Fucogalactan from the giant mushroom Macrocybe titans inhibits melanoma cells migration

An aqueous extract containing polysaccharides was obtained from the giant mushroom Macrocybe titans, and it was purified by amylase treatment, freeze-thawing process and dialysis. The purified fraction (ESP) was analyzed by HPSEC and GC–MS which showed a homogenous polysaccharide with Mw 14.2 × 103 g/mol composed by galactose and fucose.

Carbohydrate Polymers 190 (2018) 50–56 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Fucogalactan from the giant mushroom Macrocybe titans inhibits melanoma cells migration T Shayane da Silva Milhorinia, Fhernanda Ribeiro Smiderlea, Stellee Marcela Petris Biscaiab, ⁎ Fabio Rogerio Rosadoc, Edvaldo S Trindadeb, Marcello Iacominia, a Department of Biochemistry and Molecular Biology, Federal University of Parana, CEP 81531-980, Curitiba, PR, Brazil Department of Cellular Biology, Federal University of Parana, CEP 81531-980, Curitiba, PR, Brazil c Department of Biosciences, Federal University of Parana, CEP 85950-000, Palotina, PR, Brazil b A R T I C L E I N F O A B S T R A C T Keywords: Macrocybe titans Fucogalactan Chemical characterization Melanoma B16-F10 An aqueous extract containing polysaccharides was obtained from the giant mushroom Macrocybe titans, and it was purified by amylase treatment, freeze-thawing process and dialysis The purified fraction (ESP) was analyzed by HPSEC and GC–MS which showed a homogenous polysaccharide with Mw 14.2 × 103 g/mol composed by galactose and fucose NMR and methylation analysis of ESP confirmed the presence of a fucogalactan with a (1 → 6)-linked α-D-Galp main chain partially substituted at O-2 by non reducing end units of α-L-Fucp residues in the side chain Its biological activity was evaluated against murine melanoma cells B16-F10 The fucogalactan did not alter the viability, proliferative capacity and morphology of cells However, this polysaccharide was able to reduce the cell migration in vitro at 40% (100 μg/mL) and 33% (250 μg/mL) The results obtained showed that Macrocybe titans fucogalactan is a promising agent capable of altering melanoma cell migration without decrease the cell viability Introduction For millennia, mushrooms have been used in culinary and folk medicine by mankind, not only for their unique taste, but also because of their nutritional value and bioactive compounds (Patel & Goyal, 2012; Ruthes, Smiderle, & Iacomini, 2016) A great variety of bioactive molecules has been isolated from mushrooms, such as proteins (Zhang et al., 2014), glycoproteins (Cui et al., 2013), secondary metabolites (Baby, Johnson, & Govindan, 2015), lipopolysaccharides (Wasser, 2011) and polysaccharides (Moreno et al., 2016; Meng, Cheng, Han, Chen, & Wang, 2017) The latter molecules are potent antitumor and immunomodulating agents isolated from mushrooms, according to studies dated from 1976 until now (Patel & Goyal, 2012; Smiderle, Ruthes, & Iacomini, 2014) These medicinal molecules have been obtained from several mushrooms, such as Agaricus bisporus (Ruthes, Rattmann, Carbonero, Gorin, & Iacomini, 2012), Agaricus brasiliensis (Komura et al., 2010), Macrolepiota dolichaula (Samanta et al., 2015), Lactarius rufus (Ruthes et al., 2012), Flammulina velutipes (Smiderle, Carbonero, Sassaki, Gorin, & Iacomini, 2008), Lentinus edodes (Carbonero et al., 2008), Pleurotus sajor-caju (Silveira et al., 2015), among others However, there are some mushroom species that are unexplored and may have a medicinal potential that is still unknown, ⁎ such as the giant edible mushroom Macrocybe titans (H.E Bigelow & Kimbr.) Pegler, Lodge & Nakasone This species is large and its fruit body is white and firm and grows in large caespitose clusters which may exceed 30 kg of fresh weight (Pegler, Lodge, & Nakasone, 1998) Despite of its peculiar morphology and popular consumption, M titans has never been studied about its polysaccharides and there are no reports about its biological properties Therefore, considering its large size, this mushroom can be a promising source to obtain different bioactive molecules in great yields, including the polysaccharides The biologically active mushroom polysaccharides that were mostly isolated and studied from several Basidiomycetes are the β-D-glucans (Smiderle, Sassaki, Griensven, & Iacomini, 2013) Besides that, there is another class of mushroom polysaccharides that presents more than one monosaccharide, and variable anomeric configuration, linkage and branching type, including the presence of methyl groups in its structure (Ruthes et al., 2016) Among these polysaccharides, there are the heterogalactans, which are mostly composed by a main chain of (1 → 6)linked α-D-Galp units, some of them partially methylated and substituted mainly by fucose or mannose (Rosado et al., 2003; Komura et al., 2010; Ruthes et al., 2013) Heterogactans have been largely explored about their biological activities, such as immunomodulatory (Sun & Liu, 2009; Samanta et al., Corresponding author at: Department of Biochemistry and Molecular Biology, Federal University of Parana, CP 19046, Curitiba, PR, Brazil E-mail address: iacomini@ufpr.br (M Iacomini) https://doi.org/10.1016/j.carbpol.2018.02.063 Received 21 November 2017; Accepted 21 February 2018 Available online 23 February 2018 0144-8617/ © 2018 Elsevier Ltd All rights reserved Carbohydrate Polymers 190 (2018) 50–56 S.d.S Milhorini et al pressure and precipitated with ethanol (3:1; v/v) The precipitated polysaccharides were obtained by centrifugation (10.000 rpm, at °C, for 20 min) and dialyzed against distilled water (6–8 kDa) Dialysis was stopped when the eluted material contained no sugar, which was verified by phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) The polysaccharide fraction (P) was treated with αamylase (Sigma-Aldrich) and precipitated with ethanol (3:1; v/v) The precipitate was dialyzed against tap water (6–8 kDa, for 24 h), concentrated under reduced pressure and submitted to freezing and slow thawing (3×) until complete precipitation of cold-water insoluble polysaccharides (Gorin & Iacomini, 1984) After centrifugation (10.000 rpm, at °C, for 20 min), the soluble material, which contained the cold-water soluble polysaccharides (SP), was purified by dialysis against distilled water through a membrane of 1000 kDa Mw cut-off, giving rise to retained (RSP) and eluted (ESP) fractions, that were concentrated under reduced pressure and freeze-dried The yield of each fraction was calculated in comparison to the initial weight of dried mushroom 2015), anti-inflammatory and antinociceptive (Komura et al., 2010; Silveira et al., 2015) anti-sepsis (Ruthes et al., 2012), anti-oxidative (Ding, Hou, & Hou, 2012; Samanta et al., 2015) and also antitumor activities (Jeff et al., 2013; Wang, Sun, Wu, Yang, & Tan, 2014) The latter one has showed great interest by the scientific and medical community because tumors are one of the main cause of morbidity and death worldwide (Bae, Jang, Yim, & Jin, 2005; Liu, Zeng, Li, & Shi, 2016, Tian, Zhao, Zeng, Zhang, & Zheng, 2016) According to the World Health Organization (WHO/Cancer, 2018), cancer related-deaths were totalized 8.8 millions in 2015, which implies that approximately in deaths in the world is due to cancer Among the cancer types, the skin cancer classified as melanoma is one of the most aggressive with the highest rates of metastasis and mortality (Harries et al., 2016) It is the eighth most common cancer that affects people from developed countries (Hamel et al., 2016) There are some studies that demonstrated activity of mushroom polysaccharides against melanoma cells in vitro (Bae et al., 2005; Han et al., 2006) and antitumor activity of such molecules in vivo (Zhang, Yang, Chen, Hou, & Han, 2005) Based on this, the aim of this study was to obtain polysaccharides from Macrocybe titans fruiting bodies, that could exhibit some activity against melanoma cells Furthermore, the active polysaccharide was purified and its chemical structure was carefully analyzed until complete elucidation 2.3 Analysis of monosaccharide composition by GC–MS The polysaccharide fractions (1 mg) were hydrolyzed with M TFA at 100 °C for h, followed by evaporation to dryness The dried carbohydrate samples were dissolved in distilled water (100 μL) and mg NaBH4 was added The solution was held at room temperature overnight to reduce aldoses into alditols (Sassaki et al., 2008) The product was dried and excess NaBH4 was neutralized by the addition of acetic acid, and removed by the addition of methanol (×2) under a compressed air stream in a fume hood Acetylation of the alditols was performed in pyridine–Ac2O (200 μL; 1:1, v/v), heated for 30 at 100 °C The resulting alditol acetates were analyzed by GC–MS, and identified by their typical retention times and electron impact profiles The relative percentage of correspondent monosaccharides was calculated by determination of each peak area with a Varian CP-3800 gas chromatograph coupled to an Ion-Trap 4000 mass spectrometer, using a VF5 column (30 m × 0.25 mm i.d.) programmed from 100 to 280 °C at 10 °C min−1, with He as carrier gas Material and methods 2.1 Biological material The mushroom Macrocybe titans was kindly donated by Dr Fábio Rogério Rosado from the Department of Bioscience, Federal University of Paraná – Palotina-PR, Brazil 2.2 Extraction and purification procedures The dried and powdered fruiting bodies (300 g) were submitted to several purification steps as shown in Fig Firstly, the apolar compounds were extracted with CHCl3:MeOH (2:1; v/v) at 60 °C during h (3×) Subsequently, the resulting residue was dried and extracted with distilled water at room temperature under mechanical stirring during h (3×) The aqueous extract was concentrated under reduced 2.4 Methylation analysis Per-O-methylation of the purified polysaccharide (ESP; mg) was carried out using NaOH-Me2SO-MeI (Ciucanu & Kerek, 1984) After isolation of the products by neutralization (HOAc), dialysis, and evaporation, the methylation process was repeated The per-O-methylated derivatives were hydrolyzed using 45% aqueous formic acid (1 mL) for h at 100 °C followed by evaporation to dryness The residue was converted into partially O-methylated alditol acetates by reduction with NaBD4 and acetylation with pyridine–Ac2O as describe above (Section 2.3), giving rise to a mixture of partially O-methylated alditol acetates, which was analyzed by GC–MS using a VF5 capillary column as described above (Section 2.3) The derivatives were identified by their m/ z of positive ions and retention time, by comparison with standards, and the results were expressed as relative percentage of each component (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005) 2.5 Determination of homogeneity of polysaccharide fractions and molecular weight (Mw) of fucogalactan The homogeneity of polysaccharide fractions (P, SP and ESP) was determined by high-performance size-exclusion chromatography (HPSEC) coupled to refractive index detector Four gel-permeation Ultrahydrogel columns in series with exclusion sizes of × 106, × 105, × 104, and × 103 Da were used The eluent was 0.1 M aq NaNO2 containing 200 ppm aq NaN3 at 0.6 mL min−1 The sample, previously filtered through a membrane (0.22 μm), was injected (100 μL loop) at a concentration of mg mL−1 Fig Scheme of extraction and purification of M titans fucogalactan 51 Carbohydrate Polymers 190 (2018) 50–56 S.d.S Milhorini et al 2.6 Determination of relative molar mass Table Yields and monosaccharide composition of fractions obtained from M titans The retention time of fucogalactan (ESP) was compared with a curve of dextran patterns of different molecular masses (Sigma-Aldrich) (5.00 × 103; 9.40 × 103; 1.72 × 104; 4.02 × 104; 7.22 × 104; 1.24 × 105; 2.66 × 105; 4.87 × 105; 2.00 × 106), aiming to estimate its relative molecular weight Fraction P SP ESP Yield (%)a 4.1 2.0 0.8 Monosaccharides (%)b Fuc Man Gal Glc 2.7 13.7 17 6.2 7.3 – 30.3 60.2 83 60.8 18.8 – 2.7 Nuclear magnetic resonance (NMR) spectroscopy a b NMR spectra (coupled-HSQC; HSQC-DEPT; COSY; TOCSY) were obtained using a 400 MHz Bruker model Avance III spectrometer with a mm inverse probe The analyses were performed at 70 °C and samples (20 mg) were dissolved in D2O Chemical shifts are expressed in ppm (δ) relative to resonance of acetone at δ 30.2 and 2.22 corresponding to 13 C and 1H signals, respectively Yields relative to fungus dry weight Alditol acetates obtained after hydrolysis, NaBH4 reduction, and acetylation 2.8 Cell culture The cells used for cell culture were B16-F10 (BCRJ – Banco de Células Rio de Janeiro), maintained in Dulbecco’s Modified Eagle’s Medium – DMEM (ThermoFisher, Waltham, MA, EUA, CAT 12800017), supplemented with 10% (v/v) fetal bovine serum (FBS) (ThermoFisher, 12657), 10 mM Hepes (Sigma-Aldrich, H-4034), 0,25 μg/mL penicillin-streptomycin in 0,85% saline (ThermoFisher, 15140-148), 3.7 g/L sodium bicarbonate (ThermoFisher, 25080094) at 37 °C in 5% CO2 in humidified atmosphere Fig Elution profile of P, SP and ESP fraction determined by HPSEC (refractive index detector), eluted with 0.1 M NaNO2 composition (Table 1) and HPSEC (Fig 2) of the samples obtained after precipitation with ethanol (P), freeze-thawing treatment (SP), and elution through dialysis membrane (ESP) Crude polysaccharide extract (P) contained fucose, mannose, galactose and glucose This fraction (P) showed a heterogeneous elution profile on HPSEC (Fig 2) by the presence of two blunt peaks (at ∼43 and ∼53 min), confirming the necessity of further purification steps Fungi stock energy is glycogen and some species may contain high amounts of this polysaccharide, which is easily removed by α-amylase treatment (Synytsya & Novák, 2013) Therefore, this enzymatic hydrolysis was performed followed by freeze and thawing process, giving rise to a soluble polysaccharide fraction (SP), after centrifugation Its elution profile on HPSEC still contained two peaks, although the one at ∼43 was sharpened (Fig 2) and the glucose content reduced drastically (Table 1), indicating that a partial purification occurred Considering the difference of molecular weight observed on SP chromatogram (Fig 2), a dialysis through a membrane of 1000 kDa Mw cutoff was performed as purification step Only fucose and galactose were detected on the eluted fraction (ESP), and its HPSEC profile showed only one sharp peak at ∼53 (Fig 2) The molecular weight of the purified fraction ESP was estimated at 14.2 × 103 g/mol The methylation analysis (Table 2) was consistent with a polysaccharide composed of (1 → 6)-linked Galp main chain, indicated by the presence of 2,3,4-Me3-Galp (56%) derivatives This fraction also presented 3,4-Me2-Galp (21.9%) and 2,3,4-Me3-Fucp (20.7%) derivates, indicating that the main chain is branched at O-2 position by non reducing end units of Fucp in the proportion of each 2–3 units of Galp 2.9 Viability and proliferation assays The B16-F10 cells were cultured in 96-well plates (600 cells/well) After 24 h, the cells were exposed to treatment (100 or 250 μg/mL) for 24, 48 or 72 h, with fucogalactan Afterwards, the cell viability was determined using Neutral Red (Sigma-Aldrich, N6634) as described in Borenfreund & Puerner (1985), and proliferation assay was performed using protocol by Gillies, Didier and Denton (1986), in the same plate (Chiba, Kawakami, & Tohyama, 1998) with modifications All experiments were compared to cells in the absence of treatment 2.10 Morphology and migration assays The morphology and cell migration were determined using a microscope EVOS FL Auto (ThermoFisher) Therefore cells (2.5 × 104) were cultured in 6-well plates, exposed or not (control) to fucogalactan (100 and 250 μg/mL), for 72 h After treatment, images were collected using phase contrast filter for analysis of morphology The scratch was performed with a 10 μL tip, cells were washed times with medium, and finally added medium with 1% SFB The images were collected every 30 min, for 10 h This assay was performed according to the protocol describe by Liang, Park and Guan, (2007) with modifications The images were analyzed using a T-scratch program (free software by CSELab, ETH, Zurich, Switzerland) 2.11 Statistical analysis Table Partially-O-methylalditol acetates formed on methylation analysis of fucogalactan isolated from M titans Statistical analyses was performed using GraphPad Prism 5.0 software (GraphPad Software®, Inc.), being considered different p < 0.05 value, in different parametric tests, as indicated by each legend Results and discussion 3.1 Isolation and structural characterization of fucogalactan from M titans Partially O-methylated alditol acetatesa Sample (mol%)b Linkage typec 2,3,4-Me3-Fucp 2,3,4,6-Me4-Galp 2,3,4-Me3-Galp 3,4-Me2-Galp 20.7 1.4 56.0 21.9 Fucp-(1→ Galp-(1→ 6→)-Galp-(1→ 2,6→)-Galp-(1→ a Analyzed by GC–MS, after methylation, total acid hydrolysis, reduction with NABD4 and acetylation b % of peak area relative to total peak area c Based on derived O-methylalditol acetates The dried and milled mushroom was used to prepare polysaccharide extracts that were purified as described above (Section 2.2) The purification process was accompanied by analyses of monosaccharide 52 Carbohydrate Polymers 190 (2018) 50–56 S.d.S Milhorini et al Fig HSQC-DEPT spectrum of fucogalactan (ESP), in D2O at 70 °C (chemical shifts are expressed in ppm) Correlations of CH2 groups are flipped to negative phase, and are shown in grey color Fig Viability of B16-F10 cells determined by Neutral Red Assay C = control (untreated cells); Treated = 100 or 250 μg/mL of Fucogalactan; Time 24, 48 and 72 h The results are representative of three independent experiments with technical quadruplicate Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Tukey post-test, no difference compared with control group Fig Proliferation of B16-F10 cells determined by Crystal Violet Assay C = control (untreated cells); Treated = 100 or 250 μg/mL of Fucogalactan; Time: 24, 48 and 72 h The results are representative of three independent experiments with technical quadruplicate Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Tukey posttest, no difference compared with control group 53 Carbohydrate Polymers 190 (2018) 50–56 S.d.S Milhorini et al Fig Morphology of B16-F10 cells after incubation with fucogalactan Cells were treated with fucogalactan (100 μg/mL or 250 μg/mL), for 72 h and the treatment did not alter the cell morphology Control cells received no treatment which was cell migration It is known that cell migration is one of the major cellular events and it is considered a hallmark of cancer, because it is through this mechanism that tumor cell can achieve metastasis and thus generate new secondary tumors (Hanahan & Weinberg, 2000, 2011) After incubation of B16-F10 cells with fucogalactan, it was observed that this polysaccharide was able to change the migration behavior (Fig 7) As shown in Fig 7, it can be seen that after 10 h the control cells migrated until closing the scratched area (0% open area) However, the cells treated with both concentrations of fucogalactan (100 or 250 μg/mL) still presented an average open area of 40% and 33%, respectively, after the same period The inhibition of cell migration was statistically different (**** p < 0.0001) when treated groups were compared with control group By the results obtained, it was demonstrated that the fucogalactan extracted from Macrocybe titans does not alter the viability, proliferation or morphology of melanoma cells However, it was able to reduce their migration, showing that this polysaccharide presented some antitumor effect in vitro even with no toxicity against the tested cells Many authors correlate antitumor capacity of some drugs with cytotoxicity or loss of cell viability (Ivanova, Krupodorova, Barshteyn, Artamonova, & Shlyakhovenko, 2014; Shang et al., 2011; Srinivasahan & Durairaj, 2015) However, in this case, there is a great possibility of such drugs to damage or kill normal and healthy cells Thus, recently there is a search for compounds that not alter the cellular viability, but rather cause cellular modifications that lead to antitumor effects, with minimum side effects (Biscaia et al., 2017; Meng, Liang, & Luo, 2016; Novaes, Valadares, Reis, Gonỗalves, & Menezes, 2011; Tong et al., 2009) Based on this, the results presented on this study showed a promising agent against melanoma cells, that is candidate of new studies to evaluate its antitumor effect in vivo The NMR analyses (coupled-HSQC; COSY; TOCSY, data not shown) contributed to elucidate the chemical structure of the fucogalactan and the signals were assigned according to the correlation among the NMR spectra (coupled-HSQC; HSQC-DEPT; COSY; TOCSY) and to literature values of similar fucogalactans isolated from other basidiomycetes (Ruthes et al., 2012; Ruthes et al., 2013) The HSQC-DEPT (Fig 3) showed signals in the anomeric region (C-1/H-1) at δ 101.5/5.07, 98.2/ 4.98 and 98.1/5.03 corresponding to Fucp units, 6-O and 2,6-di-Osubstituted Galp units, respectively The α-configuration of Galp and Fucp units was confirmed by the values of the coupling constants JC-1/H1 13 observed in H/ C coupled-HSQC spectrum (data not shown), which was 171 Hz for Galp and 170,6 Hz for Fucp units (Perlin and Casu, 1969) The substitution at O-2 of 2,6 → )-D-Galp-(1→ was confirmed by signals at δ 77.8/3.84 and 77.8/3.82 Furthermore, the O-6 substitution of all Galp units was identified by the negative phase resonances at δ 66.8/3.71 and 66.8/3.91 The signals at δ 15.6/1.25 and 15.5/1.23 are attributed to CH3 of Fucp units The data showed that the M titans fucogalactan is composed of a (1 → 6)-linked α-D-Galp main chain that is partially substituted at O-2 position by non reducing end units of α-L-Fucp residues 3.2 Biological activity of fucogalactan from M titans The literature data has shown that several polysaccharides present antitumor activities (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer, 2011; Hung, Hsu, Chang, & Chen, 2012; Srinivasahan & Durairaj, 2015; Tong et al., 2009; Zong, Cao, & Wang, 2012), and fucogalactans of mushrooms have already been evaluated for their biological effect, presenting a reduction in late mortality rate caused by polymicrobial sepsis (Ruthes et al., 2012); antinoceptive and anti-inflammatory activities in mice (Komura et al., 2010; Ruthes et al., 2013); and an increase of the expression of TNF-α gene and NO by macrophages (Mizuno et al., 2000) In the present work, the effect of the fucogalactan isolated from M titans on melanoma tumor cells was evaluated The cells were exposed to different concentrations of fucogalactan, for different periods to determine their viability after incubation with the polysaccharide No difference was observed among the treatments and controls (Fig 4) Proliferation of tumor cells is an important target when it is desired to develop antitumor drugs (Ale et al., 2011; Hung et al., 2012) Therefore, the fucogalactan was evaluated on a cell proliferation assay, however no alteration was observed after incubation of B16-F10 cells with different concentrations of fucogalactan (Fig 5) The next step was to assess whether the treatment could affect the morphology of cells Tumor cell morphology is quite characteristic, as seen in Fig (control), showing adherent cells as well as rounded cells The treated groups (100 or 250 μg/mL) presented the same morphology of cancer cells: as spindle-shaped and epithelial-like cells and stacked up on each other (Fig 6) Thus, without changes in cell viability, proliferation and morphology, we then analyzed an important cell malignancy parameter, Conclusion A heteropolysaccharide was obtained from the giant edible mushroom Macrocybe titans by aqueous extraction It was chemically characterized and its biological activity on melanoma cells was evaluated GC–MS, NMR and HPSEC analyzes showed that this polysaccharide is a homogeneous fucogalactan with a (1 → 6)-linked α-D-Galp main chain partially substituted at O-2 by non reducing end units of α-L-Fucp residues in the side chain The activity of Macrocybe titans fucogalactan against B16-F10 cells was firstly reported in this study This polymer showed a promising antitumor effect because it did not affect the cell viability, proliferation and morphology; however it inhibited the migration of melanoma cells Considering that M titans is a mushroom that reaches around 30 kg and therefore could provide high yields of polysaccharides; and that the purified fucogalactan showed potential effect against melanoma cells, the study of this mushroom showed to be interesting and should be continued in vivo 54 Carbohydrate Polymers 190 (2018) 50–56 S.d.S Milhorini et al Fig Cell migration Assay Cells were treated with fucogalactan (100 μg/mL or 250 μg/mL), for 72 h Control cells received no treatment On time h, a scratch was produced on cells and the migration process was recorded during 10 h Fucogalactan altered cell migration in both concentrations Data are shown as mean ± SD, statistical analysis: One-way ANOVA, Dunnett post-test, **** p < 0.0001 compared with control group Acknowledgments Phytochemistry, 114, 66–101 http://dx.doi.org/10.1016/j.phytochem.2015.03.010 Bae, J S., Jang, K H., Yim, H., & Jin, H K (2005) Polysaccharides isolated from Phellinus gilvus inhibit melanoma growth in mice Cancer Letters, 218, 43–52 http://dx.doi 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Ruthes et al., 2013); and an increase of the expression of TNF-α gene and NO by macrophages (Mizuno et al., 2000) In the present work, the effect of the fucogalactan isolated from M titans on melanoma. .. inhibited the migration of melanoma cells Considering that M titans is a mushroom that reaches around 30 kg and therefore could provide high yields of polysaccharides; and that the purified fucogalactan