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.

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.

A low-anticoagulant heparin suppresses metastatic dissemination through the inhibition of tumor cell-platelets association.

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

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.

Metal ions and the extracellular matrix in tumor migration.

In this review, we explore the roles of divalent metal ions in structure and function within the extracellular matrix (ECM), specifically, their interaction with glycosaminoglycans (GAGs) during tumor progression. Metals and GAGs have been individually associated with physiological and pathological processes, however, their combined activities in regulating cell behavior and ECM remodeling have not been fully explored to date. During tumor progression, divalent metals and GAGs participate in central processes, such as cell migration and angiogenesis, either by modulating cell surface molecules, as well as soluble signaling factors. In addition, studies on metals and polysaccharides interactions have been of great value, as they provide structural information that can be correlated with function. Finally, we believe that understanding how metals are regulated in physiological and pathological conditions is paramount for the development of new treatment strategies, as well as diagnostic and exploratory tools.