Extraction, isolation, characterization and biological activity of sulfated polysaccharides present in ascidian viscera Microcosmus exasperatus

Abstract

Ascidians are marine invertebrates that synthesize sulfated glycosaminoglycans (GAGs) within their viscera. Ascidian GAGs are considered analogues of mammalian GAGs and possess great potential as bioactive compounds, presenting antitumor and anticoagulant activity. Due to its worldwide occurrence, and therefore, being a suitable organism for large-scale mariculture in many marine environments, our main objectives are to study Microcosmus exasperatus GAGs re-garding composition, structure and biological activity. We also aim to develop efficient protocols for sulfated polysaccharides extraction and purification aiming large-scale production and clinical applications. Ascidians were collected by free diving. GAGs were extracted by proteolytic diges-tion, purified by ion-exchange liquid chromatography, characterized by agarose gel electropho-resis and enzymatic treatments. Anticoagulant activity was evaluated by aPTT assays. Cell cyto-toxicity was evaluated by MTT assays using LLC tumor cell line. Clonogenic assay was performed using LLC cells as well. Tumor cell migration activity was evaluated in vitro by wound healing assays. Our results show that M. exasperatus presents three distinct polysaccharides. These pol-ysaccharides were fractionated into two fractions named SP1 and SP2. M. exasperatus produces a low anticoagulant dermatan sulfate (SP2) and a fucosylated dermatan sulfate composed of a hy-brid chain of dermatan sulfate, chondroitin sulfate and fucose residues with sulfations at position 2 and 6 of the N-acetyl-galactosamine (SP1), which, interestingly, is not susceptible to heparinases treatment and does not present significant anticoagulant activity. Glycans were tested as anti-tumoral agents and presented relevant activity as such. Short-term incubation with TP does not affect LLC tumor cells viability at low concentrations, while long-term incubation with TP de-creased LLC tumor cells growth/proliferation at different concentrations. TP affected LLC tumor cells migration at different concentrations. As a conclusion, we state that M. exasperatus presents great potential as an alternative GAG source compared to porcine or bovine heparin, producing suitable compounds that present antitumoral properties at low concentrations, that do not pos-sess anticoagulant activity, and do not enhance other aspects of malignancy, such as tumor cell migration and invasion. Our perspectives are to apply these molecules in future preclinical studies as a soluble formula, as well as in nanoparticles, for cancer treatment.