Accepted Manuscript Title: Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types Author: Gaetano Di Bari Eugenia Gentile Tiziana Latronico Giuseppe Corriero Anna Fasano Carlotta Nonnis Marzano Grazia Maria Liuzzi PII: DOI: Reference:
S1382-6689(14)00257-9 http://dx.doi.org/doi:10.1016/j.etap.2014.10.021 ENVTOX 2116
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
4-7-2014 20-10-2014 26-10-2014
Please cite this article as: Bari, Gaetano Di, Gentile, Eugenia, Latronico, Tiziana, Corriero, Giuseppe, Fasano, Anna, Marzano, Carlotta Nonnis, Liuzzi, Grazia Maria, Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2014.10.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types.
Gaetano Di Baria, Eugenia Gentilea, Tiziana Latronicoa, Giuseppe Corrierob, Anna Fasanoa, Carlotta Nonnis Marzanob, Grazia Maria Liuzzia* a
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via
Orabona 4, 70126, Bari, Italy Department of Biology, University of Bari, Via Orabona 4, 70126, Bari, Italy
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b
*Corresponding author:
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Dr. Grazia Maria Liuzzi
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Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari Via Orabona 4, 70126 Bari, Italy.
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Tel: +39 080 5443376; Fax: +39 080 5443317
Abbreviations
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BHK-21/C13, baby hamster kidney cells
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[email protected]
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BSA, bovine serum albumine
CaCo-2, human colon adenocarcinoma cells
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DMEM, Dulbecco’s modified Eagle’s medium FBS, fetal bovine serum
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GFAP, glial fibrillary acidic protein
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MTT, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide PBS, phosphate buffered saline PAS, periodic acid Schiff
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PLL, poly-L-lysine PMA, phorbol 12-myristate 13-acetate RPMI, Roswell Park Memorial Institute RT, room temperature SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis THP-1, human monocytic leukemia cell line
Abstract Marine natural products extracted from sponges represent a new source for drug discovery. Here we describe a simple method for preparing aqueous extracts from 7 Mediterranean demosponges, which allowed the extraction of water-soluble compounds, such as proteins by homogenization of sponge tissue in phosphate buffered saline (PBS). The comparative analysis by SDS-PAGE showed differences in number of bands, bandwidth and intensity among the sponges analysed. The PAS/silver staining revealed a substantial and different glycoprotein assortment among the demosponges studied. To further study the biological activities present in the sponge extracts, we determined the non-
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cytotoxic doses on four different mammalian cell types demonstrating that the optimal noncytotoxic doses were cell type- and extract-dependent.
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In conclusion, the extraction method described in this paper represents a fast and efficient procedure for the extraction of water-soluble proteins from marine sponges. Furthermore, the cell
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viability data suggest the feasibility of this method for the direct in vitro cell-based assays.
We described a simple method for preparing aqueous extracts from demosponges which
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•
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Highlights:
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allows the extraction of protein compounds.
Protein and glycoprotein profiles of sponge extracts were analysed.
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A test of cytotoxicity allowed to determine the optimal non toxic doses of each extract,
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which could be used to study their biological properties.
Keywords: demosponges; aqueous extracts; bioactive compounds; in vitro cytotoxicity; cell
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morphology.
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1. Introduction
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Several studies regarding the chemistry of molecules extracted from marine organisms have fully demonstrated that sponges provide the largest number of biologically active natural compounds (Leal et al., 2012). Over 60% of the potentially useful bioactive compounds discovered so far from living organisms have been obtained from marine fauna, 70% of which comes from sponges (Faulkner, 2002). Indeed, Porifera, due to their typically sessile condition, have evolved the ability to produce potent toxins as defensive tools against predators or competitors (Loh and Pawlik, 2014).There is a worldwide interest in marine natural products since they represent one of the few
de novo sources for drug discovery (Cragg and Newman, 2013). Moreover, it has been demonstrated that the biologically active molecules from marine organisms, possess unique pharmacological properties that are proving to be useful against cancer, AIDS, autoimmune and neurodegenerative diseases (Schmitz et al., 1993; Vinothkumar and Parameswaran, 2013). Purification procedures of these compounds usually include initial extraction with methanol, partitioning of the extract with organic solvents and chromatographic steps (Blunt et al., 2014). However, the majority of the organic solvents are toxic for cells or not well tolerated by some bioassays. Furthermore, these solvents tend to exclude water-soluble natural compounds. In this regard, it has been recently reported that sponges represent a promising resource of water-
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soluble bioactive compounds, suggesting that the therapeutic potential of molecules isolated from sponges cannot be attributed only to the secondary metabolites extracted by organic solvents, but
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also to water-soluble molecules. In this respect, bioactive peptides with anticancer potential (Suarez-Jimenez et al., 2012) and lectines with antimicrobial activities (Schröder et al., 2003) have
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been recently isolated from marine sponges. In addition, other water-soluble molecules such as the chondropsins A and B, two macrolides with anticancer properties, and the 3-alkylpyridinium
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polimers (poly-APS), which present hemolytic and cytotoxic activities, have been extracted from
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the sponges Chondropsis sp. and Haliclona sarai, respectively (Cantrell et al., 2000; Sepčić et al.,
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1997a,b). Despite the fact that the class Demospongiae is known for producing the largest number and diversity of biologically active molecules among marine invertebrates (Leal et al., 2012), the
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bioactive potential of water-soluble compounds, in particular bioactive proteins, has been little studied (Wilkesman & Schroder, 2007).
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On these grounds, in the present study we used a phosphate buffer in order to prepare aqueous extracts from 7 different Mediterranean demosponges. Some of the species chosen had been
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previously subjected to ex situ experimental rearing by this research group (Di Bari et al, in press). The method described in this paper has proved to be effective for the extraction of water-soluble proteins. By using a panel of mammalian cultured adherent cells, we assessed the cytotoxicity of the sponge extracts, which was demonstrated to be cell type-dependent. Taken together our results indicated the feasibility of the proposed method for direct in vitro cell-based assays aimed at the screening of potential bioactivity of water-soluble compounds present in the aqueous extracts.
2. Materials and methods
2.1 Chemicals Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, fetal bovine serum (FBS), penicillin and streptomycin, L-glutamine were obtained from GIBCO (Paisley, Scotland). DNase 1, poly-Llysine (PLL), trypsin, trypan blue, 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA), Schiff's reagent and bovine serum albumine (BSA) were provided by Sigma (St. Louis, MO, USA). Glial fibrillary acidic protein (GFAP) antibodies
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were purchased from Serotec (Oxford, UK).
2.2 Sponge collection
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For our experiments seven different demosponges, commonly found in the Adriatic Sea, were collected. In particular, specimens from Tethya aurantium, T. citrina, Hymeniacidon perlevis,
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Ircinia variabilis, Chondrilla nucula, Aplysina aerophoba and Sarcotragus spinosulus were chosen. Sponge species were collected by scuba diving in Southern Adriatic Sea, Italy, at depths between 1
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and 3 meters. Sponges were individually transferred to laboratory in bags filled with seawater and
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labeled for recognition. During the transport, the samples were protected against contact with air as
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well as other injuries and temperature was maintained around 18ºC. Once in laboratory, all sponges
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were numbered and listed with information like date of sampling and location, weighed and then
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frozen at –80ºC as soon as possible until the extraction.
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2.3 Preparation of aqueous extracts
Figure 1 shows a simplified flow-sheet of the procedure used for the preparation of aqueous
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extracts. Each sponge was homogenized in 10 mM PBS, 150 mM NaCl, pH 7.0 (1:4 w/v). Specimens from T. aurantium and T. citrina were grounded in PBS with mortar and pestle set in an
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iced bath. The other sponges were, instead, homogenized in the same buffer in a waring blender. The aqueous extract from T. aurantium was prepared from the endosomes, which were isolated
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from the entire sponge by removing the cortex under a steroscopic microscope. For the other species the entire sponge was used for the extract preparation. In order to facilitate the disgregation of cells, the homogenates were subjected to three cycles of freezing (20 min, -20°C) and thawing (10 min, RT). To avoid any microbial contamination due to the symbiotic bacteria, penicillin/streptomycin (5x103 U/ml) was added to the homogenate, which was then centrifuged for 60 minutes at 15000 g, 4˚C. The clear supernatant was collected and filtered through a Millipore membrane, pore size 0.22 µm.
Total protein content of each extract was determined according to Bradford method (Bradford, 1976). The protein yield of each extract was found to be approximately 50% compared to the corresponding homogenate. Extracts were then kept at -80°C until further analysis.
2.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) One dimension SDS-PAGE was carried out as described by Laemmli (1970). Sixty micrograms of total proteins were mixed with the sample buffer containing 4% SDS,10% glycerol, 10% βmercaptoethanol, 0.001% bromophenol blue and 0.5 M Tris-HCl, pH 6.8. Samples were heated at 100ºC for 2-3 minutes and then electrophorised on 12% (0.75 mm thickness) running gel overlaid
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with a 4% stacking gel. Electrophoresis was performed at 4°C, 120 V using a mini-Protean II apparatus (Bio-Rad
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Laboratories). The molecular mass of the main proteins in the sponge extracts was estimated by comparison with the standard protein kit of Bio-Rad. The protein bands were visualised by staining
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with 0.1% Coomassie brilliant blue R-250.
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2.5 Periodic acid Schiff (PAS) and subsequent silver staining of glycoproteins
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Detection of glycoproteins was assessed via periodic acid Schiff (PAS) and subsequent silver
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staining (Jay et al., 1990). Briefly, after electrophoresis, the gel were fixed in 25% isopropyl alcohol
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and 10% acetic acid overnight at 4°C and then washed in 7.5% acetic acid for 30 minutes. Gels were treated with 1% periodic acid for 1 h at 4°C and then stained in the Schiff's reagent (4.6 g/l
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pararosaniline, 7.3 g/l of sodium metabisulfite in 0.1 N HCl) until the protein bands turned to
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purple. Subsequently, the gels were further stained with silver nitrate.
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2.6 Preparation of astrocyte primary cultures Astrocytes were prepared from primary cell cultures of neocortical tissues from 1-day old rats as
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described by Latronico et al. (2013). Astrocytes were then purified by three repetitions of replating and trypsinization to deplete cultures of microglia and oligodendrocytes. The purity of the final cell
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culture was assessed by immuno-staining for GFAP. More than 98% of the cells were GFAPpositive in all the preparations. Cells were plated in PLL-coated 96 well-plates at a density of 1x105 cells/ml (100 µl/well) in complete medium (DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS) and maintained at 37°C in a 5% CO2 . 2.7 Culture of CaCo-2 cell line
Human colon adenocarcinoma CaCo-2 (wild type) cells were obtained from American Type Culture Collection (Rockville, MD, USA) at P13. The cells were grown at 37°C, 5% CO2, in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS. Confluent monolayers were subcultured by treatment with 0.25% trypsin and 0.02% EDTA in Ca2+- and Mg2+-free PBS and then plated in 96-well plates at the density of 5x105 cells/ml (100 µl/well) in complete medium and maintained at 37°C in a 5% CO2. 2.8 Culture of THP-1cells and differentiation into macrophages Human monocytic leukemia cell line, THP-1, was purchased from the American Type Culture
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Collection. The cells were maintained in RPMI1640 supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 50 µM mercaptoethanol at 37°C, 5% CO2. To differentiate
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THP-1 into macrophages, monocytes were plated in 96-well plates at the density of 5x105 cells/ml (100 µl/well) and incubated in completemedium in the presence of 100 nM PMA. After 72 h, the
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cells were washed once with serum-free RPMI 1640 and incubated overnight. at 37°C in a 5% CO2.
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2.9 Culture of BHK-21/C13 cells
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Baby hamster kidney (BHK-21/C13) cells were purchased by American Type Culture Collection
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(Rockville, MD, USA). The cells were grown as monolayers in DMEM supplemented with 100
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U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, at 37 °C, 5% CO2.One day before the treatment with the extracts, cells were plated in 96-well plates at the density of 2x105 cells/ml (100 µl/well)
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and incubated at 37ºC in a humidified atmosphere of 5% CO2. 2.10 Treatment of cultured cell types with aqueous extracts
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When the cells reached approximately 80% to 90% of confluency, they were washed twice with serum-free medium and then treated with different concentrations of each aqueous extract from the
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seven demosponges.
For our experiments, the aqueous extracts were employed at the protein concentrations of 1, 5, 10,
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30, 60 and 100 µg/ml. Cells incubated in the same experimental conditions in serum-free medium served as control. The treatment with the aqueous extracts was performed in 100 µl of serum-free medium for 24 h at 37°C, 5% CO2. At the end of the incubation period the medium was collected, centrifuged at 4200 g and culture supernatants were collected and stored at -80°C. Cells were subjected to MTT test for the assessment of cytotoxicity.
2.11 Assessment of cell viability Cytotoxicity of cells, after treatment with the different concentrations of the aqueous extracts from the seven demosponges, was detected by using the MTT assay (Mosmann, 1983). This assay is based on the reduction of MTT by the mitochondrial succinate dehydrogenase in viable cells, to a blue formazan product which can be measured spectrophotometrically using a microplate reader (Versamax-Molecular Devices). Briefly, triplicate samples of 1-5 x 105 cells/ml, plated in 96-well plates in serum-free medium, were treated with the extracts for 24 h. As a control, in other wells, cells were incubated in serumfree medium under the same conditions without the addition of the extracts. After removal of the
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culture medium, cells were rinsed in PBS and incubated at 37°C, 5% CO2 for 2 h with 0.5 mg/ml MTT.
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Reaction was stopped by removing the medium and the dye was dissolved in absolute ethanol. The difference between the absorbance of each sample at 560 and 690 nm was measured and the value
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of the untreated sample was set at 100%. The concentration of the aqueous extract that killed at
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least 60% of cells was considered toxic.
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3. Results
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3.1 Analysis by SDS-PAGE of the protein profile of aqueous extracts Protein profile of each extract was performed using SDS-polyacrylamide gels. Equal quantities of
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proteins (about 60 µg) from each sample were analysed.
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As shown in Figure 2 the comparative electrophoretic banding profile of total protein content yielded different protein bands, ranging from 120 to 15 kDa. The comparative electrophoretic
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analysis of each extract showed differences in number of bands, bandwidth and intensity. In particular, a protein band of about 50 kDa was mostly common to all the samples. With respect
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to I. variabilis (d) and C. nucula (e), a common protein band of about 31 kDa was clearly evident.Two protein bands of approximately 24 and 21 kDa were particularly evident in H. perlevis
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(lane c) while in C. nucula (lane e) a band of 29 kDa, which was absent in other sponges, was the most representative one.
3.2 Detection of glycoproteins in the aqueous extracts In order to assess the presence of glycoproteins in the aqueous extracts, in another set of experiments, after electrophoresis, the SDS-polyacrylamide gels were subjected to the PAS
staining. To this end the samples were electrophorised and horseradish peroxidase and BSA were used as positive and negative control, respectively. As shown in the representative gel in Figure 3B, the PAS staining revealed the presence of various glycoproteins in the extracts. In particular, the comparison between the gels stained with Coomassie brilliant blue (A) and with PAS/silver staining (B) showed that most of the glycoproteins present in the various extracts exibited a molecular weight different from that of the most representative proteins stained with Coomassie brillant blue. In fact, the glycoprotein profile of T. aurantium (c) and T. citrina (d) was mainly characterized by three PAS-positive protein bands of 77, 50 and 30 kDa, while in H. perlevis (e) two glycoproteins of 33 kDa and 19 kDa were clearly evident. The
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electrophoretic glycoprotein pattern of I. variabilis (f) was represented by a PAS-positive protein of about 20 kDa. In C. nucula (g) and A. aerophoba (h) the PAS staining did not reveal the presence of
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any glycosylated proteins, whereas two glycoproteins of 60 and 50 kDa were observed in S.
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spinosulus (i).
3.3 Evaluation of cytotoxicity of aqueous extracts on cultured cell types.
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Figure 4 shows the percentage of viability of the studied mammalian cell populations in response to
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the treatment with the aqueous extracts at the indicated concentrations. The concentrations of the
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aqueous extracts that yielded cell viability values < 60% in comparison to untreated cells (CTRL)
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were considered as toxic. Generally, the cell viability data were cell type- and extract-dependent. In particular, the analysis of the cell viability curves showed that the toxicity of the aqueous extracts
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from T. citrina (A) and H. perlevis (B) were highly variable depending on the cell type studied and
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had proved to be the most cytotoxic in comparison to the other extracts analysed. By contrast, the extract from C. nucula (C) was the less cytotoxic for the studied cells (C).
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The profile of cell viability curves of the different cell types treated with the extracts from T. aurantium (D), A. aerophoba (E) and S. spinosulus (F) showed similar degree of cytotoxicity,
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especially at the highest concentration employed in our experiments (100 µg/ml). The treatment with the extract from I. variabilis (G) was strictly cell type-dependent. In fact, this
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extract did not lead to cell death in BHK-21 cells and primary astrocytes, while in CaCo-2 cells and macrophages THP-1 it displayed citotoxicity at the concentrations of 10 µg/ml. The highest non-cytotoxic concentrations of each extract, calculated in the mammalian cell types studied, are summarized in Table 1.
3.4 Effect of sponge extracts on cell morphology
Figure 5 shows the effect of the highest non-cytotoxic concentrations of the extracts from the studied sponges, on cell morphology. In particular, the treatment of BHK-21 cells with all the extracts determined a change in the morphology of these cells which appeared more fasciculated, with the cell body thinner and spindly, probably due to the reduction in the free spaces between cells, in comparison to CTRL. CaCo-2 cells treated with the highest non-cytotoxic doses of the extracts displayed morphologically heterogeneous features. In particular, cells treated with T. aurantium, T. citrina and C. nucula appeared more vacuolated in comparison to CTRL, without important morphological changes. Moreover, the treatment of cells with the extracts from H. perlevis, I. variabilis, A. aerophoba and
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S. spinosulus determined a decrease in cell volume, with a lost of the polygonal morphology and acquisition of a round-shape morphology. With respect to THP-1 macrophages and primary
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astrocytes, the treatment with the aqueous extracts at the highest non-cytotoxic concentrations did not evoke significant morphological changes in comparison to CTRL. However, in the treated THP-
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1 cells an increase of the number of vacuoles was clearly appreaciable in comparison to CTRL.
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4. Discussion
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Marine sponges are a rich source of biologically active metabolites which have proved to be
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potentially effective agents for the treatment of a wide range of human diseases (Müller et al., 2004). These bioactive compounds have a broad range of pharmacological activities, such as
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antiinflammatory, antitumour, immuno- or neurosurpressive, antiviral, antimalarial and antibiotic
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properties (Sipkema et al., 2005; Suarez-Jimenez et al., 2012). Common purification procedures of such compounds include extraction with toxic polar solvents,
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such as methanol, which limitate direct biological screening on living cell culture systems. Until now, little attention has been paid to the bioactive potential of water-soluble compounds, and in
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particular of proteins. So far the studies performed on the aqueous extracts prepared form marine sponges have regarded either the isolation and characterization of proteases (Oli et al. 2014) or the
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assessment of the biological activities of compounds present in the extracts (Sepčić et al. 1997, 2010).
In this study we have prepared aqueous extracts from seven Mediterranean demosponges by using a phosphate buffer demonstrating that this procedure is efficient for the extraction of water-soluble compounds, namely proteins. The demosponges studied in this paper have been selected by taking into account their well-known richness of bioactive secondary metabolites. As example, three hydroquinones with activity against
leukemia cells have been isolated from Sarcotragus spinosulus (Abed et al., 2011) and fractionation of a crude extract of Aplysina aerophoba yielded aerophobin-2 and isofistularin-3 that can act as chemical defenses against predators (Thoms et al., 2004). The common distribution of the studied demosponges, their easy availability in the South Adriatic Sea (Apulia) and their capability of being farmed (Klöppel, et al., 2008) make them suitable for our investigations. Moreover, some of the studied sponges have been recently subjected to ex situ experimental breeding by this research group (Di Bari et al., in press). It is important to underline that sponge rearing,together with the recombinant DNA technology and peptide synthesis, rapresents a suitable approach to obtain higher amounts of pharmacologically
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active peptides and proteins. In our study we demostrated that the protein pattern of some of the demosponges analysed, such as
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H. perlevis and C. nucula, was remarkably characterized by the presence of high levels of specific protein bands, while in other samples numerous protein bands, which were poorly represented,
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characterized the pattern. Furthermore, the comparative electrophoretic analysis revealed also common proteins, which were mostly present in all the samples analyzed.
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To further characterize the protein content of the aqueous extracts, we evaluated the presence of
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glycosylated proteins by the PAS/silver staining, a high sensitive method for glycoprotein detection
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(Jay et al., 1990). The glycoprotein profile was substantially different in comparison to that
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obtained with Coomassie brilliant blue staining. This difference may be ascribed to the specificity and sensivity of the PAS staining for glycoproteins, which allow to reveal glycoproteins whose
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levels were too low to be detected in the gels stained with Coomassie brilliant blue.
Several biological activities isolated from sponge compounds have been ascribed to glycoproteins,
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and in particular to lectins. In this respect, it has been demonstrated that lectins from marine sponges possess mitogenic, chemotactic, immunobiological, antiviral and cytotoxic properties
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towards different human cells (Schröder et al., 1990; Matsumoto et al., 2012; Fenton et al., 2013). Moreover, a water-soluble glycoprotein of 21 kDa, able to lyse erytrocytes from a variety of
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organisms, has been already isolated from the Mediterranean demosponge T. aurantium (Mangel et al., 1992). In the present work we also carried out a preliminary study to determine the possible cytotoxicity of aqueous extracts from the studied demosponges on a panel of different cultured mammalian cell types. Indeed, screening of aqueous extracts from marine sponges on cells in culture is a common approach to identify compounds of biomedical importance (Beedessee et al., 2012). To study the cytotoxicity, we chose four different cell types which are generally used as in vitro models to study
a wide range of human diseases: macrophages THP-1 (Kuijk et al., 2008), CaCo-2 epithelial cells (Sambuy et al., 2005), BHK-21 fibroblasts (Aljofan et al., 2009) and primary rat astrocytes (Liuzzi et al., 2004a,b). After treatment of the selected cell types with increasing doses of the aqueous extracts, cytotoxicity was quantified by the use of the metabolic reduction of MTT by the mitochondrial enzyme succinate dehydrogenase (Mosmann, 1983). Results obtained yielded significant differences in response to the treatment with the different extracts and allowed to determine the optimal non toxic doses of each extract, which could be used to study their biological properties. The cell types selected for this study showed different sensitivity to the extracts as assessed by
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changes in cell morphology. In particular, CaCo-2 cells and BHK-21 fibroblasts showed the most clear and evident changes in cell morphology after treatment with the highest non-cytotoxic
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concentrations of extracts. These morphogical changes might be due to the presence of bioactive compounds that could be able to affect the organisation of the cytoskeleton and the assembly of
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microtubules (Prado et al., 2004).
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In conclution, these data suggest that some of the screened species produce a variety of water-
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soluble compounds that induce cellular responses. Moreover, the highest non toxic concentrations
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for the selected cell types could represent the basis for further studies on the bioactivities present in
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the aqueous extracts.
Taken together, our results encourage further work to test the bioactive properties of the compounds
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present in the aqueous extracts. Further efforts should go towards the purification and
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characterization of the water-soluble compounds, which could be responsible for bioactivity, and
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the assessment on their potential pharmacological applications.
Aknowledgments. The authors thank Gaetano Devito for his dedicated support in maintaining the
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rat colony for the preparation of astrocyte cultures.
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Loh,T. and Pawlik, J.R., 2014. Chemical defenses and resource trade-offs structure sponge communities on Caribbean coral reefs. P. Natl. Acad. Sci. USA 111, 4151-4156. Mangel, A., Leitao, J.M., Batel, R., Zimmermann, H., Müller, W.E.G., Schröder, H.C., 1992. Purification and characterization of a pore-formingprotein from the marine sponge Tethya lyncurium. Eur. J. Biochem. 210, 499-507. Matsumoto, R., Fujii, Y., Kawsar, S.M.A., Kanaly, R.A., Yasumitsu, H., Koide, Y., Hasan, I., Iwahara, C., Ogawa, Y., Im, C.H., Sugawara, S., Hosono, M., Nitta, K., Hamako, J., Matsui, T., Ozeki, Y., 2012. Cytotoxicity and glycan-binding properties of an 18 kDa lectin isolated from the marine sponge Halichondria okadai. Toxins 4, 323-338. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.
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Müller, W.E.G., Schröder, H.C., Wiens, M., Perović-Ottstadt, S., Batel, R., Müller, I.M., 2004. Traditional and modern biomedical prospecting: Part II - The benefits approaches for a sustainable exploitation of biodiversity
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(secondary metabolites and biomaterials from sponges). Evid.-based Compl. Alt., 133-144.
Oli, S., Abdelmohsen, U.R., Hentschel, U., Schirmeister, T., 2014. Identification of plakortide e from the Caribbean
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sponge Plakortishalichondroides as a trypanocidal protease inhibitor using bioactivity-guided fractionation. Mar. Drugs 12, 2614-2622.
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Prado, M.P., Torres, Y.R., Berlinck, R.G.S., Desiderà, C., Sanchez, M.A., Craveiro, M.V., Hajdu, E., da Rocha, R.M.,
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Machado-Santelli,G.M., 2004. Effects of marine organism extracts on microtubule integrity and cell cycle progression in cultured cells. J. Exp. Mar. Biol. Ecol. 313, 125-137.
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Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M.L., Stammati, A., Zucco, F., 2005. The CaCo-2 cell line as a model
Cell Biol. Toxicol. 21, 1-26.
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of the intestinal barrier: influence of cell and culture-related factors on CaCo-2 cell functional characteristics.
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Schmitz, F.J., Bowden, B.F., Toth, S.I., 1993. Antitumor and cytotoxic compounds from marine organisms, in: Attaway, D.H., Zaborsky, O.R. (Eds), Marine biotechnology, Volume 1: Pharmaceutical and Bioactive Natural
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Products. Plenum Press, New York, 197-308. Schröder, H.C., Kljajic, Z., Wegner, R., Reuter, P., Gasic, M., Uhlenbruck, G., Kurelac, B., Müller, W.E.G., 1990. The
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galactose-specific lectin from the sponge Chondrilla nucula displays anti-human immunodeficiency virus activity in vitro via stimulation of the (2`-5`) oligoadenylate metabolism. Antiviral Chem. & Chemother.1, 99-
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105.
Schröder, H.C., Ushijima, H., Krasko, A., Gamulin, V., Thakur, N.L., Diehl-Seifer, B., Müller, I.M., Müller,W.E.G., 2003. Emergence and disappearance of an immune molecule, an antimicrobial lectin, in basal metazoa. A
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tachylectin-related protein in the sponge Suberites domuncula. J. Bio. Chem. 278, 32810-32817.
Sepčić, K., Batista, U., Vacelet, J., Macek, P., Turk, T., 1997a. Biological activities of aqueous extracts from marine sponges and cytotoxic effects of 3-alkylpyridinium polymers from Reniera sarai. Comp. Biochem. Physiol. CToxicol. Pharmacol. 117, 47-53.
Sepčić, K., Guella, G., Mancini, I., Pietra, F., Serra, M.D., Menestrina, G., Tubbs, K., Maček, P., Turk, T., 1997b.
Characterization of anticholinesterase-active 3-alkylpyridinium polymers from the marine sponge Reniera sarai in aqueous solutions. J. Nat. Prod. 60, 991–996. Sepčić, K., Kauferstein, S., Mebs, D., Turk, T., 2010. Biological activities of aqueous and organic extracts from tropical marine sponges. Mar. Drugs 8, 1550–1566. Sipkema, D., Franssen, M.C., Osinga, R., Tramper, J., Wijffels, R.H., 2005. Marine sponges as pharmacy. Mar. Biotechnol. 7, 142-162.
anticancer potential: sources from marine animals. Mar.Drugs 10, 963-986.
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Suarez-Jimenez, G.M., Burgos-Hernandez, A., Ezquerra-Brauer, J.M., 2012. Bioactive peptides and depsipeptides with
Thoms, C., Wolff, M., Padmakumar, K., Ebel, R., Proksch, P., 2004. Chemical Defense of Mediterranean Sponges
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Aplysina cavernicola and Aplysina aerophoba. Zeitschrift fur Naturforschung - Section C Journal of Biosciences, 59, 113-122.
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Vinothkumar, S., Parameswaran, P.S., 2013.Recent advances in marine drug research. Biotechnol. Adv. 31, 1826-1845. Wilkesman, J.G., Schröder, H.C., 2007. Analysis of serine proteases from marine sponges by 2-D zymography.
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Electrophoresis 28, 429-436.
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Legends
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Figure 1. Flowchart of the procedure used for the preparation of the aqueous extracts. Figure 2. Representative SDS-PAGE gel electrophoretic analysis of the protein profiles of aqueous extracts from
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different Mediterranean demosponges. a: Tethya aurantium; b: Tethya citrina; c: Hymeniacidon perlevis; d: Ircinia variabilis; e: Chondrilla nucula; f: Aplysina aerophoba; g: Sarcotragus spinosulus; h: standard proteins. 60 µg of total proteins were analysed by SDS-PAGE on 12% polyacrilamide electrophoretic gels. The most representative protein
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bands of each extract are indicated by the arrows.
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Figure 3. Detection of glycoproteins in the aqueous extracts from different Mediterranean demosponges by PAS/silver staining. a: standard proteins; b: Horseradish peroxidase (positive control); c: Tethya aurantium; d: Tethya citrina; e: Hymeniacidon perlevis; f: Ircinia variabilis; g: Chondrilla nucula; h: Aplysina aerophoba; i: Sarcotragus spinosulus; l: ovine serum albumine (BSA) - negative control. 60 µg of total proteins were analysed by SDS-PAGE on 12% polyacrilamide electrophoretic gels. Gels were stained with either Coomassie brillant blue (A) or PAS/silver staining (B). In B the most representative glycoproteins of each extract are indicated by the arrows. The specificity of the PAS/silver staining toward glycoproteins is demostrated by the lack of BSA band whereas the sensitivity is demostrated by the remarkable staining of the glycoprotein horseradish peroxidase.
Figure.4. Effect of aqueous extracts from Tethya citrina (A), Hymeniacidon perlevis (B), Chondrilla nucula (C), Tethya aurantium (D), Aplysina aerophoba (E), Sarcotragus spinosulus (F) and Ircinia variabilis (G), on cell viability as determined by the MTT assay. The results are expressed as percentage of surviving cells over untreated cells (CTRL). Data are presented as mean ± SD from three different experiments with independent cell populations. The horizontal dashed line, set at 60 %, indicates the threshold of cell viability. Concentrations of the aqueous extracts that yielded cell viability values < 60% of control were considered as toxic doses.
Figure 5. Morphological features of cells treated with the highest no toxic concentrations of the different extracts. Panel of cells observed under phase-contrast microscope after 24 hrs of treatment at 37°C, 5% CO2, (Magnification 50×). Representative photomicrographs from three independent experiments show the morphological
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changes evoked by the different sponge extracts at the highest non cytotoxic concentrations.
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Table 1- Highest non-cytotoxic concentrations (µg/ml) of the aqueous extracts from Mediterranean demosponges on different cell types.
CaCo-2
THP-1
Astrocytes
Tethya aurantium
10
60
A
30
10
Tethya citrina
10
60
10
10
Hymeniacidon perlevis
5
10
5
30
5
5
≥100
≥100
≥100
30
60
Aplysina aerophoba
10
30
30
30
Sarcotragus spinosulus
10
30
10
60
Ircinia variabilis
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≥100
A
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Chondrilla nucula
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BHK-21
D
Sponge
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Cell types
Table 1- Highest non-cytotoxic concentrations (µg/ml) of the aqueous extracts from Mediterranean demosponges on different cell types.
Cell types BHK-21
CaCo-2
THP-1
Astrocytes
Tethya aurantium
10
60
30
10
Tethya citrina
10
60
10
10
Hymeniacidon perlevis
5
10
5
30
Ircinia variabilis
≥100
5
5
≥100
Chondrilla nucula
≥100
≥100
30
60
Aplysina aerophoba
10
30
30
30
Sarcotragus spinosulus
10
30
10
60
A
CC
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TE
D
M
A
N
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SC
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Sponge
S1382-6689(14)00257-9 http://dx.doi.org/doi:10.1016/j.etap.2014.10.021 ENVTOX 2116
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
4-7-2014 20-10-2014 26-10-2014
Please cite this article as: Bari, Gaetano Di, Gentile, Eugenia, Latronico, Tiziana, Corriero, Giuseppe, Fasano, Anna, Marzano, Carlotta Nonnis, Liuzzi, Grazia Maria, Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2014.10.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types.
Gaetano Di Baria, Eugenia Gentilea, Tiziana Latronicoa, Giuseppe Corrierob, Anna Fasanoa, Carlotta Nonnis Marzanob, Grazia Maria Liuzzia* a
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Via
Orabona 4, 70126, Bari, Italy Department of Biology, University of Bari, Via Orabona 4, 70126, Bari, Italy
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b
*Corresponding author:
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Dr. Grazia Maria Liuzzi
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Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari Via Orabona 4, 70126 Bari, Italy.
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Tel: +39 080 5443376; Fax: +39 080 5443317
Abbreviations
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BHK-21/C13, baby hamster kidney cells
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[email protected]
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BSA, bovine serum albumine
CaCo-2, human colon adenocarcinoma cells
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DMEM, Dulbecco’s modified Eagle’s medium FBS, fetal bovine serum
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GFAP, glial fibrillary acidic protein
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MTT, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide PBS, phosphate buffered saline PAS, periodic acid Schiff
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PLL, poly-L-lysine PMA, phorbol 12-myristate 13-acetate RPMI, Roswell Park Memorial Institute RT, room temperature SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis THP-1, human monocytic leukemia cell line
Abstract Marine natural products extracted from sponges represent a new source for drug discovery. Here we describe a simple method for preparing aqueous extracts from 7 Mediterranean demosponges, which allowed the extraction of water-soluble compounds, such as proteins by homogenization of sponge tissue in phosphate buffered saline (PBS). The comparative analysis by SDS-PAGE showed differences in number of bands, bandwidth and intensity among the sponges analysed. The PAS/silver staining revealed a substantial and different glycoprotein assortment among the demosponges studied. To further study the biological activities present in the sponge extracts, we determined the non-
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cytotoxic doses on four different mammalian cell types demonstrating that the optimal noncytotoxic doses were cell type- and extract-dependent.
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In conclusion, the extraction method described in this paper represents a fast and efficient procedure for the extraction of water-soluble proteins from marine sponges. Furthermore, the cell
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viability data suggest the feasibility of this method for the direct in vitro cell-based assays.
We described a simple method for preparing aqueous extracts from demosponges which
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•
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Highlights:
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allows the extraction of protein compounds.
Protein and glycoprotein profiles of sponge extracts were analysed.
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A test of cytotoxicity allowed to determine the optimal non toxic doses of each extract,
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•
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which could be used to study their biological properties.
Keywords: demosponges; aqueous extracts; bioactive compounds; in vitro cytotoxicity; cell
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morphology.
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1. Introduction
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Several studies regarding the chemistry of molecules extracted from marine organisms have fully demonstrated that sponges provide the largest number of biologically active natural compounds (Leal et al., 2012). Over 60% of the potentially useful bioactive compounds discovered so far from living organisms have been obtained from marine fauna, 70% of which comes from sponges (Faulkner, 2002). Indeed, Porifera, due to their typically sessile condition, have evolved the ability to produce potent toxins as defensive tools against predators or competitors (Loh and Pawlik, 2014).There is a worldwide interest in marine natural products since they represent one of the few
de novo sources for drug discovery (Cragg and Newman, 2013). Moreover, it has been demonstrated that the biologically active molecules from marine organisms, possess unique pharmacological properties that are proving to be useful against cancer, AIDS, autoimmune and neurodegenerative diseases (Schmitz et al., 1993; Vinothkumar and Parameswaran, 2013). Purification procedures of these compounds usually include initial extraction with methanol, partitioning of the extract with organic solvents and chromatographic steps (Blunt et al., 2014). However, the majority of the organic solvents are toxic for cells or not well tolerated by some bioassays. Furthermore, these solvents tend to exclude water-soluble natural compounds. In this regard, it has been recently reported that sponges represent a promising resource of water-
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soluble bioactive compounds, suggesting that the therapeutic potential of molecules isolated from sponges cannot be attributed only to the secondary metabolites extracted by organic solvents, but
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also to water-soluble molecules. In this respect, bioactive peptides with anticancer potential (Suarez-Jimenez et al., 2012) and lectines with antimicrobial activities (Schröder et al., 2003) have
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been recently isolated from marine sponges. In addition, other water-soluble molecules such as the chondropsins A and B, two macrolides with anticancer properties, and the 3-alkylpyridinium
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polimers (poly-APS), which present hemolytic and cytotoxic activities, have been extracted from
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the sponges Chondropsis sp. and Haliclona sarai, respectively (Cantrell et al., 2000; Sepčić et al.,
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1997a,b). Despite the fact that the class Demospongiae is known for producing the largest number and diversity of biologically active molecules among marine invertebrates (Leal et al., 2012), the
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bioactive potential of water-soluble compounds, in particular bioactive proteins, has been little studied (Wilkesman & Schroder, 2007).
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On these grounds, in the present study we used a phosphate buffer in order to prepare aqueous extracts from 7 different Mediterranean demosponges. Some of the species chosen had been
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previously subjected to ex situ experimental rearing by this research group (Di Bari et al, in press). The method described in this paper has proved to be effective for the extraction of water-soluble proteins. By using a panel of mammalian cultured adherent cells, we assessed the cytotoxicity of the sponge extracts, which was demonstrated to be cell type-dependent. Taken together our results indicated the feasibility of the proposed method for direct in vitro cell-based assays aimed at the screening of potential bioactivity of water-soluble compounds present in the aqueous extracts.
2. Materials and methods
2.1 Chemicals Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, fetal bovine serum (FBS), penicillin and streptomycin, L-glutamine were obtained from GIBCO (Paisley, Scotland). DNase 1, poly-Llysine (PLL), trypsin, trypan blue, 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA), Schiff's reagent and bovine serum albumine (BSA) were provided by Sigma (St. Louis, MO, USA). Glial fibrillary acidic protein (GFAP) antibodies
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were purchased from Serotec (Oxford, UK).
2.2 Sponge collection
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For our experiments seven different demosponges, commonly found in the Adriatic Sea, were collected. In particular, specimens from Tethya aurantium, T. citrina, Hymeniacidon perlevis,
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Ircinia variabilis, Chondrilla nucula, Aplysina aerophoba and Sarcotragus spinosulus were chosen. Sponge species were collected by scuba diving in Southern Adriatic Sea, Italy, at depths between 1
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and 3 meters. Sponges were individually transferred to laboratory in bags filled with seawater and
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labeled for recognition. During the transport, the samples were protected against contact with air as
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well as other injuries and temperature was maintained around 18ºC. Once in laboratory, all sponges
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were numbered and listed with information like date of sampling and location, weighed and then
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frozen at –80ºC as soon as possible until the extraction.
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2.3 Preparation of aqueous extracts
Figure 1 shows a simplified flow-sheet of the procedure used for the preparation of aqueous
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extracts. Each sponge was homogenized in 10 mM PBS, 150 mM NaCl, pH 7.0 (1:4 w/v). Specimens from T. aurantium and T. citrina were grounded in PBS with mortar and pestle set in an
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iced bath. The other sponges were, instead, homogenized in the same buffer in a waring blender. The aqueous extract from T. aurantium was prepared from the endosomes, which were isolated
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from the entire sponge by removing the cortex under a steroscopic microscope. For the other species the entire sponge was used for the extract preparation. In order to facilitate the disgregation of cells, the homogenates were subjected to three cycles of freezing (20 min, -20°C) and thawing (10 min, RT). To avoid any microbial contamination due to the symbiotic bacteria, penicillin/streptomycin (5x103 U/ml) was added to the homogenate, which was then centrifuged for 60 minutes at 15000 g, 4˚C. The clear supernatant was collected and filtered through a Millipore membrane, pore size 0.22 µm.
Total protein content of each extract was determined according to Bradford method (Bradford, 1976). The protein yield of each extract was found to be approximately 50% compared to the corresponding homogenate. Extracts were then kept at -80°C until further analysis.
2.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) One dimension SDS-PAGE was carried out as described by Laemmli (1970). Sixty micrograms of total proteins were mixed with the sample buffer containing 4% SDS,10% glycerol, 10% βmercaptoethanol, 0.001% bromophenol blue and 0.5 M Tris-HCl, pH 6.8. Samples were heated at 100ºC for 2-3 minutes and then electrophorised on 12% (0.75 mm thickness) running gel overlaid
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with a 4% stacking gel. Electrophoresis was performed at 4°C, 120 V using a mini-Protean II apparatus (Bio-Rad
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Laboratories). The molecular mass of the main proteins in the sponge extracts was estimated by comparison with the standard protein kit of Bio-Rad. The protein bands were visualised by staining
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with 0.1% Coomassie brilliant blue R-250.
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2.5 Periodic acid Schiff (PAS) and subsequent silver staining of glycoproteins
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Detection of glycoproteins was assessed via periodic acid Schiff (PAS) and subsequent silver
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staining (Jay et al., 1990). Briefly, after electrophoresis, the gel were fixed in 25% isopropyl alcohol
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and 10% acetic acid overnight at 4°C and then washed in 7.5% acetic acid for 30 minutes. Gels were treated with 1% periodic acid for 1 h at 4°C and then stained in the Schiff's reagent (4.6 g/l
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pararosaniline, 7.3 g/l of sodium metabisulfite in 0.1 N HCl) until the protein bands turned to
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purple. Subsequently, the gels were further stained with silver nitrate.
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2.6 Preparation of astrocyte primary cultures Astrocytes were prepared from primary cell cultures of neocortical tissues from 1-day old rats as
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described by Latronico et al. (2013). Astrocytes were then purified by three repetitions of replating and trypsinization to deplete cultures of microglia and oligodendrocytes. The purity of the final cell
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culture was assessed by immuno-staining for GFAP. More than 98% of the cells were GFAPpositive in all the preparations. Cells were plated in PLL-coated 96 well-plates at a density of 1x105 cells/ml (100 µl/well) in complete medium (DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS) and maintained at 37°C in a 5% CO2 . 2.7 Culture of CaCo-2 cell line
Human colon adenocarcinoma CaCo-2 (wild type) cells were obtained from American Type Culture Collection (Rockville, MD, USA) at P13. The cells were grown at 37°C, 5% CO2, in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS. Confluent monolayers were subcultured by treatment with 0.25% trypsin and 0.02% EDTA in Ca2+- and Mg2+-free PBS and then plated in 96-well plates at the density of 5x105 cells/ml (100 µl/well) in complete medium and maintained at 37°C in a 5% CO2. 2.8 Culture of THP-1cells and differentiation into macrophages Human monocytic leukemia cell line, THP-1, was purchased from the American Type Culture
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Collection. The cells were maintained in RPMI1640 supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 50 µM mercaptoethanol at 37°C, 5% CO2. To differentiate
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THP-1 into macrophages, monocytes were plated in 96-well plates at the density of 5x105 cells/ml (100 µl/well) and incubated in completemedium in the presence of 100 nM PMA. After 72 h, the
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cells were washed once with serum-free RPMI 1640 and incubated overnight. at 37°C in a 5% CO2.
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2.9 Culture of BHK-21/C13 cells
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Baby hamster kidney (BHK-21/C13) cells were purchased by American Type Culture Collection
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(Rockville, MD, USA). The cells were grown as monolayers in DMEM supplemented with 100
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U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, at 37 °C, 5% CO2.One day before the treatment with the extracts, cells were plated in 96-well plates at the density of 2x105 cells/ml (100 µl/well)
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and incubated at 37ºC in a humidified atmosphere of 5% CO2. 2.10 Treatment of cultured cell types with aqueous extracts
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When the cells reached approximately 80% to 90% of confluency, they were washed twice with serum-free medium and then treated with different concentrations of each aqueous extract from the
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seven demosponges.
For our experiments, the aqueous extracts were employed at the protein concentrations of 1, 5, 10,
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30, 60 and 100 µg/ml. Cells incubated in the same experimental conditions in serum-free medium served as control. The treatment with the aqueous extracts was performed in 100 µl of serum-free medium for 24 h at 37°C, 5% CO2. At the end of the incubation period the medium was collected, centrifuged at 4200 g and culture supernatants were collected and stored at -80°C. Cells were subjected to MTT test for the assessment of cytotoxicity.
2.11 Assessment of cell viability Cytotoxicity of cells, after treatment with the different concentrations of the aqueous extracts from the seven demosponges, was detected by using the MTT assay (Mosmann, 1983). This assay is based on the reduction of MTT by the mitochondrial succinate dehydrogenase in viable cells, to a blue formazan product which can be measured spectrophotometrically using a microplate reader (Versamax-Molecular Devices). Briefly, triplicate samples of 1-5 x 105 cells/ml, plated in 96-well plates in serum-free medium, were treated with the extracts for 24 h. As a control, in other wells, cells were incubated in serumfree medium under the same conditions without the addition of the extracts. After removal of the
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culture medium, cells were rinsed in PBS and incubated at 37°C, 5% CO2 for 2 h with 0.5 mg/ml MTT.
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Reaction was stopped by removing the medium and the dye was dissolved in absolute ethanol. The difference between the absorbance of each sample at 560 and 690 nm was measured and the value
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of the untreated sample was set at 100%. The concentration of the aqueous extract that killed at
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least 60% of cells was considered toxic.
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3. Results
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3.1 Analysis by SDS-PAGE of the protein profile of aqueous extracts Protein profile of each extract was performed using SDS-polyacrylamide gels. Equal quantities of
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proteins (about 60 µg) from each sample were analysed.
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As shown in Figure 2 the comparative electrophoretic banding profile of total protein content yielded different protein bands, ranging from 120 to 15 kDa. The comparative electrophoretic
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analysis of each extract showed differences in number of bands, bandwidth and intensity. In particular, a protein band of about 50 kDa was mostly common to all the samples. With respect
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to I. variabilis (d) and C. nucula (e), a common protein band of about 31 kDa was clearly evident.Two protein bands of approximately 24 and 21 kDa were particularly evident in H. perlevis
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(lane c) while in C. nucula (lane e) a band of 29 kDa, which was absent in other sponges, was the most representative one.
3.2 Detection of glycoproteins in the aqueous extracts In order to assess the presence of glycoproteins in the aqueous extracts, in another set of experiments, after electrophoresis, the SDS-polyacrylamide gels were subjected to the PAS
staining. To this end the samples were electrophorised and horseradish peroxidase and BSA were used as positive and negative control, respectively. As shown in the representative gel in Figure 3B, the PAS staining revealed the presence of various glycoproteins in the extracts. In particular, the comparison between the gels stained with Coomassie brilliant blue (A) and with PAS/silver staining (B) showed that most of the glycoproteins present in the various extracts exibited a molecular weight different from that of the most representative proteins stained with Coomassie brillant blue. In fact, the glycoprotein profile of T. aurantium (c) and T. citrina (d) was mainly characterized by three PAS-positive protein bands of 77, 50 and 30 kDa, while in H. perlevis (e) two glycoproteins of 33 kDa and 19 kDa were clearly evident. The
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electrophoretic glycoprotein pattern of I. variabilis (f) was represented by a PAS-positive protein of about 20 kDa. In C. nucula (g) and A. aerophoba (h) the PAS staining did not reveal the presence of
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any glycosylated proteins, whereas two glycoproteins of 60 and 50 kDa were observed in S.
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spinosulus (i).
3.3 Evaluation of cytotoxicity of aqueous extracts on cultured cell types.
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Figure 4 shows the percentage of viability of the studied mammalian cell populations in response to
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the treatment with the aqueous extracts at the indicated concentrations. The concentrations of the
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aqueous extracts that yielded cell viability values < 60% in comparison to untreated cells (CTRL)
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were considered as toxic. Generally, the cell viability data were cell type- and extract-dependent. In particular, the analysis of the cell viability curves showed that the toxicity of the aqueous extracts
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from T. citrina (A) and H. perlevis (B) were highly variable depending on the cell type studied and
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had proved to be the most cytotoxic in comparison to the other extracts analysed. By contrast, the extract from C. nucula (C) was the less cytotoxic for the studied cells (C).
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The profile of cell viability curves of the different cell types treated with the extracts from T. aurantium (D), A. aerophoba (E) and S. spinosulus (F) showed similar degree of cytotoxicity,
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especially at the highest concentration employed in our experiments (100 µg/ml). The treatment with the extract from I. variabilis (G) was strictly cell type-dependent. In fact, this
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extract did not lead to cell death in BHK-21 cells and primary astrocytes, while in CaCo-2 cells and macrophages THP-1 it displayed citotoxicity at the concentrations of 10 µg/ml. The highest non-cytotoxic concentrations of each extract, calculated in the mammalian cell types studied, are summarized in Table 1.
3.4 Effect of sponge extracts on cell morphology
Figure 5 shows the effect of the highest non-cytotoxic concentrations of the extracts from the studied sponges, on cell morphology. In particular, the treatment of BHK-21 cells with all the extracts determined a change in the morphology of these cells which appeared more fasciculated, with the cell body thinner and spindly, probably due to the reduction in the free spaces between cells, in comparison to CTRL. CaCo-2 cells treated with the highest non-cytotoxic doses of the extracts displayed morphologically heterogeneous features. In particular, cells treated with T. aurantium, T. citrina and C. nucula appeared more vacuolated in comparison to CTRL, without important morphological changes. Moreover, the treatment of cells with the extracts from H. perlevis, I. variabilis, A. aerophoba and
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S. spinosulus determined a decrease in cell volume, with a lost of the polygonal morphology and acquisition of a round-shape morphology. With respect to THP-1 macrophages and primary
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astrocytes, the treatment with the aqueous extracts at the highest non-cytotoxic concentrations did not evoke significant morphological changes in comparison to CTRL. However, in the treated THP-
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1 cells an increase of the number of vacuoles was clearly appreaciable in comparison to CTRL.
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4. Discussion
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Marine sponges are a rich source of biologically active metabolites which have proved to be
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potentially effective agents for the treatment of a wide range of human diseases (Müller et al., 2004). These bioactive compounds have a broad range of pharmacological activities, such as
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antiinflammatory, antitumour, immuno- or neurosurpressive, antiviral, antimalarial and antibiotic
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properties (Sipkema et al., 2005; Suarez-Jimenez et al., 2012). Common purification procedures of such compounds include extraction with toxic polar solvents,
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such as methanol, which limitate direct biological screening on living cell culture systems. Until now, little attention has been paid to the bioactive potential of water-soluble compounds, and in
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particular of proteins. So far the studies performed on the aqueous extracts prepared form marine sponges have regarded either the isolation and characterization of proteases (Oli et al. 2014) or the
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assessment of the biological activities of compounds present in the extracts (Sepčić et al. 1997, 2010).
In this study we have prepared aqueous extracts from seven Mediterranean demosponges by using a phosphate buffer demonstrating that this procedure is efficient for the extraction of water-soluble compounds, namely proteins. The demosponges studied in this paper have been selected by taking into account their well-known richness of bioactive secondary metabolites. As example, three hydroquinones with activity against
leukemia cells have been isolated from Sarcotragus spinosulus (Abed et al., 2011) and fractionation of a crude extract of Aplysina aerophoba yielded aerophobin-2 and isofistularin-3 that can act as chemical defenses against predators (Thoms et al., 2004). The common distribution of the studied demosponges, their easy availability in the South Adriatic Sea (Apulia) and their capability of being farmed (Klöppel, et al., 2008) make them suitable for our investigations. Moreover, some of the studied sponges have been recently subjected to ex situ experimental breeding by this research group (Di Bari et al., in press). It is important to underline that sponge rearing,together with the recombinant DNA technology and peptide synthesis, rapresents a suitable approach to obtain higher amounts of pharmacologically
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active peptides and proteins. In our study we demostrated that the protein pattern of some of the demosponges analysed, such as
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H. perlevis and C. nucula, was remarkably characterized by the presence of high levels of specific protein bands, while in other samples numerous protein bands, which were poorly represented,
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characterized the pattern. Furthermore, the comparative electrophoretic analysis revealed also common proteins, which were mostly present in all the samples analyzed.
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To further characterize the protein content of the aqueous extracts, we evaluated the presence of
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glycosylated proteins by the PAS/silver staining, a high sensitive method for glycoprotein detection
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(Jay et al., 1990). The glycoprotein profile was substantially different in comparison to that
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obtained with Coomassie brilliant blue staining. This difference may be ascribed to the specificity and sensivity of the PAS staining for glycoproteins, which allow to reveal glycoproteins whose
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levels were too low to be detected in the gels stained with Coomassie brilliant blue.
Several biological activities isolated from sponge compounds have been ascribed to glycoproteins,
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and in particular to lectins. In this respect, it has been demonstrated that lectins from marine sponges possess mitogenic, chemotactic, immunobiological, antiviral and cytotoxic properties
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towards different human cells (Schröder et al., 1990; Matsumoto et al., 2012; Fenton et al., 2013). Moreover, a water-soluble glycoprotein of 21 kDa, able to lyse erytrocytes from a variety of
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organisms, has been already isolated from the Mediterranean demosponge T. aurantium (Mangel et al., 1992). In the present work we also carried out a preliminary study to determine the possible cytotoxicity of aqueous extracts from the studied demosponges on a panel of different cultured mammalian cell types. Indeed, screening of aqueous extracts from marine sponges on cells in culture is a common approach to identify compounds of biomedical importance (Beedessee et al., 2012). To study the cytotoxicity, we chose four different cell types which are generally used as in vitro models to study
a wide range of human diseases: macrophages THP-1 (Kuijk et al., 2008), CaCo-2 epithelial cells (Sambuy et al., 2005), BHK-21 fibroblasts (Aljofan et al., 2009) and primary rat astrocytes (Liuzzi et al., 2004a,b). After treatment of the selected cell types with increasing doses of the aqueous extracts, cytotoxicity was quantified by the use of the metabolic reduction of MTT by the mitochondrial enzyme succinate dehydrogenase (Mosmann, 1983). Results obtained yielded significant differences in response to the treatment with the different extracts and allowed to determine the optimal non toxic doses of each extract, which could be used to study their biological properties. The cell types selected for this study showed different sensitivity to the extracts as assessed by
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changes in cell morphology. In particular, CaCo-2 cells and BHK-21 fibroblasts showed the most clear and evident changes in cell morphology after treatment with the highest non-cytotoxic
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concentrations of extracts. These morphogical changes might be due to the presence of bioactive compounds that could be able to affect the organisation of the cytoskeleton and the assembly of
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microtubules (Prado et al., 2004).
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In conclution, these data suggest that some of the screened species produce a variety of water-
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soluble compounds that induce cellular responses. Moreover, the highest non toxic concentrations
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for the selected cell types could represent the basis for further studies on the bioactivities present in
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the aqueous extracts.
Taken together, our results encourage further work to test the bioactive properties of the compounds
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present in the aqueous extracts. Further efforts should go towards the purification and
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characterization of the water-soluble compounds, which could be responsible for bioactivity, and
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the assessment on their potential pharmacological applications.
Aknowledgments. The authors thank Gaetano Devito for his dedicated support in maintaining the
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rat colony for the preparation of astrocyte cultures.
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Legends
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Figure 1. Flowchart of the procedure used for the preparation of the aqueous extracts. Figure 2. Representative SDS-PAGE gel electrophoretic analysis of the protein profiles of aqueous extracts from
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different Mediterranean demosponges. a: Tethya aurantium; b: Tethya citrina; c: Hymeniacidon perlevis; d: Ircinia variabilis; e: Chondrilla nucula; f: Aplysina aerophoba; g: Sarcotragus spinosulus; h: standard proteins. 60 µg of total proteins were analysed by SDS-PAGE on 12% polyacrilamide electrophoretic gels. The most representative protein
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bands of each extract are indicated by the arrows.
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Figure 3. Detection of glycoproteins in the aqueous extracts from different Mediterranean demosponges by PAS/silver staining. a: standard proteins; b: Horseradish peroxidase (positive control); c: Tethya aurantium; d: Tethya citrina; e: Hymeniacidon perlevis; f: Ircinia variabilis; g: Chondrilla nucula; h: Aplysina aerophoba; i: Sarcotragus spinosulus; l: ovine serum albumine (BSA) - negative control. 60 µg of total proteins were analysed by SDS-PAGE on 12% polyacrilamide electrophoretic gels. Gels were stained with either Coomassie brillant blue (A) or PAS/silver staining (B). In B the most representative glycoproteins of each extract are indicated by the arrows. The specificity of the PAS/silver staining toward glycoproteins is demostrated by the lack of BSA band whereas the sensitivity is demostrated by the remarkable staining of the glycoprotein horseradish peroxidase.
Figure.4. Effect of aqueous extracts from Tethya citrina (A), Hymeniacidon perlevis (B), Chondrilla nucula (C), Tethya aurantium (D), Aplysina aerophoba (E), Sarcotragus spinosulus (F) and Ircinia variabilis (G), on cell viability as determined by the MTT assay. The results are expressed as percentage of surviving cells over untreated cells (CTRL). Data are presented as mean ± SD from three different experiments with independent cell populations. The horizontal dashed line, set at 60 %, indicates the threshold of cell viability. Concentrations of the aqueous extracts that yielded cell viability values < 60% of control were considered as toxic doses.
Figure 5. Morphological features of cells treated with the highest no toxic concentrations of the different extracts. Panel of cells observed under phase-contrast microscope after 24 hrs of treatment at 37°C, 5% CO2, (Magnification 50×). Representative photomicrographs from three independent experiments show the morphological
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changes evoked by the different sponge extracts at the highest non cytotoxic concentrations.
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Table 1- Highest non-cytotoxic concentrations (µg/ml) of the aqueous extracts from Mediterranean demosponges on different cell types.
CaCo-2
THP-1
Astrocytes
Tethya aurantium
10
60
A
30
10
Tethya citrina
10
60
10
10
Hymeniacidon perlevis
5
10
5
30
5
5
≥100
≥100
≥100
30
60
Aplysina aerophoba
10
30
30
30
Sarcotragus spinosulus
10
30
10
60
Ircinia variabilis
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≥100
A
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Chondrilla nucula
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BHK-21
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Sponge
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Cell types
Table 1- Highest non-cytotoxic concentrations (µg/ml) of the aqueous extracts from Mediterranean demosponges on different cell types.
Cell types BHK-21
CaCo-2
THP-1
Astrocytes
Tethya aurantium
10
60
30
10
Tethya citrina
10
60
10
10
Hymeniacidon perlevis
5
10
5
30
Ircinia variabilis
≥100
5
5
≥100
Chondrilla nucula
≥100
≥100
30
60
Aplysina aerophoba
10
30
30
30
Sarcotragus spinosulus
10
30
10
60
A
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D
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A
N
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Sponge
