Eliene O. Kozlowski

Extracellular Galectin-3 in Tumor Progression and Metastasis

Galectin-3, the only chimera galectin found in vertebrates, is one of the best-studied galectins. It is expressed in several cell types and is involved in a broad range of physiological and pathological processes, such as cell adhesion, cell activation and chemoattraction, cell cycle, apoptosis, and cell growth and differentiation. However, this molecule raises special interest due to its role in regulating cancer cell activities. Galectin-3 has high affinity for β-1,6-N-acetylglucosamine branched glycans, which are formed by the action of the β1,6-N-acetylglucosaminyltransferase V (Mgat5). Mgat5-related changes in protein/lipid glycosylation on cell surface lead to alterations in the clustering of membrane proteins through lattice formation, resulting in functional advantages for tumor cells. Galectin-3 presence enhances migration and/or invasion of many tumors. Galectin-3-dependent clustering of integrins promotes ligand-induced integrin activation, leading to cell motility. Galectin-3 binding to mucin-1 increases transendothelial invasion, decreasing metastasis-free survival in an experimental metastasis model. Galectin-3 also affects endothelial cell behavior by regulating capillary tube formation. This lectin is found in the tumor stroma, suggesting a role for microenvironmental galectin-3 in tumor progression. Galectin-3 also seems to be involved in the recruitment of tumor-associated macrophages, possibly contributing to angiogenesis and tumor growth. This lectin can be a relevant factor in turning bone marrow in a sanctuary for leukemia cells, favoring resistance to therapy. Finally, galectin-3 seems to play a relevant role in orchestrating distinct cell events in tumor microenvironment and for this reason, it can be considered a target in tumor therapies. In conclusion, this review aims to describe the processes of tumor progression and metastasis involving extracellular galectin-3 and its expression and regulation.

Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion.

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing ∼4.6 mg g−1 of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional 1H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg−1, 5-fold lower than porcine heparin (180 IU mg−1), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC50 = 0.835 μg ml−1) and thrombin (IC50 = 9.3 μg ml−1) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg−1, the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.

Antitumor properties of a new non-anticoagulant heparin analog from the mollusk Nodipecten nodosus: Effect on P-selectin, heparanase, metastasis and cellular recruitment

Inflammation and cancer are related pathologies acting synergistically to promote tumor progression. In both, hematogenous metastasis and inflammation, P-selectin participates in interactions involving tumor cells, platelets, leukocytes and endothelium. Heparin has been shown to inhibit P-selectin and as a consequence it blunts metastasis and inflammation. Some heparin analogs obtained from marine invertebrates are P-selectin inhibitors and do not induce bleeding effects. The present work focuses on the P-selectin blocking activity of a unique heparan sulfate (HS) from the bivalve mollusk Nodipecten nodosus. Initially, we showed that the mollusk HS inhibited LS180 colon carcinoma cell adhesion to immobilized P-selectin in a dose-dependent manner. In addition, we demonstrated that this glycan attenuates leukocyte rolling on activated endothelium and inflammatory cell recruitment in thioglycollate-induced peritonitis in mice. Biochemical analysis indicated that the invertebrate glycan also inhibits heparanase, a key player in cell invasion and metastasis. Experimental metastasis of Lewis lung carcinoma cells was drastically attenuated by the mollusk HS through a mechanism involving inhibition of platelet-tumor-cell complex formation in blood vessels. These data suggest that the mollusk HS is a potential alternative to heparin for inhibiting P-selectin-mediated events such as metastasis and inflammatory cell recruitment.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO4) (59.4%), ΔUA-1→3-GalNAc(6SO4) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

Fucosylated Chondroitin Sulfate inhibits Plasmodium falciparum cytoadhesion and merozoite invasion

ABSTRACT Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.

Structure and Biological Activities of Glycosaminoglycan Analogs from Marine Invertebrates: New Therapeutic Agents?

Although the oceans are habited by about 230,000 different animal species, only 1% of these organisms have been studied so far. Thus, the sea constitutes an immense reservoir of unique natural molecules with potential biological interest. Among the countless described compounds of marine origin, the sulfated glycosaminoglycans are a particularly interesting group of molecules. These polymers are widely distributed among different marine phyla, occurring in elevated quantities. Normally, the glycosaminoglycans from marine sources contain a high negative charge density due to the presence of sulfate groups in different positions. In addition, unique structural motifs, such as glucose and sulfated fucose branches, rarely described in glycosaminoglycans from terrestrial organisms, are also found. Various pharmacological properties ranging from anticoagulant and antithrombotic to antimetastatic and anti-inflammatory have been attributed to these molecules. The interaction and modulation of plasma coagulation proteases and inhibitors, adhesion molecules, and growth factors, among others, are the basis for their pharmacological effects. In this chapter, we review the phylogenetic distribution, the structure, and the biological effects of the marine glycosaminoglycans, as well as the molecular mechanisms involved in some of their biological activities. We also briefly discuss the possibility of using these glycans as therapeutic agents.

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

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.

Abstract B44: Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity

The primary interaction between tumor and endothelial cells is mediated by endothelial selectins and sialyl-Lewis carbohydrates found on cancer cell surface. Heparin, a sulfated polysaccharide, binds to P- and L-selectins, an effect that has been associated with attenuation of metastasis. However, due to its strong anticoagulant activity, its use in anti-tumor therapy is restricted. Sulfated fucans from sea urchins have simple, unique structures of linear chains of sulfated α-L-fucose in well-defined repetitive patterns. In this study, we tested five sulfated fucans from different species of sea urchins for their ability to interfere with the tumor-endothelium interaction and compared our findings to those obtained with heparin. Our results show that sulfated fucan from L. variegatus (FucSulf I), bearing an unique 2,4-disulfated fucose residue and low anticoagulant activity, inhibits the adhesion of tumor cells to endothelium, tumor transendothelial migration and endothelial cell tubulogenesis, both by inhibiting P-selectin mediated tumor cell adhesion, and VEGF-dependent signaling in endothelial cells. FucSulf I also inhibited proliferation of several types of tumor cell lines in vitro and tumor growth in vivo . Our results indicate this sulfated fucan is a potential antitumor agent and a possible therapeutic alternative to heparin. Citation Format: Viviane Mignone, Camila Castro Figueiredo, Aline Oliveira, Eliene Kozlowski, Lubor Borsig, Mauro Pavao, Paulo Mourao, Veronica Morandi. Sulfated fucan inhibits tumor interaction with endothelial cells and tumor growth: Possible contribution of an antivascular endothelial growth factor (VEGF) neutralizing activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B44.

Abstract 2363: Dual effects of unique oversulfated dermatan sulfates from ascidians (Chordata-Tunicata) on tumor invasion: inhibition of ephitelial-mesenchymal transition and hematogeneous metastasis

Metastatic disease is responsible for most cancer-associated deaths and epithelial-mesenchymal transition (EMT) along with hematogeneous metastasis are critical steps in cancer progression. Therefore, inhibition of these events could be an effective approach to reduce the metastatic disease. Heparin has been show to modulate EMT-associated growth factors and also to inhibit P-selectin, leading to attenuation of metastasis. Previously, we showed that unique dermatan sulfates (DS) from Styela plicata and Pallusia nigra, composed by 2,4-O-sulfated and 2,6-O-sulfated disaccharide units, respectively, bind with high affinity (KD of ∼ 33nM) to the EMT-related growth factor, hepatocyte growth factor (HGF), modulating its MET-dependent intracellular signaling. In the present work, the effect of the ascidian DSs on EMT-mediated cell migration and P-selectin-mediated metastasis in experiments in vitro and in vivo were evaluated. To investigate migration, confluent human mammary cells were scraped with a pipette tip and cultured for 3 days in the presence of the ascidian DSs. The migration of human mammary cells in the wound-healing cell migration assay was drastically inhibited by the glycans. The anti-selectin activity was evaluated by the binding of LS180 cells to immobilized P-selectin in the presence of the ascidian DSs. The ascidian DSs inhibited the binding of tumor cells to P-selectin with IC50 values of 13.51 μg/mL (2,4-DS) and 12.19 μg/mL (2,6-DS) and were better inhibitors than unfractionated heparin (IC50 of 24.51 μg/mL). DS from porcine skin had no effect. To investigate the effect of ascidian DSs in vivo, mice were injected with LS180 cells 10 minutes after treatment with PBS, UFH (1 mg/mouse) or ascidian DSs (100 μg/mouse) and the presence of platelets-tumor cells aggregates in the lung microvasculature was evaluated. The ascidian DSs inhibited the adhesion of platelets to tumor cells in vivo in a concentration 10-fold lower than heparin. The inhibitory effect of glycans could be observed as early as 30 minutes upon cells injection or 3 hours later. Metastasis mice in vivo was investigated by injecting 100 μg of each ascidian DSs or PBS 10 minutes prior injection of MC-38 cells. Twenty-eight days later, lungs were harvested for macroscopic evaluation of metastasis. Both ascidian DSs drastically reduced metastasis of MC-38 cells. PBS had no effect. Metastasis was drastically reduced in P-sel-/- mice and neither 2,4- nor 2,6-DS had any additional effect. In conclusion, ascidian DSs inhibit cell migration and reduce hematogeneous metastasis by a EMT- and P-selectin mediated events and could be used therapeutically to prevent tumor invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2011-2363