Direct antitumoral effects of sulfated fucans isolated from echinoderms: a possible role of neuropilin-1/ß1 integrin endocytosis and focal adhesion kinase degradation.

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in preclinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50%), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of ß1 integrin and neuropilin-1 chains, two cell receptors implicated in fibronectin-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated focal adhesion kinase levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.

Elemental profiles in distant tissues during tumor progression.

BACKGROUND: Essential elements have functions in tumor progression by promoting protumoral cellular processes, such as proliferation, and migration, among others. Obtaining an understanding of how these elements relate to tumor progression processes is of great importance for research. Elemental profile studies in distant tissues, which can be modulated by tumor cells to promote metastasis, have not been sufficiently investigated. The main goal of this study is to evaluate multielemental distribution during tumor progression, focusing on tumor tissue and distant tissues that may be affected. METHODS: Tumor progression in vivo was simulated by inoculating C57BL/6 mice with Lewis Lung Carcinoma (LLC) cells. Samples of the primary tumor and distant tissues were collected during 5 weeks of tumor progression for the control and experimental (tumor-bearing) groups. The biological samples were analyzed using the synchrotron radiation X-Ray fluorescence technique. Data on the concentration of P, S, K, Ca, Mn, Fe, Cu, and Zn in the samples were obtained and statistically analyzed to evaluate the distribution of the elements during tumor progression in the primary tumor as well as distant tissues. RESULTS: It was possible to observe significant changes in the concentrations' distribution of P, S, K, Ca, Mn, Fe, and Cu in distant tissues caused by the presence of tumor cells. It was also possible to detect a greater similarity between tumor tissue (which has the lung as tissue of origin) and a tissue of non-origin, such as the liver, which is an unprecedented result. Moreover, changes in the distributions of concentrations were detected and studied over time for the different tissues analyzed, such as primary tumor, liver and lung, in Control and Tumor groups. CONCLUSIONS: Among other results, this paper could explore the modulation of distant tissues caused by the presence of a primary tumor. This could be achieved by the evaluation of several elements of known biological importance allowing the study of different biological processes involved in cancer. The role of essential elements as modulators of the tumor microenvironment is a relevant aspect of tumor progression and this work is a contribution to the field of tumoral metallomics.

Effects of Dermatan Sulfate from Marine Invertebrate Styela plicata in the Wound Healing Pathway: A Natural Resource Applied to Regenerative Therapy.

Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.

Prognostic significance of hedgehog signaling network-related gene expression in breast cancer patients.

Breast cancer continues to be a serious public health problem. The role of the hedgehog pathway in normal development of the mammary gland as well as in carcinogenesis and progression of breast cancer is the subject of intense investigation, revealing functional interactions with cell surface heparan sulfate. Nevertheless, its influence on breast cancer prognosis, and its relation to specific sulfation motifs in heparan sulfate have only been poorly studied in large patient cohorts. Using the public database KMplotter that includes gene expression and survival data of 3951 patients, we found that the higher expression of SHH, HHAT, PTCH1, GLI1, GLI2, and GLI3 positively influences breast cancer prognosis. Stratifying patients according to the expression of hormone receptors, histological grade, lymph node metastasis, and systemic therapy, we observed that GLI1, GLI2, and GLI3 expression, as well as co-expression of SHH and ELP1 were associated with worse relapse-free survival in patients with HER2-positive tumors. Moreover, GLI1 expression in progesterone receptor-negative tumors and GLI3 expression in grade 3 tumors correlated with poor prognosis. SHH, in a panel of cell lines representing different breast cancer subtypes, and HHAT, PTCH1, GLI1, GLI2, and GLI3 were mostly expressed in cell lines classified as HER2-positive and basal-like. Expression of SHH, HHAT, GLI2, and GLI3 was differentially affected by overexpression of the heparan sulfate sulfotransferases HS2ST1 and HS3ST2 in vitro. Although high HS2ST1 expression was associated with poor prognosis in KMplotter analysis, high levels of HS3ST2 were associated with a good prognosis, except for ER-positive breast cancer. We suggest the GLI transcription factors as possible markers for the diagnosis, treatment, and prognosis of breast cancer especially in HER2-positive tumors, but also in progesterone receptor-negative and grade-3 tumors. The pathway interaction and prognostic impact of specific heparan sulfate sulfotransferases provide novel perspectives regarding a therapeutical targeting of the hedgehog pathway in breast cancer.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Tunicate Heparan Sulfate Enriched in 2-Sulfated ß-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin.

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

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.

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.

Mammary Branching Morphogenesis Requires Reciprocal Signaling by Heparanase and MMP-14.

The development of the mammary gland involves formation of a branched arboreal structure resulting from the penetration and proliferation of epithelial cells into the fat pad. The mammary cells invade by remodeling their surrounding extracellular matrix (ECM), which are rich in proteins, and glycans such as heparan sulfate proteoglycans (HSPGs). There is increasing literature on how the interaction between signaling by ECM and matrix metalloproteinases (MMPs) is relevant to morphogenetic and physiological contexts. Here we sought to understand how heparanase, the sole mammalian heparan sulfate-degrading endoglycosidase may regulate mammary gland development. We found a robust localization of heparanase within growing end buds during branching in vivo. Using three-dimensional (3D) organotypic cultures, we showed that heparanase expression and activity are required for mammary epithelial invasion/branching within dense collagen I gels. Morphometric analysis of glands from both heparanase-overexpressing and knockout mice showed a direct correlation between degree of branching and the heparanase levels, confirming our 3D organotypic culture observations. Finally, we uncovered a reciprocal association between levels of heparanase and MMP14, a membrane-bound MMP, shedding further light on how branching occurs within developing mammary glands.

HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

Fucosylated chondroitin sulfate inhibits Plasmodium falciparum cytoadhesion and merozoite invasion.

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.

Ascidian (Chordata-Tunicata) Glycosaminoglycans: Extraction, Purification, Biochemical, and Spectroscopic Analysis.

Sulfate polysaccharides with unique structures of the chondroitin/dermatan and heparin/heparan families of sulfated glycosaminoglycans have been described in several species of ascidians (Chordata-Tunicata). These unique sulfated glycans have been isolated from the ascidians and characterized by biochemical and spectroscopic methods. The ascidian glycans can be extracted by different tissues or cells by proteolytic digestion followed by cetylpyridinium chloride/ethanol precipitation. The total glycans are then fractionated by ion-exchange chromatography on DEAE-cellulose and/or Mono Q (HR 5/5) columns. Alternatively, precipitation with different ethanol concentrations can be employed. An initial analysis of the purified ascidian glycans is carried out by agarose gel electrophoresis on diaminopropane/acetate buffer, before or after digestion with specific glycosaminoglycan lyases or deaminative cleavage with nitrous acid. The disaccharides formed by exhaustive degradation of the glycans are purified by gel-filtration chromatography on a Superdex Peptide column and analyzed by HPLC on a strong ion-exchange Sax Spherisorb column. 1H- or 13C-nuclear magnetic resonance spectroscopy in one or two dimensions is used to confirm the structure of the intact glycans.

Continuous design and economic analysis of a Sargassum muticum biorefinery process.

This work assesses scale effects in designing a biorefinery from Sargassum muticum seaweed by applying a detailed process modeling methodology. Two process conversion units were simulated: one considering anaerobic digestion steps for producing biogas and generating electricity (base project), and the other with residual seaweed solids sold as fertilizer (alternative project). A comprehensive economic analysis was performed to estimate the minimum selling price of the process's main product (fucoidan extract). Results indicated that capital expenditures are up to 12.7% times higher in the base project. Minimum selling prices of the fucoidan extract product demonstrate the biorefinery's economies of scale for both projects. Seaweed's low methane potential reduces the economic attractiveness of electricity generation from biogas in the base project. Conversely, organic fertilizer price was more influential in the alternative project. Nonetheless, risk analyses show similar results for both scenarios, mainly due to fucoidan extract selling price and CAPEX estimates uncertainties.

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 approximately 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 (1)H, 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 (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg 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.

Dermatan sulfate in tunicate phylogeny: order-specific sulfation pattern and the effect of [→4IdoA(2-sulfate)ß-1→3GalNAc(4-sulfate)ß-1→] motifs in dermatan sulfate on heparin cofactor II activity.

BACKGROUND: Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)ß-1→3GalNAcß-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. RESULTS: Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)ß-1→3GalNAcß-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units. CONCLUSIONS: Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)ß-1→3GalNAcß-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)ß-1→3GalNAc(4S)ß-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.

Fucosylated chondroitin sulfate attenuates renal fibrosis in animals submitted to unilateral ureteral obstruction: a P-selectin-mediated event?

Fibrosis is the end point of most renal diseases, and several glycosaminoglycans have been shown to attenuate this process. Marine invertebrate glycosaminoglycans with unique structures have opened the possibility to test these new compounds on renal fibrosis. The effect of a fucosylated chondroitin sulfate from an echinoderm marine species is reported with the use of a model of renal fibrosis in rats, termed unilateral ureteral obstruction. Animals were given 4 mg/kg body wt of fucosylated chondroitin sulfate intraperitoneally, once a day. After 14 days, their kidneys were examined by histological, immunohistochemical, and biochemical methods. Compared with control mice, collagen deposition decreased in the course of renal fibrosis in the animals receiving fucosylated chondroitin sulfate, as revealed by Sirius red staining and hydroxyproline content. The cellularity related to myofibroblasts and macrophages was also reduced, as was the production of transforming growth factor (TGF)-ß. The glycosaminoglycan content increased in the renal interstitium of animals submitted to unilateral ureteral obstruction compared with the control contralateral kidney, mostly due to an increase of chondroitin sulfate content. Interestingly, no change in the pattern of glycosaminoglycan deposition was observed after administration of fucosylated chondroitin sulfate. Fibrosis induced by unilateral ureteral obstruction is attenuated in P-selectin-deficient mice, which also do not respond to the invertebrate glycosaminoglycan. In conclusion, fucosylated chondroitin sulfate attenuates renal fibrosis on a ureteral obstruction model in mice preponderantly through a P-selectin-mediated mechanism.

The Heparan Sulfate Sulfotransferases HS2ST1 and HS3ST2 Are Novel Regulators of Breast Cancer Stem-Cell Properties.

Heparan sulfate (HS) is a glycosaminoglycan found mainly in its protein-conjugated form at the cell surface and the extracellular matrix. Its high sulfation degree mediates functional interactions with positively charged amino acids in proteins. 2-O sulfation of iduronic acid and 3-O sulfation of glucosamine in HS are mediated by the sulfotransferases HS2ST and HS3ST, respectively, which are dysregulated in several cancers. Both sulfotransferases regulate breast cancer cell viability and invasion, but their role in cancer stem cells (CSCs) is unknown. Breast CSCs express characteristic markers such as CD44+/CD24-/low , CD133 and ALDH1 and are involved in tumor initiation, formation, and recurrence. We studied the influence of HS2ST1 and HS3ST2 overexpression on the CSC phenotype in breast cancer cell lines representative of the triple-negative (MDA-MB-231) and hormone-receptor positive subtype (MCF-7). The CD44+/CD24-/low phenotype was significantly reduced in MDA-MB-231 cells after overexpression of both enzymes, remaining unaltered in MCF-7 cells. ALDH1 activity was increased after HS2ST1 and HS3ST2 overexpression in MDA-MB-231 cells and reduced after HS2ST1 overexpression in MCF-7 cells. Colony and spheroid formation were increased after HS2ST1 and HS3ST2 overexpression in MCF-7 cells. Moreover, MDA-MB-231 cells overexpressing HS2ST1 formed more colonies and could not generate spheres. The phenotypic changes were associated with complex changes in the expression of the stemness-associated notch and Wnt-signaling pathways constituents, syndecans, heparanase and Sulf1. The results improve our understanding of breast CSC function and mark a subtype-specific impact of HS modifications on the CSC phenotype of triple-negative and hormone receptor positive breast cancer model cell lines.

Non-Anticoagulant Heparan Sulfate from the Ascidian Phallusia nigra Prevents Colon Carcinoma Metastasis in Mice by Disrupting Platelet-Tumor Cell Interaction.

Although metastasis is the primary cause of death in patients with malignant solid tumors, efficient anti-metastatic therapies are not clinically available currently. Sulfated glycosaminoglycans from marine sources have shown promising pharmacological effects, acting on different steps of the metastatic process. Oversulfated dermatan sulfates from ascidians are effective in preventing metastasis by inhibition of P-selectin, a platelet surface protein involved in the platelet-tumor cell emboli formation. We report in this work that the heparan sulfate isolated from the viscera of the ascidian Phallusia nigra drastically attenuates metastases of colon carcinoma cells in mice. Our in vitro and in vivo assessments demonstrate that the P. nigra glycan has very low anticoagulant and antithrombotic activities and a reduced hypotension potential, although it efficiently prevented metastasis. Therefore, it may be a promising candidate for the development of a novel anti-metastatic drug.

Methods for Isolation and Characterization of Sulfated Glycosaminoglycans from Marine Invertebrates.

Marine invertebrates produce different kinds of sulfated polysaccharides. These glycans play essential roles in several biological processes and the study of these molecules is promising in a variety of fields. In the following sections, we describe the materials and methods used for the extraction, purification, and characterization of marine invertebrate-derived glycosaminoglycans.

Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis.

INTRODUCTION: A heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin was isolated from the body of the ascidian Styela plicata (Chordata-Tunicata). The disaccharide composition and the effect of the invertebrate glycan on venous and arterial models of thrombosis was investigated. METHODS AND RESULTS: High performance liquid chromatography of the products formed by a mixture of heparin lyases showed that the ascidian heparin is composed mainly by delta UA(2SO4)-1-->4-beta-d-GlcN(SO4) (47.5%), delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(6SO4) (38.3%) disaccharides and smaller amounts of the disaccharides delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4)(6SO4) (2.8%) and delta UA(2SO4)-1-->4-beta-d-GlcN(SO4)(3SO4) (8.0%). The invertebrate heparin has an aPTT activity of 18 IU/mg and an antithrombin-mediated antithrombin and anti-factor Xa activities 10-fold lower than that of mammalian heparin. In a venous model of thrombosis in the vena cava, S. plicata heparin inhibits only 80% of thrombosis at a dose 10-fold higher than that of the mammalian heparin that inhibits 100% of thrombosis. However, in an arterio-shunt model of arterial thrombosis, both S. plicata and mammalian heparin possess equivalent antithrombotic activities. It is also shown that at equivalent doses, ascidian heparin has a lower bleeding effect than mammalian heparin. CONCLUSION: The antithrombin-mediated anticoagulant activity of heparin polymers is not directly related to antithrombotic potency in the arterio-venous shunt. The results of the present work suggest that heparin preparations obtained from the body of S. plicata may have a safer therapeutic action in the treatment of arterial thrombosis than mammalian heparin.