration to the peritoneum. Fig. 5. View largeDownload slide Cell recruitment in a thioglycolate-induced peritonitis model is inhibited by sea urchin polysaccharides. C57BL/6 mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. After 3 h, we performed a peritoneal wash. The percentages of polymorphonuclear leukocytes (PMN) were obtained based on cell morphology and total cell count was measured with a hemocytometer. (A) The cells in the peritoneal wash were stained with Wright’s Stain and differentially counted for mononuclear and polymorphonuclear cells. (B) Percentage of PMN cells per group. (C) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Fig. 5. View largeDownload slide Cell recruitment in a thioglycolate-induced peritonitis model is inhibited by sea urchin polysaccharides. C57BL/6 mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. After 3 h, we performed a peritoneal wash. The percentages of polymorphonuclear leukocytes (PMN) were obtained based on cell morphology and total cell count was measured with a hemocytometer. (A) The cells in the peritoneal wash were stained with Wright’s Stain and differentially counted for mononuclear and polymorphonuclear cells. (B) Percentage of PMN cells per group. (C) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. On the other hand, E. lucunter’s galactan and S.droebachiensis’ fucan both had a powerful effect in reducing PMN recruitment, despite the lack of significant difference between total cell recruitment after treatment with this fucan, comparing to the positive control. Interestingly, Figure 6 shows that P-sel−/− mice treated with E. lucunter’s galactan and S.droebachiensis’ fucan had also fewer PMN recruitment in comparison to both wild-type or untreated P-sel−/− mice, suggesting a possible inhibition of another type of lectin in this model, such as L-selectin present on leukocytes. Despite of this result, the total cell count in the peritoneal lavage was not altered after treatment of P-sel−/− mice. Taken together these data strongly suggest that these polysaccharides block selectin interaction and thereby inhibit metastasis and inflammation. Fig. 6. View largeDownload slide Polymorphonuclear leukocyte recruitment inhibited by sea urchin polysaccharides in a thioglycolate-induced peritonitis model in Psel−/− mice. C57BL/6 P-selectin knockout mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. Results were obtained as described in the materials and methods section. (A) Percentage of PMN cells per group. (B) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Fig. 6. View largeDownload slide Polymorphonuclear leukocyte recruitment inhibited by sea urchin polysaccharides in a thioglycolate-induced peritonitis model in Psel−/− mice. C57BL/6 P-selectin knockout mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. Results were obtained as described in the materials and methods section. (A) Percentage of PMN cells per group. (B) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Discussion Sulfated polysaccharides include a diverse set of naturally occurring molecules that have been implicated in numerous therapeutic effects. However, the interest in sulfated fucans and sulfated galactans and their applications in medicine has increased strongly only in the past few years. The development of drugs based on naturally occurring carbohydrates is the primary goal of these projects, mainly because of their wide-ranging benefits to human health and their relatively simple chemical structure. In marine invertebrates, sulfated fucans and galactans are components of the extracellular matrix (ECM). In contrast to those extracted from ascidians and sea cucumbers, which are usually components of the tunic and body wall, respectively (Pavão et al. 1989; Mulloy et al. 1994), the sulfated polysaccharides from sea urchins form a complex ECM in the jelly layer surrounding the egg, in which they interact with many proteins (Vacquier and Moy 1997). Thus, it is not a coincidence that the specific patterns of sulfation, the glycosidic linkage, the branching and the type of sugar have central roles in triggering the acrosome reaction during fertilization (Vilela-silva et al. 2008; Pomin 2015). Similarly, when tested for pharmacological activities, they usually have effects that differ according to their structures. The analysis of the biological activity of the three polysaccharides studied here has raised interesting questions. For instance, the sulfated fucan from S. franciscanus and the sulfated galactan from E. lucunter have the same sulfation pattern (2-O-sulfated), glycosidic linkage (1→3), and α anomeric configuration. However, the single difference in the sugar type results in a significant difference in their anticoagulant properties. The fucan from S. franciscanus is approximately 10 times less effective as an anticoagulant than the E. lucunter galactan, as indicated by activated partial thromboplastin time assay (aPTT) values (Table I). Moreover, the effect of the fucan is exclusively based on its potentiation of the antithrombin-mediated factor Xa inhibition. This shows that this single difference is sufficient to dictate how the polysaccharide interacts with many different proteins (Pereira et al. 2002). In further work using molecular dynamics and docking experiments, these two molecules seemed to have similar conformations in solution but different binding orientations in the anticoagulant ternary complexes composed by the oligosaccharide, thrombin and antithrombin (Becker et al. 2007). Another example is the acrosome reaction. Although the sulfated fucans from S. franciscanus and S. droebachiensis are structurally almost identical, differing only in the glycosidic linkage (1→3 and 1→4, respectively), they are not able to induce interspecies acrosome reactions (Hirohashi et al. 2002). This suggests a requirement for a specific glycosidic linkage on the 2-O-sulfated fucan. On the other hand, cross-acrosome reaction between two other fucans from the sea urchins Strongylocentrotus purpuratus and Strongylocentrotus pallidus has been observed. In this case, the two species possess α(1→3) linked sulfated fucans with different sulfation patterns, but this feature alone could not impair the induction of acrosome reaction between species (Vilela-silva et al. 2008). This cross-acrosome reaction is also observed between the fucan from S. franciscanus and the galactan from E. lucunter, which shows that the single difference in the sixth carbon between the two molecules is not important for proper recognition by the sperm receptors (Hirohashi et al. 2002). The ability of different types of sulfated polysaccharides, to inhibit P- and L-selectin is well reported and assumed to be directly related to the anti-inflammatory and antimetastatic activity of this type of molecules (Borsig et al. 2001; Borsig, Stevenson, et al. 2007, 2007b; Borsig 2010; Wang et al., 2002; Fritzsche et al. 2006; Alban et al. 2009). For example, the anti-inflammatory activity of unfractionated heparin requires glucosamine 6-sulfation and is mediated by blockage of P- and L-selectin (Wang et al., 2002); fucosylated chondroitin sulfate from sea cucumber also inhibits P- and L-selectin binding, resulting in attenuation of experimental metastasis and neutrophyls recruitment (Borsig, Wang, et al. 2007); β-1,3-Glucan sulfate diminishes contact hypersensitivity responses through Inhibition of L- and P-selectin functions (Alban et al. 2009); semisynthetic sulfated glucans, derived from the linear glucans phycarin, curdlan or pullulan, decreases leukocyte rolling on P-selectin layers (Fritzsche et al. 2006). Therefore, in the present work we assumed that the molecular mechanism involved in the anti-inflammatory and antimetastatic activity of heparin is similar to that of other nonheparin sulfated polysaccharides, being mediated by P- and L-selectin blockage. Whereas the fucan from S. droebachiensis and the galactan from E. lucunter could prevent the formation of experimental metastasis and inflammation-mediated leukocyte recruitment, the fucan from S. franciscanus lacks any activity. In this case, a complex set of features was necessary to ensure antiselectin activity. The only difference between active galactan from E. lucunter and the inactive fucan from S. franciscanus is the missing hydroxyl group on carbon 6 in the fucan. However, the different linkage of the sulfated fucan 1→4 found in S. droebachiensis restores the antimetastatic activity. One possible explanation for this difference is that the 3D conformation alters the ligand presentation between these polysaccharides. The fucan from S. franciscanus and the galactan from E. lucunter are known to have similar 3D conformations (Becker et al. 2007), but a recent NMR study showed that they have slightly differential dynamics. Specifically, the α-L-fucan is a more flexible molecule in solution, and this can relate to their differential binding properties (Queiroz et al. 2016). Regarding the antimetastatic activity, we can infer that the S. droebachiensis fucan and E. lucunter galactan prevented metastasis most efficiently in our model. The E. lucunter galactan, however, has a higher anticoagulant activity, which could be associated with undesirable side effects during treatment. Additionally, the galactan’s higher efficacy in preventing cell recruitment (Figure 5) could be explained by the interaction with other molecules such as L-selectin, integrins or chemokines (Marki et al. 2015). Finally, in this work, we presented a new application for fucans and galactans on the basis of their demonstrated potential to inhibit P-selectin and prevent metastasis and cell recruitment during inflammation. We also showed that the structure–function relationship between them is complex, and future studies within this context might provide new insights for the glycobiology field, as specific features of these sulfated polysaccharides directly regulated several pharmacological effects. Materials and methods Cell lines and reagents Human colon carcinoma cells (LS180; ATCC, Manassas, VA) were grown in minimum essential medium-α (MEM-α) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). Lewis LLC (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (Vitrocell) supplemented with 10% FBS. All reagents were from Sigma (St Louis, MO), unless otherwise stated. UFH (Liquemine) was obtained from Roche Pharma (Reinach, Switzerland). Isolation of the sulfated fucans and galactan from sea urchins Echinometra lucunter specimens were collected at Guanabara Bay, Rio de Janeiro, Brazil, and the egg jelly was obtained as described (Vacquier and Moy 1997). The egg jelly from S. droebachiensis and S. franciscanus were kindly provided by professor Christiane Biermann (Portland State University, USA) and professor Victor Vacquier (University of California San Diego, USA) respectively. Polysaccharide purification was performed by anion exchange chromatography as previously described (Vilela-Silva et al. 1999, 2002). P-selectin inhibition assay in vitro Calcein-AM-labeled LS180 cells and increasing concentrations of the sulfated polysaccharides were added in triplicate to a 96-well plate with immobilized P-selectin chimeras, as described previously (Hostettler et al. 2007). Animal models An animal use protocol was obtained (05/2016), and procedures were followed in strict accordance with guidelines established by the Comissão de Ética no Uso de Animais/Instituto de Pesquisas Biomédicas/Hospital Naval Marcílio Dias (CEUA-IPB). Platelet–tumor cell association in vivo Lung sections were obtained from the mice 30 min after the intravenous injection of the tumor cells as described previously (Borsig et al. 2001), with slight modifications. Briefly, LLC cells were labeled with calcein-AM and intravenously injected into C57BL/6 mice with or without previous intravenous injection of 100 μg of the sulfated polysaccharides. After 30 min, the lungs were harvested for analysis. The lung sections were immunofluorescently stained with goat anti-integrin αIIb (CD41) (Santa Cruz Biotechnology) followed by anti-goat Cy3-conjugated antibody and analyzed by immunofluorescence microscopy. The platelet–tumor cell association was quantified by evaluating the calcein-AM labeled cells present in 10 fields of each lung section (10 sections per animal, 3 animals per experimental group). Experimental metastasis model C57BL/6 mice were intravenously injected with 106 LLC cells via the tail vein. Mice were injected either with PBS (vehicle) or 100 μg of the sulfated polysaccharides 15 min prior to tumor cell injection and killed after 21 days. PBS perfused lungs were macroscopically evaluated for the presence of metastatic foci. Thioglycolate-induced acute peritonitis C57BL/6 mice were intravenously injected with 100 μg of the sulfated polysaccharides. After 15 min, the animals were injected intraperitoneally with 1 mL of 4% thioglycolate. The mice were sacrificed after 3 h, and peritoneal lavage was collected using 8 mL ice-cold PBS. The total number of cells in peritoneal lavage was counted with a hemocytometer. This lavage was analyzed after cytospin centrifugation and staining with Wright’s stain. Differential counting was performed to evaluate the percentage of polymorphonuclear cells present in the peritoneal cavity. P-sel−/− and wild-type mice were used in this experiment. Funding Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Ary Frauzino para Pesquisa e Controle do Câncer. Conflict of interest statement None declared. Acknowledgements The authors would like to thank Professor Eliene Kozlowski (in memoriam), who participated from the beginning of the project design to the data analysis of all experiments in this article. The authors would also like to thank professor Mariana Stelling for substantial revisions of the article and the students Mariana Soares and Tamires Gerhardt for technical support. Abbreviations aPTT activated partial thromboplastin time assay ECM extracellular matrix FBS fetal bovine serum Fucp fucopyranose GAG glycosaminoglycan Galp galactopyranose LLC Lewis lung carcinoma PBS phosphate-buffered saline PMN polymorphonuclear leukocytes sLea sialyl-lewisa sLex sialyl-lewisx UFH unfractionated heparin References Alban S , Ludwig RJ , Bendas G , Scho¨n MP , Oostingh GJ , Radeke HH , Fritzsche J , Pfeilschifter J , Kaufmann R , Boehncke W . 2009 . PS3, A semisynthetic b-1,3-glucan sulfate, diminishes contact hypersensitivity responses through inhibition of L- and P-selectin functions . J Invest Dermatol . 129 : 1192 – 1202 . Google Scholar CrossRef Search ADS PubMed Alves AP , Mulloy B , Diniz JA , Mourão PAS . 1997 . Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins . J Biol Chem . 272 : 6965 – 6971 . 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For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis

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Abstract

Abstract Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell–platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet–tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure–function relationship or even for future treatments. antimetastatic activity, p-selectin, sulfated fucan, sulfated galactan Introduction Sulfated fucans and sulfated galactans are high molecular weight polyanionic molecules. They are mainly isolated from marine invertebrates and algae. They consist of repeating O-sulfated α-l-fucopyranose (Fucp) or α-l-, α-d-, β-d-galactopyranose (Galp) units, respectively (Pomin 2012), with well-defined sulfation patterns, especially those isolated from sea urchins (Pomin 2015). This structural feature is rare among other well-described sulfated polysaccharides, which permits diverse structure–function studies. In fact, many applications in medicine and biomedical research have been reported for these compounds, mainly in coagulation and fertilization (Pereira et al. 2002; Vilela-silva et al. 2008). Recently, studies on similar classes of polysaccharides from marine sources have also revealed effects on hemostasis, immune modulation and tumor biology (Fitton et al. 2015; Mourão 2015; Fernando et al. 2016), suggesting more extensive medical applications for these molecules. Tumor progression is an integrative process, involving not only cancer cells but also the tumor microenvironment and stromal cells. The capacity of tumor cells to overcome immunological barrier and reach other sites is essential for metastasis (Zeeshan and Mutahir 2017). Hematogenous metastasis is one of the most important pathways for cancer progression and its efficiency is associated with more than 90% of cancer-related deaths (Valastyan and Weinberg 2011). However, shear forces and the presence of immune cells in the bloodstream provide an adverse environment that results in attenuated metastasis. Tumor cells can activate platelets and coagulation, leading to the formation of a tumor cell–platelets–fibrin clot that protects the tumor cells from physical stress and immune surveillance (Borsig et al. 2001). These tumor microemboli is dependent on the platelets’ P-selectin expression, and the absence of tumor-derived glycans or the inhibition of P-selectin has been shown to attenuate metastasis (Kim et al. 1998; Ludwig et al. 2007). Selectins are a family of transmembrane glycoproteins involved in carbohydrate-mediated cell adhesion and are expressed by many cell types. P-selectin is mainly stored within the Weibel-Palade bodies of endothelial cells and in platelet α-granules, thus allowing a rapid presentation upon activation (Kansas 1996). All members of the selectin family recognize the core tetrasaccharide sialyl-Lewisx (sLex) and its isomer sLea, whereas the protein and the carbohydrate backbone to which this ligand is conjugated dictates the specific affinity to the selectins (Kansas 1996). Abnormal glycosylation of cancer cells is considered to be one of the most significant changes in the enhancement of the efficiency of tumor progression. The sialylation of membrane-bound mucins, which raises the affinity to P-selectin, is a striking example (Kim and Varki 1997). Platelets directly interact with tumor cells via P-selectin during hematogenous dissemination, which increases the potential for the tumor cells to reach a distant site (Leblanc and Peyruchaud 2016). Furthermore, since platelets can interact with the endothelium (Ruggeri and Mendolicchio 2007), platelet–tumor cell emboli formation is also important to ensure the effective arrest in capillaries at metastatic sites and to facilitate the extravasation of the tumor cells (Schumacher et al. 2013). Heparin is a glycosaminoglycan (GAG) known to inhibit P-selectin and, consequently, metastasis. However, due to its high anticoagulant activity, it can potentially cause hemorrhage, which greatly limits its use as an antimetastatic drug (Borsig 2010). Therefore, the search for other sulfated polysaccharides with high anti P-selectin activity and an inhibitory effect on metastasis is an interesting area of investigation. Marine invertebrates are a rich source of heparin-like molecules and sulfated polysaccharides have high therapeutic potential (Pavão 2014). Although several studies have been carried out on the pharmacological activity of heparin analogs from marine invertebrates, less is known about the anti P-selectin activity of sulfated fucans and sulfated galactans from sea urchins. Most sulfated fucans and sulfated galactans isolated from the egg jelly of sea urchins have large, simple linear structures, but they can vary in the pattern of sulfation and the position of glycosidic linkage. As shown in Figure 1, the very similar sulfated polysaccharides isolated from Echinometra lucunter, Strongylocentrotus droebachiensis and Strongylocentrotus franciscanus are composed of 2-O-sulfated monosaccharide units that vary both in their glycosidic linkages and their constituent monosaccharides. Whereas E. lucunter contains exclusively 3-linked α-L-Galp (Alves et al. 1997), S. droebachiensis and S. franciscanus contain exclusively 4-linked and 3-linked α-L-Fucp molecules, respectively (Vilela-Silva et al. 1999, 2002). The average molecular mass of the S. droebachiensis fucan is 80 kDa, while the other two are 100 kDa (Pereira et al. 2002). Despite their similarities, these molecules can ensure species-specificity in sea urchin fertilization, and they also have different anticoagulant effects (Pomin and Mourão 2008). This information is summarized in Table I. Fig. 1. View largeDownload slide Structures of the 2-O-sulfated α-l-fucans and α-l-galactan from different species of sea urchins. The three fully characterized structures of the sulfated polysaccharides isolated from the egg jelly coats of sea urchins. They show the same sulfation pattern but differ with respect to the glycosidic linkages and the constituent monosaccharides. Fig. 1. View largeDownload slide Structures of the 2-O-sulfated α-l-fucans and α-l-galactan from different species of sea urchins. The three fully characterized structures of the sulfated polysaccharides isolated from the egg jelly coats of sea urchins. They show the same sulfation pattern but differ with respect to the glycosidic linkages and the constituent monosaccharides. Table I. Structure, average molecular mass and effects on the coagulation of the 2-O-sulfated polysaccharides from the sea urchin species Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 aDetermined by polyacrylamide gel electrophoresis. bThe activity is expressed as international units/mg using a parallel standard curve based on the International Heparin Standard (193 U/mg). a,bData from Pereira et al. (2002). View Large Table I. Structure, average molecular mass and effects on the coagulation of the 2-O-sulfated polysaccharides from the sea urchin species Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 Species Structure Average molecular massa (kDa) aPTTb (IU/mg) Echinometra lucunter [→3)-α-L-Galp-2(OSO3−)-(1→]n 100 20 Strongylocentrotus droebachiensis [→4)-α-L-Fucp-2(OSO3−)-(1→]n 80 <1 Strongylocentrotus franciscanus [→3)-α-L-Fucp-2(OSO3−)-(1→]n 100 ~2 aDetermined by polyacrylamide gel electrophoresis. bThe activity is expressed as international units/mg using a parallel standard curve based on the International Heparin Standard (193 U/mg). a,bData from Pereira et al. (2002). View Large This work aimed to investigate whether specific features of the chemical structure of sulfated fucans and galactans from sea urchins could affect their ability to prevent selectin-mediated metastasis. For this purpose, the very similar sulfated fucans from S. droebachiensis and S. franciscanus and the galactan from E. lucunter were tested in mouse models of P-selectin-dependent tumor progression and inflammation. Results Sulfated polysaccharides inhibit tumor cell binding to P-selectin Recent reports have shown the ability of sulfated polysaccharides to inhibit P-selectin binding to its ligands (Kozlowski et al. 2011; Gomes et al. 2015). This is crucial for the antimetastatic activity of these compounds, which not only prevents colonization of distant sites but also extends the patient’s lifespan in many types of cancer (Borsig, Stevenson, et al. 2007). Due to the similar structures of the three sea urchin sulfated polysaccharides, we assessed their potential to inhibit the adhesion of a human colon adenocarcinoma cell line (LS180) to immobilized P-selectin in vitro. Figure 2 shows the representative curves of P-selectin inhibition with increasing concentrations of the sea urchin sulfated polysaccharides and unfractioned heparin (UFH), which was used as a positive control. All glycans inhibited the binding of tumor cells to the immobilized P-selectin in a dose-dependent manner. In particular, the inhibitory activities of the fucan from S. droebachiensis and the galactan from E. lucunter were more potent (IC50 = 11 and 9,6 μg/mL, respectively) than that for UFH (IC50 = 24,5 μg/mL). On the other hand, the fucan from S. franciscanus only inhibited the binding at significantly higher concentrations (IC50 = 170 μg/mL). Fig. 2. View largeDownload slide In vitro tumor cell binding to immobilized P-selectin is inhibited by sea urchin sulfated polysaccharides. The LS180 cell adhesion to immobilized P-selectin chimeras was measured in the presence of increasing concentrations of unfractionated heparin; E. lucunter 1→3 linked galactan; S. franciscanus 1→3 linked fucan; and S. droebachiensis 1→4 linked fucan. Each curve represents 3 independent experiments. The IC50 values, expressed in μg/mL, were 24,5 for UFH; 9,6 for the E. lucunter galactan; 170 for the S. franciscanus fucan and 11 for the S. droebachiensis fucan. Fig. 2. View largeDownload slide In vitro tumor cell binding to immobilized P-selectin is inhibited by sea urchin sulfated polysaccharides. The LS180 cell adhesion to immobilized P-selectin chimeras was measured in the presence of increasing concentrations of unfractionated heparin; E. lucunter 1→3 linked galactan; S. franciscanus 1→3 linked fucan; and S. droebachiensis 1→4 linked fucan. Each curve represents 3 independent experiments. The IC50 values, expressed in μg/mL, were 24,5 for UFH; 9,6 for the E. lucunter galactan; 170 for the S. franciscanus fucan and 11 for the S. droebachiensis fucan. Sea urchin sulfated polysaccharides prevent tumor cell association with platelets in vivo P-selectin-mediated binding of platelets to the surface of the tumor cells is one of the most important steps for successful hematogenous metastasis. Without the formation of tumor cell–platelet complex, the tumor cells become exposed to physical stress and are susceptible to the immune cells in the bloodstream, which results in reduced metastasis. To determine whether sea urchin compounds can prevent the formation of the tumor cell–platelet complex in vivo, calcein-labeled mouse Lewis lung adenocarcinoma cells (LLC) were injected into C57BL/6 mice via the tail vein after injection of the polysaccharides. The interaction of the tumor cells with platelets was quantified via anti-CD41 immunofluorescence in lung sections excised 30 min after this procedure. As shown in Figure 3, in animals injected with the E. lucunter galactan, 24% of the tumor cells in the pulmonary capillaries were associated with platelets, whereas in those injected with S. droebachiensis fucan, the association was 27%. On the other hand, in the animals that had been injected with phosphate-buffered saline (PBS), 69% of the tumor cells were present in the platelet complexes. These numbers are consistent with the results obtained in vitro. As expected from the previous result, the S. franciscanus fucan did not significantly reduce this association. Fig. 3. View largeDownload slide Inhibition of LLC cells binding to platelets in vivo by the sulfated polysaccharides. (A) Representative images of platelet (red)-tumor cell (green) association in lung sections (DNA in blue) 30 min after injection of the tumor cells into mice that had been pre-treated with the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. (B) 3 × 105 LLC cells were injected into C57BL/6 mice after previous injection of 100 μg per animal of the polysaccharides or injection of PBS as a control group. ***P < 0.001. **P < 0.01. Fig. 3. View largeDownload slide Inhibition of LLC cells binding to platelets in vivo by the sulfated polysaccharides. (A) Representative images of platelet (red)-tumor cell (green) association in lung sections (DNA in blue) 30 min after injection of the tumor cells into mice that had been pre-treated with the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. (B) 3 × 105 LLC cells were injected into C57BL/6 mice after previous injection of 100 μg per animal of the polysaccharides or injection of PBS as a control group. ***P < 0.001. **P < 0.01. Fucan from S. franciscanus does not present antimetastatic activity Since these polysaccharides demonstrated potential to inhibit P-selectin and platelet–tumor cell aggregation and given the major importance of these two factors in metastasis, we decided to determine whether the sulfated polysaccharides had any effect on tumor cell metastatic potential. To investigate this, C57BL/6 mice were injected with the polysaccharides, followed by injection of LLC cells. After 21 days, the lungs were harvested and the macrometastasis quantified. Figure 4 shows that treatment with the E. lucunter galactan or the S. droebachiensis fucan completely prevented metastasis in our model. As expected, the S. franciscanus fucan had no significant antimetastatic activity, despite slightly decreasing the formation of metastasis, with an average of 3,5 metastatic foci per mouse vs. 5.6 in the control group. Fig. 4. View largeDownload slide Sea urchin sulfated polysaccharides attenuate experimental metastasis of LLC adenocarcinoma cells. A total of 106 LLC cells were injected into C57BL/6 mice that had been pre-treated with 100 μg per mouse of the following compounds: PBS (Control); the S. franciscanus fucan; the S. droebachiensis fucan; or the E. lucunter galactan. After 21 days, the mice were euthanized and perfused with PBS, and the macrometastatic foci were quantified. ***P < 0.001. Fig. 4. View largeDownload slide Sea urchin sulfated polysaccharides attenuate experimental metastasis of LLC adenocarcinoma cells. A total of 106 LLC cells were injected into C57BL/6 mice that had been pre-treated with 100 μg per mouse of the following compounds: PBS (Control); the S. franciscanus fucan; the S. droebachiensis fucan; or the E. lucunter galactan. After 21 days, the mice were euthanized and perfused with PBS, and the macrometastatic foci were quantified. ***P < 0.001. Sea urchin polysaccharides prevented P-selectin-dependent inflammatory cell recruitment Activated endothelial cells express surface P-selectin, which binds to leukocytes and is essential for cellular recruitment to the inflamed sites (Mayadas et al. 1993). Since our compounds demonstrated a P-selectin dependent antimetastatic activity, we decided to evaluate whether they have effects on P-selectin dependent mediated leukocyte recruitment in an inflammatory model, using a thioglycolate-induced acute peritonitis model in mice. After 3 h of inflammatory stimulus, differential counting of leukocytes was performed in the peritoneal wash of mice and the polymorphonuclear leukocytes (PMN) were quantified. Additionally, the total cell recruitment to the peritoneal cavity was also counted. Figure 5 shows that, in accordance with the previous results (Figures 2–4), the S. franciscanus fucan was not able to significantly prevent PMN recruitment or reduce total cell migration to the peritoneum. Fig. 5. View largeDownload slide Cell recruitment in a thioglycolate-induced peritonitis model is inhibited by sea urchin polysaccharides. C57BL/6 mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. After 3 h, we performed a peritoneal wash. The percentages of polymorphonuclear leukocytes (PMN) were obtained based on cell morphology and total cell count was measured with a hemocytometer. (A) The cells in the peritoneal wash were stained with Wright’s Stain and differentially counted for mononuclear and polymorphonuclear cells. (B) Percentage of PMN cells per group. (C) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Fig. 5. View largeDownload slide Cell recruitment in a thioglycolate-induced peritonitis model is inhibited by sea urchin polysaccharides. C57BL/6 mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. After 3 h, we performed a peritoneal wash. The percentages of polymorphonuclear leukocytes (PMN) were obtained based on cell morphology and total cell count was measured with a hemocytometer. (A) The cells in the peritoneal wash were stained with Wright’s Stain and differentially counted for mononuclear and polymorphonuclear cells. (B) Percentage of PMN cells per group. (C) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. On the other hand, E. lucunter’s galactan and S.droebachiensis’ fucan both had a powerful effect in reducing PMN recruitment, despite the lack of significant difference between total cell recruitment after treatment with this fucan, comparing to the positive control. Interestingly, Figure 6 shows that P-sel−/− mice treated with E. lucunter’s galactan and S.droebachiensis’ fucan had also fewer PMN recruitment in comparison to both wild-type or untreated P-sel−/− mice, suggesting a possible inhibition of another type of lectin in this model, such as L-selectin present on leukocytes. Despite of this result, the total cell count in the peritoneal lavage was not altered after treatment of P-sel−/− mice. Taken together these data strongly suggest that these polysaccharides block selectin interaction and thereby inhibit metastasis and inflammation. Fig. 6. View largeDownload slide Polymorphonuclear leukocyte recruitment inhibited by sea urchin polysaccharides in a thioglycolate-induced peritonitis model in Psel−/− mice. C57BL/6 P-selectin knockout mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. Results were obtained as described in the materials and methods section. (A) Percentage of PMN cells per group. (B) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Fig. 6. View largeDownload slide Polymorphonuclear leukocyte recruitment inhibited by sea urchin polysaccharides in a thioglycolate-induced peritonitis model in Psel−/− mice. C57BL/6 P-selectin knockout mice were intraperitoneally injected with 1 mL of thioglycolate (4%) 15 min after intravenous injection of the S. franciscanus fucan, the S. droebachiensis fucan, the E. lucunter galactan or PBS. The negative control group was injected with PBS only. Results were obtained as described in the materials and methods section. (A) Percentage of PMN cells per group. (B) Total cell count in the peritoneal lavage. Statistical significance was determined using ANOVA. ***P < 0.001. Discussion Sulfated polysaccharides include a diverse set of naturally occurring molecules that have been implicated in numerous therapeutic effects. However, the interest in sulfated fucans and sulfated galactans and their applications in medicine has increased strongly only in the past few years. The development of drugs based on naturally occurring carbohydrates is the primary goal of these projects, mainly because of their wide-ranging benefits to human health and their relatively simple chemical structure. In marine invertebrates, sulfated fucans and galactans are components of the extracellular matrix (ECM). In contrast to those extracted from ascidians and sea cucumbers, which are usually components of the tunic and body wall, respectively (Pavão et al. 1989; Mulloy et al. 1994), the sulfated polysaccharides from sea urchins form a complex ECM in the jelly layer surrounding the egg, in which they interact with many proteins (Vacquier and Moy 1997). Thus, it is not a coincidence that the specific patterns of sulfation, the glycosidic linkage, the branching and the type of sugar have central roles in triggering the acrosome reaction during fertilization (Vilela-silva et al. 2008; Pomin 2015). Similarly, when tested for pharmacological activities, they usually have effects that differ according to their structures. The analysis of the biological activity of the three polysaccharides studied here has raised interesting questions. For instance, the sulfated fucan from S. franciscanus and the sulfated galactan from E. lucunter have the same sulfation pattern (2-O-sulfated), glycosidic linkage (1→3), and α anomeric configuration. However, the single difference in the sugar type results in a significant difference in their anticoagulant properties. The fucan from S. franciscanus is approximately 10 times less effective as an anticoagulant than the E. lucunter galactan, as indicated by activated partial thromboplastin time assay (aPTT) values (Table I). Moreover, the effect of the fucan is exclusively based on its potentiation of the antithrombin-mediated factor Xa inhibition. This shows that this single difference is sufficient to dictate how the polysaccharide interacts with many different proteins (Pereira et al. 2002). In further work using molecular dynamics and docking experiments, these two molecules seemed to have similar conformations in solution but different binding orientations in the anticoagulant ternary complexes composed by the oligosaccharide, thrombin and antithrombin (Becker et al. 2007). Another example is the acrosome reaction. Although the sulfated fucans from S. franciscanus and S. droebachiensis are structurally almost identical, differing only in the glycosidic linkage (1→3 and 1→4, respectively), they are not able to induce interspecies acrosome reactions (Hirohashi et al. 2002). This suggests a requirement for a specific glycosidic linkage on the 2-O-sulfated fucan. On the other hand, cross-acrosome reaction between two other fucans from the sea urchins Strongylocentrotus purpuratus and Strongylocentrotus pallidus has been observed. In this case, the two species possess α(1→3) linked sulfated fucans with different sulfation patterns, but this feature alone could not impair the induction of acrosome reaction between species (Vilela-silva et al. 2008). This cross-acrosome reaction is also observed between the fucan from S. franciscanus and the galactan from E. lucunter, which shows that the single difference in the sixth carbon between the two molecules is not important for proper recognition by the sperm receptors (Hirohashi et al. 2002). The ability of different types of sulfated polysaccharides, to inhibit P- and L-selectin is well reported and assumed to be directly related to the anti-inflammatory and antimetastatic activity of this type of molecules (Borsig et al. 2001; Borsig, Stevenson, et al. 2007, 2007b; Borsig 2010; Wang et al., 2002; Fritzsche et al. 2006; Alban et al. 2009). For example, the anti-inflammatory activity of unfractionated heparin requires glucosamine 6-sulfation and is mediated by blockage of P- and L-selectin (Wang et al., 2002); fucosylated chondroitin sulfate from sea cucumber also inhibits P- and L-selectin binding, resulting in attenuation of experimental metastasis and neutrophyls recruitment (Borsig, Wang, et al. 2007); β-1,3-Glucan sulfate diminishes contact hypersensitivity responses through Inhibition of L- and P-selectin functions (Alban et al. 2009); semisynthetic sulfated glucans, derived from the linear glucans phycarin, curdlan or pullulan, decreases leukocyte rolling on P-selectin layers (Fritzsche et al. 2006). Therefore, in the present work we assumed that the molecular mechanism involved in the anti-inflammatory and antimetastatic activity of heparin is similar to that of other nonheparin sulfated polysaccharides, being mediated by P- and L-selectin blockage. Whereas the fucan from S. droebachiensis and the galactan from E. lucunter could prevent the formation of experimental metastasis and inflammation-mediated leukocyte recruitment, the fucan from S. franciscanus lacks any activity. In this case, a complex set of features was necessary to ensure antiselectin activity. The only difference between active galactan from E. lucunter and the inactive fucan from S. franciscanus is the missing hydroxyl group on carbon 6 in the fucan. However, the different linkage of the sulfated fucan 1→4 found in S. droebachiensis restores the antimetastatic activity. One possible explanation for this difference is that the 3D conformation alters the ligand presentation between these polysaccharides. The fucan from S. franciscanus and the galactan from E. lucunter are known to have similar 3D conformations (Becker et al. 2007), but a recent NMR study showed that they have slightly differential dynamics. Specifically, the α-L-fucan is a more flexible molecule in solution, and this can relate to their differential binding properties (Queiroz et al. 2016). Regarding the antimetastatic activity, we can infer that the S. droebachiensis fucan and E. lucunter galactan prevented metastasis most efficiently in our model. The E. lucunter galactan, however, has a higher anticoagulant activity, which could be associated with undesirable side effects during treatment. Additionally, the galactan’s higher efficacy in preventing cell recruitment (Figure 5) could be explained by the interaction with other molecules such as L-selectin, integrins or chemokines (Marki et al. 2015). Finally, in this work, we presented a new application for fucans and galactans on the basis of their demonstrated potential to inhibit P-selectin and prevent metastasis and cell recruitment during inflammation. We also showed that the structure–function relationship between them is complex, and future studies within this context might provide new insights for the glycobiology field, as specific features of these sulfated polysaccharides directly regulated several pharmacological effects. Materials and methods Cell lines and reagents Human colon carcinoma cells (LS180; ATCC, Manassas, VA) were grown in minimum essential medium-α (MEM-α) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). Lewis LLC (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (Vitrocell) supplemented with 10% FBS. All reagents were from Sigma (St Louis, MO), unless otherwise stated. UFH (Liquemine) was obtained from Roche Pharma (Reinach, Switzerland). Isolation of the sulfated fucans and galactan from sea urchins Echinometra lucunter specimens were collected at Guanabara Bay, Rio de Janeiro, Brazil, and the egg jelly was obtained as described (Vacquier and Moy 1997). The egg jelly from S. droebachiensis and S. franciscanus were kindly provided by professor Christiane Biermann (Portland State University, USA) and professor Victor Vacquier (University of California San Diego, USA) respectively. Polysaccharide purification was performed by anion exchange chromatography as previously described (Vilela-Silva et al. 1999, 2002). P-selectin inhibition assay in vitro Calcein-AM-labeled LS180 cells and increasing concentrations of the sulfated polysaccharides were added in triplicate to a 96-well plate with immobilized P-selectin chimeras, as described previously (Hostettler et al. 2007). Animal models An animal use protocol was obtained (05/2016), and procedures were followed in strict accordance with guidelines established by the Comissão de Ética no Uso de Animais/Instituto de Pesquisas Biomédicas/Hospital Naval Marcílio Dias (CEUA-IPB). Platelet–tumor cell association in vivo Lung sections were obtained from the mice 30 min after the intravenous injection of the tumor cells as described previously (Borsig et al. 2001), with slight modifications. Briefly, LLC cells were labeled with calcein-AM and intravenously injected into C57BL/6 mice with or without previous intravenous injection of 100 μg of the sulfated polysaccharides. After 30 min, the lungs were harvested for analysis. The lung sections were immunofluorescently stained with goat anti-integrin αIIb (CD41) (Santa Cruz Biotechnology) followed by anti-goat Cy3-conjugated antibody and analyzed by immunofluorescence microscopy. The platelet–tumor cell association was quantified by evaluating the calcein-AM labeled cells present in 10 fields of each lung section (10 sections per animal, 3 animals per experimental group). Experimental metastasis model C57BL/6 mice were intravenously injected with 106 LLC cells via the tail vein. Mice were injected either with PBS (vehicle) or 100 μg of the sulfated polysaccharides 15 min prior to tumor cell injection and killed after 21 days. PBS perfused lungs were macroscopically evaluated for the presence of metastatic foci. Thioglycolate-induced acute peritonitis C57BL/6 mice were intravenously injected with 100 μg of the sulfated polysaccharides. After 15 min, the animals were injected intraperitoneally with 1 mL of 4% thioglycolate. The mice were sacrificed after 3 h, and peritoneal lavage was collected using 8 mL ice-cold PBS. The total number of cells in peritoneal lavage was counted with a hemocytometer. This lavage was analyzed after cytospin centrifugation and staining with Wright’s stain. Differential counting was performed to evaluate the percentage of polymorphonuclear cells present in the peritoneal cavity. P-sel−/− and wild-type mice were used in this experiment. Funding Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Ary Frauzino para Pesquisa e Controle do Câncer. Conflict of interest statement None declared. Acknowledgements The authors would like to thank Professor Eliene Kozlowski (in memoriam), who participated from the beginning of the project design to the data analysis of all experiments in this article. The authors would also like to thank professor Mariana Stelling for substantial revisions of the article and the students Mariana Soares and Tamires Gerhardt for technical support. 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GlycobiologyOxford University Press

Published: Mar 7, 2018

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