Antioxidant and antiproliferative effect of a glycosaminoglycan extract from Rapana venosa marine snail

Abstract

Marine glycosaminoglycans (GAG) isolated from different invertebrates, such as molluscs, starfish or jellyfish, have been described as unique molecules with important pharmacological applications. Scarce information is available on GAG extract from Rapana venosa marine snail. The aim of this study was to isolate a GAG extract from R. venosa marine snail and to investigate its physicochemical, antioxidant and antiproliferative properties for further biomedical use. The morphology, chemical and elemental composition of the extract were established as well as the sulfate content and N- to O-sulfation ratio. Fourier transform infrared (FTIR) spectra indicated that GAG extract presented similar structural characteristics to bovine heparan sulfate and chondroitin sulfate. The pattern of extract migration in agarose gel electrophoresis and specific digestion with chondroitinase ABC and heparinase III indicated the presence of a mixture of chondroitin sulfate-type GAG, as main component, and heparan sulfate-type GAG. Free radical scavenging and ferric ion reducing assays showed that GAG extract had high antioxidant activity, which slightly decreased after enzymatic treatment. In vitro MTT and Live/Dead assays showed that GAG extract had the ability to inhibit cell proliferation in human Hep-2 cell cultures, at cytocompatible concentrations in normal NCTC clone L929 fibroblasts. This capacity decreased after enzymatic digestion, in accordance to the antioxidant activity of the products. Tumoral cell migration was also inhibited by GAG extract and its digestion products. Overall, GAG extract from R. venosa marine snail exhibited antioxidant and antiproliferative activities, suggesting its potential use as novel bioactive compound for biomedical applications.

Fig 1. EDX spectra of GAG extract isolated from Rapana venosa marine snail.

Fig 2. FTIR spectra of GAG extracted from Rapana venosa marine snail, bovine condroitin sulfate (CS) and heparan sulfate (HS).

Fig 3. Agarose gel electrophoresis of GAG extract from Rapana venosa marine snail before and after incubation with heparinase III (Hep III) and chondroitinase ABC (Chase ABC).

Standards of bovine condroitin sulfate (CS) and heparan sulfate (HS) migrated in similar conditions.

Fig 4

Cell viability of normal L929 cells (A) and tumor Hep-2 (B) cells after cultivation with different concentrations of marine GAG extract from Rapana venosa, before and after enzymatic digestion with heparinase III (Hep III) and chondroitinase ABC (Chase ABC) for 72 h, assessed by MTT assay. The results are expressed as mean values ± SD (n = 6). *p<0.05, compared to negative control (C-).

Fig 5

Micrographs of live (green) and dead (red) Hep-2 cells treated with different concentrations of Rapana venosa GAG extract, before and after digestion with heparinase III (Hep III) and chondroitinase ABC (Chase ABC), for 72 h, assessed by Live/Dead assay (A). Scale bar = 50 μm. Histogram of cell viability determined by ImageJ analysis (n = 3) (B).

Fig 6

Micrographs of cell migration of Hep-2 scratched cells treated with Rapana venosa GAG extract and its digestion products with heparinase III (HepIII) and chondroitinase ABC (Chase ABC) for 24 h (A). Scale bar = 100 μm. The rate of cell migration determined by ImageJ analysis (B). The results were expressed as mean values ± SD (n = 3). *p<0.05, compared to negative control.