Safe therapeutics of murine melanoma model using a novel antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii

A heteropolysaccharide was isolated by cold aqueous extraction from edible mushroom Pleurotus eryngii (“King Oyster”) basidiocarps and its biological properties were evaluated. Structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses.

Carbohydrate Polymers 178 (2017) 95–104 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Research Paper Safe therapeutics of murine melanoma model using a novel antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii MARK S.M.P Biscaiaa, E.R Carbonerob, D.L Bellana, B.S Borgesa, C.R Costaa, G.R Rossia, J.P Gonỗalvesa, C.M Meloc, F.A.R Líverod, A.C Ruthese, R Zotzf, E.V Silvab, C.C Oliveiraa, ⁎ ⁎ A Accod, H.B Naderg, R Chammasc, M Iacominih, C.R.C Francoa, , E.S Trindadea, a Departamento de Biologia Celular, Universidade Federal Paraná (UFPR), CEP 81351-980, Curitiba, Paraná, Brazil Departamento de Bioquímica, Universidade Federal de Goiás, CEP 75704-020, Catalóo, Goiỏs, Brazil c Centro de Investigaỗóo Translacional em Oncologia (CTO), Instituto Câncer Estado de São Paulo (ICESP), Faculdade de Medicina, Universidade de São Paulo (USP), CEP 01246903, São Paulo, São Paulo, Brazil d Departamento de Farmacologia, UFPR, CEP 81531-980, Curitiba, Paraná, Brazil e Division of Glycoscience, AlbaNova University Centre, Royal Institute of Technology, 106 91 Stockholm, Sweden f Pontifícia Universidade Católica Paraná, Animal Facility, CEP 80215-901, Curitiba, Paraná, Brazil g Departamento de Bioquímica, Universidade Federal de São Paulo, CEP 04044-020 São Paulo, São Paulo, Brazil h Departamento de Bioquímica e Biologia Molecular, UFPR, CEP 81531-980, Curitiba, Paraná, Brazil b A R T I C L E I N F O A B S T R A C T Keywords: Pleurotus eryngii (“King Oyster”) Mannogalactan Chemical structure Antitumor Melanoma B16-F10 Non-cytotoxic A heteropolysaccharide was isolated by cold aqueous extraction from edible mushroom Pleurotus eryngii (“King Oyster”) basidiocarps and its biological properties were evaluated Structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses A mannogalactan having a main chain of (1 → 6)-linked α-D-galactopyranosyl and 3-O-methyl-α-D-galactopyranosyl residues, both partially substituted at OH-2 by β-D-Manp (MG-Pe) single-unit was found Biological effects of mannogalactan from P eryngii (MG-Pe) were tested against murine melanoma cells MG-Pe was non-cytotoxic, but reduced in vitro melanoma cells invasion Also, 50 mg/kg MG-Pe administration to melanoma-bearing C57BL/6 mice up to 10 days decreased in 60% the tumor volume compared to control Additionally, no changes were observed when biochemical profile, complete blood cells count (CBC), organs, and body weight were analyzed Mg-Pe was shown to be a promising anti-melanoma molecule capable of switching melanoma cells to a non-invasive phenotype with no toxicity to melanoma-bearing mice Introduction Cancer is ranked as the second deadliest disease worldwide, with 8.8 million deaths reported in 2015 Millions of new diagnostics emerge every year, and it is anticipated that this number should increase by 70% over the next 20 years (“WHO | Cancer”, 2016) One third of all diagnosed cancers are skin cancers, being malignant melanoma the most aggressive and of fast development, causing metastases (“WHO | Skin Cancers,” 2016) Melanoma is difficult to treat because it presents several heterogeneous cell subpopulation Thus, antitumor agents are not effective, making necessary drugs combination for better efficacy (Somasundaram, Villanueva, & Herlyn, 2012) Popular treatment protocols include the use of dacarbazine, and more recently immunotherapy with ipilimumab has been adopted (Harries et al., 2016) ⁎ Often, antitumor drugs result in resistant cells populations (Somasundaram, Villanueva, & Herlyn, 2012), and the patients survival rates are still low (American Cancer Society, 2016) Melanoma hallmarks, as well as for other types of cancer, include cell invasion and metastasis (Hanahan & Weinberg, 2011) Both events occur after extracellular matrix alterations, creating a microenvironment favorable to disease development (Theocharis, Skandalis, Gialeli, & Karamanos, 2016) Thus, novel therapeutic approaches that block invasion and metastasis activation, aiming to prolong tumor-free life and reduce metastasis formation are desirable (Moro, Mauch, & Zigrino, 2014) Within this context of tumor microenvironment complex and its molecules, the search for new treatments is essential, as well as the search for new molecules with anti-tumor action Polysaccharides are among the molecules exploited as therapeutic agents, and are Corresponding authors E-mail addresses: crcfranc@terra.com.br (C.R.C Franco), edstrindad@gmail.com, estrindade@ufpr.br (E.S Trindade) http://dx.doi.org/10.1016/j.carbpol.2017.08.117 Received May 2017; Received in revised form 22 August 2017; Accepted 27 August 2017 Available online 06 September 2017 0144-8617/ © 2017 Elsevier Ltd All rights reserved Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al purified by treatment with Fehling solution material (FPCW-Pe) centrifuged off (9000 rpm at 20 °C for 10 min) The insoluble Cu2+ complex (FPCW-Pe) was neutralized with HOAc, dialyzed against tap water, deionized with mixed ion exchange resins, and freeze-dried Cu2+ solution treatment was repeated for the FPCW-Pe, which yielded the FP2CW-Pe fraction that was nominated as MG-Pe A flowchart on extraction and purification is available as supplementary material (Supplementary 1) considered a breakthrough in anticancer therapy in recent years (Patel & Goyal, 2012) Mushroom derived polysaccharides, especially of Pleurotus genus, are of particular interest as antitumor activity has been demonstrated Polysaccharides from Pleurotus eryngii were extracted, purified, and characterized by different methods (Zhang, Zhang, Yang, & Sun, 2013) Antitumor activity of polysaccharides from Pleurotus eryngii (Fu, Liu, & Zhang, 2016) was demonstrated by heteropolysaccharides mainly composed of glucose (Ma et al., 2014; Ren, Wang, Guo, Yuan, & Yang, 2016), trough reduction of tumor cell viability in dose-dependent manner Other heteropolysaccharide composed of mannose, galactose, and glucose, from Pleurotus ostreatus, was demonstrated to inhibit tumor cells without cytotoxicity (Tong et al., 2009) Several others non-toxic polysaccharides and polysaccharideprotein complexes with antitumor activity were described, as reviewed by Zong, Cao, and Wang (2012) Also, antimelanoma activity was demonstrated by Pleurotus ferula ethanol extract, inhibiting cell migration and proliferation (Wang et al., 2014) Thus this study aimed to characterize water soluble polysaccharide extracted from the edible mushroom Pleurotus eryngii, and to evaluate its in vitro and in vivo biological effects on melanoma 2.4 Characterization of the isolated polysaccharide 2.4.1 Gas liquid chromatography–mass spectrometry (GC–MS) Gas liquid chromatography–mass spectrometry (GC–MS) was performed using Agilent 7820A gas chromatograph with Agilent 5975E Ion Trap mass spectrometer, with He as carrier gas A capillary column (30 m × 0.25 mm i.d.) of HP-5 [(5%-Phenyl)-methylpolysiloxane; Agilent J & W] was used for quantitative analysis of alditol acetates and partially O-methylated alditol acetates 2.4.2 NMR spectra NMR spectra (1H, 13C, HSQC-DEPT, HSQC-TOCSY, and HSQCNOESY) were obtained using 500 MHz Bruker Avance spectrometer incorporating Fourier transform Analyses were performed at 50 °C on sample dissolved in D2O Chemical shifts are expressed in δ relative to the internal standard tetramethylsilane (TMS) (δ = 0.0 for 13C and 1H) Materials and methods 2.1 Chemicals and reagents Dulbecco’s Modified Eagle’s Medium – DMEM (12800-017), fetal bovine serum – FBS (12657), penicillin-streptomycin (15140-148), sodium bicarbonate (25080094), trypan blue (15250), and Alexa Fluor™ 546 Phalloidin (A22283) were obtained from ThermoFisher (Waltham, MA, EUA) Hepes (H-4034), thyazolyl blue tetrazolium bromide – MTT (M5655), and neutral red (N6634) were obatined from Sigma-Aldrich (Saint Louis, MO, USA) Matrigel™ matrix (354234) and 7AAD (559763) were obtained from BD Biosciences (Franklin Lakes, NJ, EUA) Paraformaldehyde (15714) and DAPI-Fluoromount-G (17984-24) were obtained from Electron Microscopy Sciences (Hatfield, PA, USA) Ethanol (100983) was obtained from Merck (Darmstadt, DE) Toluidine blue (V000820), and sodium citrate (116) were obtained from Vetec (Duque de Caxias, RJ, BR) 2.4.3 Determination of homogeneity and molar mass (Mw) MG-Pe fraction homogeneity and molar mass (Mw) determination were performed on a Waters high-performance size-exclusion chromatography (HPSEC) apparatus coupled to a differential refractometer (RI) and a Wyatt Technology Dawn-F Multi-Angle Laser Light Scattering detector (MALLS) Waters Ultrahydrogel columns (2000, 500, 250 and 120) were connected in series and coupled with multidetection equipment, using a NaNO2 solution (0.1 M) as eluent, containing 0.5 g/l NaN3 2.4.4 Monosaccharide composition Polysaccharides monosaccharide components were identified and their ratios were determined following hydrolysis with M TFA for h at 100 °C, and conversion to alditol acetates (GC–MS) by successive NaBH4 and/or reduction, and acetylation with Ac2O-pyridine (1:1, v/v) for 12 h at room temperature (Wolfrom & Thompson, 1963a, 1963b) 2.2 Source of Pleurotus eryngii Fresh Pleurotus eryngii was obtained from Yuki Cogumelos Company, located in Araỗoiaba da Serra, State of Sóo Paulo, Brazil A culture of Pleurotus eryngii was deposited at CCIBt: Collection of Algae, Cyanobacteria and Fungi Cultures of the Botany Institute, Botanical Garden of São Paulo (CCIBt voucher 4257) 2.5 Preparation of O-methylated mannogalactan Per-O-methylation of the FP2CW-Pe fraction (10 mg) was carried out using NaOH-Me2SO-MeI (Ciucanu & Kerek, 1984) The per-O-methylated derivatives (1 mg) were hydrolyzed with 45% aqueous formic acid (200 μl) for 14 h at 100 °C, followed by reduction and acetylation as described above (item 2.1.3), to give a mixture of partially O-methylated alditol acetates, which was analyzed by GC–MS 2.3 Extraction and purification of polysaccharide Fresh Pleurotus eryngii fruiting bodies (3.8 kg) were dried by lyophilization The yield (630 g) was pulverized and the content polysaccharides were water extracted at 10 °C for h (×2, 3000 ml) Extracts were filtered and the filtrate was collected, and centrifuged at 9000 rpm at 10 °C for 10 to obtain a clear solution The combined aqueous extracts were evaporated to a small volume, precipitated by addition to excess EtOH (3:1; v/v), and collected by centrifugation under the same centrifugation conditions The resulting polysaccharide precipitates were dissolved in H2O, dialyzed (Spectra/Por®; 12–14 kDa MWCO) against distilled water for 20 h to remove low-molecularweight carbohydrates, giving rise to fraction CW-Pe It was then dissolved in H2O and the solution submitted to freezing followed by mild thawing at °C, cold water-soluble (SCW-Pe) and insoluble fractions (ICW-Pe), which were separated by centrifugation (9000 rpm at 10 °C for 20 min) The soluble portion (SCW-Pe) was dialyzed through a membrane of 1000 kDa Mw cut-off (Spectra/Por® PVDF), giving rise to retained (RSCW-Pe) and eluted (ESCW-Pe) ESCW-Pe was further 2.6 In vitro biological effects 2.6.1 Cell culture B16-F10 murine melanoma cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium – DMEM, supplemented with 10% (v/v) fetal bovine serum (FBS), 10 mM Hepes, 0,25 μg/mL penicillinstreptomycin in 0,85% saline, 3.7 g/L sodium bicarbonate at 37 °C in 5% CO2 in humidified atmosphere No antibiotics were used in the cells culture used to animal inoculation 2.6.2 Cytotoxicity, cell viability, and proliferation assays B16-F10 cells were exposed to the MG-Pe polysaccharide, in a timeconcentration dependent manner and cytotoxicity was determined using MTT (Thyazolyl Blue Tetrazolium Bromide), as described by 96 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al eosinophil, and lymphocytes) was performed Further, plasma was obtained after blood centrifugation at 3000 × g for 10 min; these samples were used to determine biochemical profile (alanine aminotransferase – ALT, aspartate aminotransferase – AST, alkaline phosphatase, cholesterol, triglycerides, creatinine, and urea) These parameters were detected using a chemistry analyzer (Mindray BS-200), according to the kit manufacturer’s instructions (Kovalent, Reagelabor) Mosmann (1983) Different experimental approaches were employed to verify cell viability, such as neutral red as described by Borenfreund and Puerner (1985), trypan blue as described by Phillips (1973), and 7AAD according to the manufacturer’s instructions Proliferation assay was performed using the protocol described by Gillies, Didier, and Denton (1986), with modifications, as follows: the cells were fixed with 1% paraformaldehyde, washed with phosphate buffer saline (PBS), stained with crystal violet 0.25 mg/mL, washed with PBS, eluted with 33% acetic acid in water, incubated for 30 at room temperature, and reading the absorbance in 570 nm All experiments were compared to cells in the absence of MG-Pe (control condition) 2.8 Statistical analysis Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software®, Inc.) Parametric tests such one-way ANOVA; two-tailed ANOVA and unpaired T-test two-tailed were used (details can be found in each figure) Data are reported as mean ± SD, with p < 0.05 considered for statistical significances 2.6.3 Morphological examination with confocal and scanning electron microscopies Confocal and electron microscopies were used to determine cell morphology Cells (1 × 104) cultured in 24-well plates over glass coverslips (Corning), exposed or not for 72 h to 100 μg/mL MG-Pe, were fixed in 2% paraformaldehyde, for 30 min, at 22 °C, washed with PBS, and stained with Alexa Fluor® 546 Phalloidin – (1:500 in 0.01% saponin-PBS) for 30 at 22 °C Coverslips were washed and mounted in DAPI-Fluoromount-G, and examined by A1R MP+ Nikon laser scanning confocal microscope Cells visualization was assessed using differential interference contrast (DIC) Scanning electron microscopy (SEM) was performed according to the protocol described by Guimarães et al (2009), and observed using JEOL JSM- 6360 LV SEM Results and discussion 3.1 Mannogalactan characterization Pleurotus eryngii, also known as king oyster mushroom, was shown to contain 84% moisture on desiccation in a freeze dryer, and the dried material was submitted to aqueous extraction at 10 °C Cold aqueous extract fractionation (CW-Pe, 30.6 g) by freezing/thawing process provided water-soluble (SCW-Pe, 20.8 g) and insoluble (ICW-Pe, 9.8 g) polysaccharidic fractions, which were separated by centrifugation In order to separate a viscous fraction (RSCW-Pe, 3.3 g) the SCW-Pe fraction was submitted to closed dialysis through a 1000 kDa Mw cut-off membrane (Spectra/Por® PVDF) To obtain a purified sample, an aliquot of the elution fraction (ESCW-Pe, 10 g) was treated with Fehling solution two times sequentially, yielding a Cu+2 precipitate (FP2CW-Pe, 3.6 g), that was homogeneous in HPSEC-MALLS analysis (Supplementary 2), a Mw 20.9 × 103 g mol−1 This contained mannose (32.9%), 3-O-methyl-galactose (15.0%) (confirmed by the presence of ions at m/z 130 and 190, after reduction and acetylation), and galactose (52.1%) as monosaccharide components (Supplementary 3), suggesting the presence of a mannogalactan, which was named MG-Pe In order to characterize MG-Pe glycosidic linkages, it was submitted to methylation analysis, which showed a branched heterogalactan due to the presence of 2,3,4,6-Me4Man (29.6%), 2,3,4-Me3Gal (43.5%), and 3,4-Me2Gal (26.9%) (Supplementary 4) NMR analysis [13C- (Fig 1A), HSQC-DEPT (Fig 1B), COSY (Supplementary 5), HSQC-TOCSY (Supplementary 6) and HSQC-NOESY (Supplementary 7)] was also helpful to elucidate MG-Pe structure, since the coupling of protons observed in COSY and HSQC- TOCSY spectra, made possible the assignments of heterogalactan respective carbons using HSQC-DEPT analysis (Fig 1B; Table 1; Supplementary 8), which were confirmed by connectivities observed in HSQC-TOCSY spectrum In addition, HSQC-NOESY experiment was carried out to determine the polymer units sequence The HSQC-DEPT spectrum (Fig 1B), recorded in D2O at 50 °C, showed the presence of mainly eight H1/C1 signals in the anomeric region at δ 5.142/101.14, 5.137/101.46, 5.126/100.92, 4.998/100.83, 4.994/101.04, 4.990/100.68, 4.805/104.35, and 4.780/104.44 Monosaccharides residues were designated as A to H according to their decreasing chemical shift values, which were attributed to 2,6-di-Osubstituted α-Galp (A: δ 5.142; B: δ 5.137) and 3-O-Me-α-Galp (C: δ 5.126), 6-O-substituted of α-Galp (D: δ 4.998; E: δ 4.994) and 3-O-Meα-Galp units (F: δ 4.990), and non-reducing end groups (G: δ 4.805; H: δ 4.780) The above methylation analysis indicated the presence of 3-O, 6-Oand 2-O-substituted linkages, these being confirmed by NMR spectroscopy O-substituted C-3 signals for 3-O-Me-Galp units were at δ 81.82 and 81.02, and substituted C-2 of Galp and 3-O-Me-Galp residues were at δ 79.84 and 79.79, and δ 78.69, respectively (Fig 1A and B; Table 1) Linked eCH2 groups of the 6-O- and 2,6-di-O-substituted of Galp (δ 2.6.4 Invasion assay B16-F10 cells were pre-treated with 100 μg/mL MG-Pe for 72 h Invasion assay was performed as previously described (Zhao et al., 2001), with some modifications Briefly, μg/filter of Matrigel Matrix was allowed to polymerize (incubator, 37 °C) in Transwell inserts and pre-treated cells were seeded in serum-free medium directly into the superior filter surface Inserts were then placed in medium containing 10% FBS and 10 μg/mL fibronectin, to create a chemotactic gradient After 20 h of incubation, cells were fixed with 4% paraformaldehyde, and stained with 2% toluidine blue for h The superior filters surface was washed out to remove non-invading cells Inserts bottom surface were imaged (SONY, DSC-H20) and invading cells were distained by elution with 0.1 M sodium citrate in ethanol for 10 The absorbance was measured in 550 nm (Biotek, Epoch Microplate Spectrophotometer) 2.7 Animal experiment C57BL/6 male mice (8–12 weeks old) were maintained and treated in accordance with ethical principles established by the Experimental Animal Brazilian Council (COBEA) This study was approved by the Ethics Committee on Animal Experimentation of UFPR (certificate #746/2013, process 23075.040348/2013-94) B16-F10 cells (5 × 105) were subcutaneously injected into C57BL/6 mice After days of tumor growth, the animals (5 per group) received daily intraperitoneal (IP) injections of 100 μL PBS (control) or 50 mg/ kg MG-Pe, for 10 days On the 15th experimental day animals were anesthetized (10 mg/kg xylazine and 100 mg/kg ketamine) and euthanized (cervical dislocation after anesthesia) Tumors were daily measured with a digital caliper (FORD), and on the last day tumors were removed Animals were weighted before tumor inoculation and on the last experimental day Body weight difference, before and after fifteen days of treatment, was calculated After anesthesia, animals blood and organs were analyzed as described by Martins et al (2015) Blood was collect from the cava vein with heparinized syringes; and organs (adrenal gland, spleen, kidney and lungs) were collected and weighed The organ-to-body weight ratios were taken into consideration in grams (g) and transformed to relative weight (%) Complete blood count (CBC) (total leukocytes, red blood cells, hemoglobin, hematocrit, platelets, segmented, rods, 97 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al Fig (A) 13 C NMR or (B) HSQC-DEPT spectrum of mannogalactan (MG-Pe) from P eryngii in D2O at 50 °C 3-O-Me-Galp units of the main chain (residue C) showed an interresidue correlation with C-1/H-1 at δ 104.44/4.780 of β-Manp (residue H) The C1/H-1 signal from 6-O- (residues A and B) and 2,6-di-O-Galp units (residues D and E) had interresidue crosspeaks with C-6 linked signal at δ 69.45 (residues D and E) and 69.87 (residues A and B), respectively, which could not be distinguished due to overlapping signals In summary, the results of MG-Pe monosaccharide composition, methylation data, and NMR spectroscopic analysis, showed that it is a branched mannogalactan containing a (1 → 6)-linked main chain, composed of 3-O-Me-α-D-galactopyranosyl and α-D-galactopyranosyl units, partially substituted at O-2 by β-D-Manp single-unit side chains 69.45 and 69.87, respectively) and 3-O-Me-Galp units (δ 69.59 and 70.00, respectively) of the main chain were confirmed by inverted signals in the HSQC-DEPT spectrum (Fig 1B; Table 1) Interresidues correlations observed in the HSQC-NOESY experiment were important to confirm the glycosidic linkages between monosaccharides, but due to overlapping signals it was not possible to determine all units sequences in this polymer The units of β-Manp (residue G) have an interresidue correlation from H-1 (δ 4.805) to C-2 (δ 79.84 and 79.79) of Galp units (residues A and B, respectively) that had signals of C-1/H-1 at δ 101.14/5.142 (A) and 101.46/5.137 (B) assigned from HSQC-TOCSY The O-substituted C-2 signals (δ 78.69) from 98 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al Table 13 C and 1H assignments of mannogalactan from P eryngii.a Units 6a →2,6)-α-D-Galp-(1→ (Residue A) 13 C H C H 13 C H 13 C H 13 C H 13 C H 13 C H 13 C H →2,6)-α-D-Galp-(1→ (Residue B) →2,6)-3-O-Me-α-D-Galp-(1→ (Residue C) →6)-α-D-Galp-(1→ (Residue D) →6)-α-D-Galp-(1→ (Residue E) →6)-3-O-Me-α-D-Galp-(1→ (Residue F) β-D-Manp-(1→ (Residue G) β-D-Manp-(1→ (Residue H) a b 13 101.14 5.142 101.46 5.137 100.92 5.126 100.83 4.998 101.04 4.994 100.68 4.990 104.35 4.805 104.44 4.780 79.84 3.96 79.79 4.00 78.69 4.02 71.22 3.87 71.22 3.82 70.22 3.88 73.24 4.11 73.35 4.09 71.36 3.98 71.36 4.00 81.02 3.68 72.34 3.89 72.27 3.91 81.82 3.55 75.88 3.64 75.77 3.67 72.44 4.02 72.56 4.07 68.83 4.30 72.06 4.10 72.00 4.05 68.36 4.28 69.76 3.64 69.76 3.62 71.76 4.18 71.78 4.14 71.70 4.20 71.70 4.20 71.70 4.20 71.56 4.17 79.05 3.39 79.06 3.39 −O-CH3 69.87 3.72 69.87 3.72 70.00 3.73 69.45 3.69 69.45 3.69 69.59 3.70 64.00 3.77 64.00 3.78 6b 3.89 3.89 3.88 59.5 3.48 3.91 3.92 3.90 59.2 3.46 3.92 3.95 13 Assignments are based on H, C, COSY, HSQC-TOCSY, and HSQC-DEPT examination The values of chemical shifts were recorded with reference to TMS as internal standard 3.2 MG-Pe is non-cytotoxic in in vitro assays (Supplementary 9) Partially methylated mannogalactans are typical heterogalactans of the Pleurotus spp [P pulmonarius (Smiderle et al., 2008), P ostreatus (Jakovljevic, Miljkovic-Stojanovic, Radulovic, & Hranisavljevic-Jakovljevic, 1998), P ostreatoroseus and P ostreatus var florida (Rosado et al., 2003),with differences in the degree of substitution and the levels of methyl groups The present study also purposed to evaluate possible polysaccharide effects on malignancy parameters, using a safe concentration (neither cytotoxic nor lethal) After purification, different concentrations of MGPe and incubation times were used to evaluate in vitro cytotoxicity Fig MG-Pe is non-cytotoxic to B16-F10 cells Cells were treated with up to 250 μg/mL MG-Pe and different techniques were used to determine cell damage: (A) MTT; (B) Neutral Red, (C) Tripan Blue, (D) 7AAD assays; or cell proliferation by crystal violet (E) C = control, untreated cells; Treated = 1, 10, 50, 100, and 250 μg/mL of MG-Pe The results are representative of three independent experiments with technical quintuplicate Data are shown as mean ± SD, statistical analysis: ANOVA One-way, Tukey post-test, p > 0.05 compared with control group 99 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al Fig MG-Pe does not change B16-F10 cells morphology Cells were treated with 100 μg/mL MG-Pe and morphology was assessed by different techniques: (A, B) DIC; (C and D) confocal microscopy; (E–H) SEM Cytoskeleton is visualized in red and nuclei in blue in C and D (left panel: control; right panel: treated) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) concentrations ranging from up to 250 μg/mL were tested Mitochondria functionality was found to be preserved, as detected by MTT method (Fig 2A) Furthermore, MG-Pe did not induce loss of cell viability, as shown by NR method (Fig 2B) Based on these initial against B16-F10 cells Data from literature suggest polysaccharides antitumoral activity at variable concentrations (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer, 2011; Hung, Hsu, Chang, & Chen, 2012), thus 100 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al studies that showed tumor reducing effects using a range of 20 up to 80 mg/kg of polysaccharides, IP, for 10–13 days, with daily or alternate days of treatment (Abu et al., 2015; Borchers, Keen, & Gershwin, 2004; Hou et al., 2013) Our results showed that melanoma-bearing mice, treated daily for 10 days with 50 mg/kg MG-Pe, presented tumors 60% smaller (volume) (**p = 0.0039) than tumors of control group (Fig 5A and B) This amazing finding of tumor growth impairment, together with the decreased capacity of cell invasiveness and the lack of cytotoxicity may suggest that MG-Pe could be a promising therapeutic agent Thus the last step was to evaluate mice physiological parameters, in order to verify possible in vivo MG-Pe toxic effects screening results (MTT and NR assays), as we visualized non-cytotoxicity at all concentrations, we sought an effect with a lower dosage possible, thinking of better efficiency with fewer molecules, thus generating less cost, thus we have chosen to follow the experimentations with 100 μg/mL Trypan blue exclusion dye (Fig 2C) and 7AAD assay (Fig 2D) confirmed no cell membrane damage after MG-Pe cells treatment Also, cell proliferation was not affected by treatment (Fig 2E) Polysaccharides antitumor activity was reported (Zong et al., 2012), and correlated not only to tumor cells proliferation decrease (Ale et al., 2011; Hung et al., 2012) but also to tumor cells viability decrease (Shang et al., 2011), and consequently increased cytotoxicity (Ivanova, Krupodorova, Barshteyn, Artamonova, & Shlyakhovenko, 2014; Srinivasahan & Durairaj, 2015) On the other hand, a recently common sense has emerged Antitumor activity would be more effective if the medication used is non-cytotoxic, as cytotoxicity affects both normal and tumor cells Indirect effects against tumor cells, as seen by the polysaccharide from Pleurotus ostreatus composed of mannose, galactose, and glucose (Tong et al., 2009) that modulate the host immune system (Meng, Liang, & Luo, 2016), thus reducing side effects (Novaes, Valadares, Reis, Gonỗalves, & da Cunha Menezes, 2011) is highly desirable These polysaccharides are promising molecules to treat cancer, and in some cases they are already being used as adjuvants in surgery, chemotherapy, and radiotherapy, demonstrating to soften side effects and therefore enhancing patient life quality (Wasser, 2014) Based on these and on the evidences that MG-Pe is non-cytotoxic in vitro, we next sought to investigate MG-Pe effects on malignant melanoma cells features, such as cell morphology and invasion assays 3.6 MG-Pe does not modify mice physiological parameters Given the fact that MG-Pe did not present in vitro cytotoxicity we have also investigated mice physiological parameters, through biochemical and hematological testing of experienced animals 3.3 B16-F10 morphology is maintained after MG-Pe treatment The next step was to evaluate if the polysaccharide could cause morphological changes in B16-F10 cells Using DIC by optical microscopy, it was observed that these cells, even after treatment, maintained its morphological characteristics (spindle-shaped and epithelial-like cells) (Fig 3A and B) Cytoskeleton organization of control or treated cells (Fig 3C and D) confirms these findings, as it evidences cells with organized stress fiber, as well as cells without standard actin organization Ultra structural analysis in SEM shows cells with no contact inhibition, stacking up on each other (Fig 3E–H) Altogether, morphological analysis confirms no MG-Pe cytotoxic effects 3.4 MG-Pe decreases B16-F10 cells invasion on matrigel Cell invasion is a key event for tumor progression and metastasis initiation (Hanahan & Weinberg, 2011) Thus we sought to investigate if MG-Pe could interfere with this parameter, by investigating in vitro invasive cells capacity after treatment In this work, a significant reduction in invasion was evident after treatment (Fig 4A and B) Absorbance analysis revealed an inhibition of 42% compared to control group (*p = 0.0351) (Fig 4C) It was reported by others that polysaccharides have reduced cell invasion capacity, including of melanoma cells (Lee, Lee, Kim, Song, & Hong, 2014; Zhang et al., 2009) In addition, excellent results (tumor reduction) were obtained when polysaccharides where administered in vivo (Abu et al., 2015; Niu, Liu, Zhao, & Cao, 2009) 3.5 MG-Pe has in vivo antimelanoma action Facing promising results like reduction of cell invasion and noncytotoxicity, the next step was to investigate its effects on melanomabearing mice Several polysaccharides from mushrooms were described to possess antitumor activity (Ivanova et al., 2014; Meng et al., 2016; Novaes et al., 2011; Ren, Perera, & Hemar, 2012; Tong et al., 2009; Wasser, 2014; Zhang et al., 2009; Zong et al., 2012) We chose daily doses of 50 mg/kg for 10 days based on other in vivo Fig MG-Pe decrease cell invasion on matrigel Transwell inserts bottom surface of (A) control and (B) pre-treated for 72 h with 100 μg/mL MG-Pe groups (C) Absorbance values are shown as mean ± SD The results are representative of two independent experiments with technical triplicates Statistical analysis: Unpaired T-test (*p = 0.0351) Arrow: B16-F10; arrow head: Transwell pores 101 Carbohydrate Polymers 178 (2017) 95–104 S.M.P Biscaia et al within the reference levels reported for healthy mice (41.97–60.02 mg/ dL) (Almeida, Faleiros, Teixeira, Cota, & Chica, 2008) The apparent hyperesplenism induction by treatment deserves further investigation Cancer cachexia syndrome is often observed in tumor patients This progressive and untreatable weight loss, mainly by muscle mass reduction is responsible for 20–30% of cancer deaths (He et al., 2013; NIH, 2017) Here, neither animal organs nor body weight were affected by treatment (Fig 6A and B) It is worth noting that control animals weight was inferior to MG-Pe treated animals Whereas untreated animals developed this common disease feature of weight loss, treated animals life quality was maintained Other characteristics such as slower motility, piloerection, and eyes partially closed throughout the experiment, were observed in control animals but not in the treated animals (data not shown) Side effects, such as diarrhea, were not observed in either group Thus the treatment with MG-Pe showed decreased tumor progression with no indication of acute systemic toxicity Taken together the results clearly show that MG-Pe is a potent inhibitor of in vivo melanoma growth, without systemic toxicity Also, tumor metastases are probably less likely to occur as MG-Pe decreased in vitro invasive cells capacity Conclusion The structure of the novel antimelanoma compound described here was shown to be a 20.9 × 103 g mol−1, partially methylated mannogalactan obtained from Pleurotus eryngii (King Oyster) Biological effects on melanoma cells and melanoma-bearing mice were described In vitro cytotoxicity as well as alteration of animal biochemical and hematological parameters on an acute evaluation were not observed Changes in B16-F10 in vitro invasive phenotype and tumor growth impairment on melanoma-bearing mice were found after MG-Pe-treatment Importantly, we have demonstrated MG-Pe antitumor action without cytotoxicity or animals physiological parameters alterations Further analyses are necessary in order to unravel MG-Pe mechanism of action responsible for the biological effects shown here Novel anticancer molecules that promote tumor suppression with diminished or no side effects to patients are the targets for developing new therapies In this context, MG-Pe could be a good candidate Fig Impairment of tumor size progression after treatment with MG-Pe (A) Solid tumor growth after B16-F10 cells injection in C57BL/6 mice and subsequent treatment with MGPe (50 mg/kg) Treatment was initiated days post-inoculation and was repeated daily for 10 days (B) Images representative of tumor size from control (A) and treated (B) groups The results are representative of three independent experiments with animals each group Data was analyzed by T-test (Wilcoxon matched pairs test) two tailed (**p = 0.0039) No significant differences between treated and control groups were found for most parameters tested, except for a decrease in urea levels in the treated group (Table 2) However, in both groups the urea was Table Analysis of parameters of biochemical profile and complete blood count Biochemical profile −1 ALT (U L ) AST (U L−1) Alkaline phosphatase (U L−1) Creatinine (mg dL−1) Urea (mg dL−1) Cholesterol (mg dL−1) Triglycerides (mg dL−1) Control 35.53 155.4 79.33 0.233 60.87 72.23 56.05 ± ± ± ± ± ± ± Complete Blood Count (CBC) Control White blood cells (WBC) (×103/uL) Red blood cells (RBC) (×106/uL) Hemoglobin (HGB) (g/dL) Hematocrit (HCT) (%) Plates (PLT) (×103/uL) Segmented (%) Band cells (%) Lymphocytes (%) Eosinophil (%) Monocytes (%) 5.525 6.883 10.65 29.70 312.5 32.25 1.500 63.00 1.500 2.250 2.924 31.67 9.034 0.03333 4.656 9.773 7.194 ± ± ± ± ± ± ± ± ± ± Treated p value 29.00 ± 2.806 164.8 ± 21.03 69.44 ± 2.887 0.2400 ± 0.0400 48.18 ± 1.718 62.52 ± 3.432 70.86 ± 5.225 0.1802 0.8067 0.2428 0.9132 0.0213* 0.3374 0.1310 Treated 1.095 1.425 1.030 6.029 61.03 11.27 0.2887 11.05 0.5000 0.2500 5.720 7.114 9.660 29.08 355.4 31.20 1.400 63.20 1.600 2.200 ± ± ± ± ± ± ± ± ± ± p value 0.6053 0.2717 0.3906 1.140 45.56 4.964 0.2449 4.862 0.4000 0.4899 0.8736 0.8625 0.3584 0.9126 0.5825 0.9291 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human breast cancer cells Oncology Reports, 25, 267–272 http://dx.doi.org/10.3892/or Smiderle, F R., Olsen, L M., Carbonero, E R., Marcon, R., Baggio, C H., Freitas, C S., Iacomini, M (2008) A 3-O-methylated mannogalactan from Pleurotus pulmonarius: Fig (A) Relative organs weight (ratio organ-to-body weight) and (B) animals body weight (difference before and after the treatment) C57BL/6 mice were injected with B16F10 cells and subsequently treated for 10 days with either 50 mg/kg MG-Pe (Treated) or vehicle (Control) Data were analyzed by unpaired T-test for each organ No statistical significances were found Acknowledgments We thank Brazilian funding agencies CAPES (PROAP and PROCAD 2013), and CNPq for financial support, and Sthefany R.F Viana (Yuki Cogumelos Company, Araỗoiaba da Serra, Sóo Paulo, Brasil) for Pleurotus eryngii basidiocarps donation We also thank the UFPR Electron Microscopy Center; the Multi-User Confocal Microscopy Center of UFPR; Prof Dra Rosangela Locatelli 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Pleurotus eryngii was obtained from Yuki Cogumelos Company, located in Araỗoiaba da Serra, State of Sóo Paulo, Brazil A culture of Pleurotus eryngii was deposited at CCIBt: Collection of Algae,... Cyanobacteria and Fungi Cultures of the Botany Institute, Botanical Garden of São Paulo (CCIBt voucher 4257) 2.5 Preparation of O -methylated mannogalactan Per-O-methylation of the FP2CW-Pe fraction... biochemical profile (alanine aminotransferase – ALT, aspartate aminotransferase – AST, alkaline phosphatase, cholesterol, triglycerides, creatinine, and urea) These parameters were detected using a chemistry