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.

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Safe therapeutics of murine melanoma model using a novel antineoplasic,

the partially methylated mannogalactan from Pleurotus eryngii

S.M.P Biscaiaa, E.R Carbonerob, D.L Bellana, B.S Borgesa, C.R Costaa, G.R Rossia,

J.P Gonçalvesa, 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 do Paraná (UFPR), CEP 81351-980, Curitiba, Paraná, Brazil

b Departamento de Bioquímica, Universidade Federal de Goiás, CEP 75704-020, Catalão, Goiás, Brazil

c Centro de Investigação Translacional em Oncologia (CTO), Instituto do Câncer do 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 do 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

A R T I C L E I N F O

Keywords:

Pleurotus eryngii (“King Oyster”)

Mannogalactan

Chemical structure

Antitumor

Melanoma B16-F10

Non-cytotoxic

A B S T R A C T

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 man-nogalactan 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 byβ-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

1 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

hetero-geneous cell subpopulation Thus, antitumor agents are not effective,

making necessary drugs combination for better efficacy

(Somasundaram, Villanueva, & Herlyn, 2012) Popular treatment

pro-tocols include the use of dacarbazine, and more recently

im-munotherapy 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 microenviron-ment favorable to disease developmicroenviron-ment (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

http://dx.doi.org/10.1016/j.carbpol.2017.08.117

Received 2 May 2017; Received in revised form 22 August 2017; Accepted 27 August 2017

⁎ Corresponding authors.

E-mail addresses: crcfranc@terra.com.br (C.R.C Franco), edstrindad@gmail.com , estrindade@ufpr.br (E.S Trindade).

Available online 06 September 2017

0144-8617/ © 2017 Elsevier Ltd All rights reserved.

MARK

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

Pleur-otus eryngii (Fu, Liu, & Zhang, 2016) was demonstrated by

hetero-polysaccharides mainly composed of glucose (Ma et al., 2014; Ren,

Wang, Guo, Yuan, & Yang, 2016), trough reduction of tumor cell

via-bility in dose-dependent manner Other heteropolysaccharide

com-posed 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

polysaccharide-protein complexes with antitumor activity were described, as reviewed

byZong, Cao, and Wang (2012) Also, antimelanoma activity was

de-monstrated 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 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),

so-dium 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)

2.2 Source of Pleurotus eryngii

Fresh Pleurotus eryngii was obtained from Yuki Cogumelos

Company, located in Araçoiaba da Serra, State of São 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)

2.3 Extraction and purification of polysaccharide

Fresh Pleurotus eryngii fruiting bodies (3.8 kg) were dried by

lyo-philization The yield (630 g) was pulverized and the content

poly-saccharides were water extracted at 10 °C for 6 h (×2, 3000 ml)

Extracts werefiltered and the filtrate was collected, and centrifuged at

9000 rpm at 10 °C for 10 min 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-molecular-weight carbohydrates, giving rise to fraction CW-Pe It was then

dis-solved in H2O and the solution submitted to freezing followed by mild

thawing at 4 °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 Mwcut-off (Spectra/Por®PVDF), giving rise to

retained (RSCW-Pe) and eluted (ESCW-Pe) ESCW-Pe was further

purified by treatment with Fehling solution material (FPCW-Pe) cen-trifuged off (9000 rpm at 20 °C for 10 min) The insoluble Cu2+

com-plex (FPCW-Pe) was neutralized with HOAc, dialyzed against tap water, deionized with mixed ion exchange resins, and freeze-dried Cu2+ so-lution treatment was repeated for the FPCW-Pe, which yielded the

FP2CW-Pe fraction that was nominated as MG-Pe Aflowchart on ex-traction and purification is available as supplementary material (Supplementary 1)

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 per-formed 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, DEPT, TOCSY, and HSQC-NOESY) 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 inδ relative to the internal standard tetramethylsilane (TMS) (δ = 0.0 for13C and1H) 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 chroma-tography (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 equip-ment, 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 1 M TFA for 8 h

at 100 °C, and conversion to alditol acetates (GC–MS) by successive NaBH4and/or reduction, and acetylation with Ac2O-pyridine (1:1, v/v) for 12 h at room temperature (Wolfrom & Thompson, 1963a, 1963b) 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-methy-lated derivatives (1 mg) were hydrolyzed with 45% aqueous formic acid (200μl) 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-me-thylated alditol acetates, which was analyzed by GC–MS

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, 0,25μg/mL penicillin-streptomycin in 0,85% saline, 3.7 g/L sodium bicarbonate at 37 °C in 5% CO2in 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 time-concentration dependent manner and cytotoxicity was determined using MTT (Thyazolyl Blue Tetrazolium Bromide), as described by

Mosmann (1983) Different experimental approaches were employed to

verify cell viability, such as neutral red as described byBorenfreund

and Puerner (1985), trypan blue as described byPhillips (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 min at room temperature,

and reading the absorbance in 570 nm All experiments were compared

to cells in the absence of MG-Pe (control condition)

2.6.3 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 100μg/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%

sa-ponin-PBS) for 30 min 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 byGuimarães

et al (2009), and observed using JEOL JSM- 6360 LV SEM

2.6.4 Invasion assay

B16-F10 cells were pre-treated with 100μg/mL MG-Pe for 72 h

Invasion assay was performed as previously described (Zhao et al.,

2001), with some modifications Briefly, 5 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

superiorfilter surface Inserts were then placed in medium containing

10% FBS and 10μg/mL fibronectin, to create a chemotactic gradient

After 20 h of incubation, cells werefixed with 4% paraformaldehyde,

and stained with 2% toluidine blue for 1 h The superiorfilters 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 min The

absor-bance was measured in 550 nm (Biotek, Epoch Microplate

Spectro-photometer)

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 5 days of tumor growth, the animals (5 per group) received

daily intraperitoneal (IP) injections of 100μL 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

eu-thanized (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

de-scribed byMartins 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

ra-tios 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,

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 amino-transferase – ALT, aspartate aminotransferase – AST, alkaline phos-phatase, cholesterol, triglycerides, creatinine, and urea) These para-meters were detected using a chemistry analyzer (Mindray BS-200), according to the kit manufacturer’s instructions (Kovalent, Reagelabor) 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 eachfigure) Data are reported as mean ± SD, with p < 0.05 considered for statistical significances

3 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 Mwcut-off membrane (Spectra/Por®PVDF) To obtain a purified sample, an ali-quot of the elution fraction (ESCW-Pe, 10 g) was treated with Fehling solution two times sequentially, yielding a Cu+2precipitate (FP2CW-Pe, 3.6 g), that was homogeneous in HPSEC-MALLS analysis (Supplementary 2), a Mw20.9 × 103g 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 (Sup-plementary 5), HSQC-TOCSY (Sup(Sup-plementary 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 atδ 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-O-substitutedα-Galp (A: δ 5.142; B: δ 5.137) and 3-O-Me-α-Galp (C: δ 5.126), 6-O-substituted ofα-Galp (D: δ 4.998; E: δ 4.994) and 3-O-Me-α-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-O-and 2-O-substituted linkages, these being confirmed by NMR spectro-scopy O-substituted C-3 signals for 3-O-Me-Galp units were atδ 81.82 and 81.02, and substituted C-2 of Galp and 3-O-Me-Galp residues were

atδ 79.84 and 79.79, and δ 78.69, respectively (Fig 1A and B;Table 1) LinkedeCH2 groups of the 6-O- and 2,6-di-O-substituted of Galp (δ

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

mono-saccharides, but due to overlapping signals it was not possible to

de-termine all units sequences in this polymer The units ofβ-Manp

(re-sidue G) have an interre(re-sidue 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)

as-signed from HSQC-TOCSY The O-substituted C-2 signals (δ 78.69) from

3-O-Me-Galp units of the main chain (residue C) showed an interresidue correlation with C-1/H-1 atδ 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 byβ-D-Manp single-unit side chains

Fig 1 (A) 13 C NMR or (B) HSQC-DEPT spectrum of mannogalactan (MG-Pe) from P eryngii in D 2 O at 50 °C.

(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

3.2 MG-Pe is non-cytotoxic in in vitro assays 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

MG-Pe and incubation times were used to evaluate in vitro cytotoxicity

Table 1

13 C and 1 H assignments of mannogalactan from P eryngii a

a Assignments are based on 1 H, 13 C, COSY, HSQC-TOCSY, and HSQC-DEPT examination.

b The values of chemical shifts were recorded with reference to TMS as internal standard.

Fig 2 MG-Pe is non-cytotoxic to B16-F10 cells Cells were treated with 1 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.

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

concentrations ranging from 1 up to 250μg/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

Fig 3 MG-Pe does not change B16-F10 cells morphology Cells were treated with 100 μg/mL MG-Pe and morphology was assessed by dif-ferent 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.)

screening results (MTT and NR assays), as we visualized

non-cytotoxi-city at all concentrations, we sought an effect with a lower dosage

possible, thinking of better efficiency with fewer molecules, thus

gen-erating less cost, thus we have chosen to follow the experimentations

with 100μg/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

poly-saccharide 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,

Gonçalves, & 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

fea-tures, such as cell morphology and invasion assays

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

micro-scopy, 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 stressfiber, as well as cells without standard actin

organi-zation Ultra structural analysis in SEM shows cells with no contact

inhibition, stacking up on each other (Fig 3E–H) Altogether,

mor-phological 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

re-ported by others that polysaccharides have reduced cell invasion

ca-pacity, 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

non-cytotoxicity, the next step was to investigate its effects on

melanoma-bearing 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

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 cyto-toxicity 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

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 bio-chemical and hematological testing of experienced animals

Fig 4 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 va-lues are shown as mean ± SD The results are representative of two independent ex-periments with technical triplicates Statistical analysis: Unpaired T-test (*p = 0.0351) Arrow: B16-F10; arrow head: Transwell pores.

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

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 re-duction 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 ani-mals 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 in-hibitor 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

4 Conclusion The structure of the novel antimelanoma compound described here was shown to be a 20.9 × 103g mol−1, partially methylated manno-galactan 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 hemato-logical 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-treat-ment 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 antic-ancer 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 5 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

MG-Pe (50 mg/kg) Treatment was initiated 5 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 5 animals

each group Data was analyzed by T-test (Wilcoxon matched pairs test) two tailed

(**p = 0.0039).

Table 2

Analysis of parameters of biochemical profile and complete blood count.

The table shows the analysis of parameters of biochemical profile and Complete Blood Count (CBC), after testing with C57BL/6 mice (n = 5)

injected with B16F10 cells, and subsequent treatment (day 5–day 14) with MG-Pe (50 mg/kg of animal) Data was analyzed by unpaired

T-test, * p < 0.05.

We thank Brazilian funding agencies CAPES (PROAP and PROCAD

2013), and CNPq forfinancial support, and Sthefany R.F Viana (Yuki

Cogumelos Company, Araçoiaba da Serra, São 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 Dittrich, and MSc Olair Carlos

Beltrame from the UFPR Veterinary Hospital

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the

online version, athttp://dx.doi.org/10.1016/j.carbpol.2017.08.117

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