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

Carbohydrate Polymers 178 (2017) 95–104

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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 do Paraná (UFPR), CEP 81351-980, Curitiba, Paraná, Brazil
Departamento de Bioquímica, Universidade Federal de Goiỏs, CEP 75704-020, Catalóo, Goiỏs, Brazil
c
Centro de Investigaỗóo 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
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.

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 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: (C.R.C. Franco), , (E.S. Trindade).

/>Received 2 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).

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), 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 1 M TFA for 8 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 6 h (×2, 3000 ml).
Extracts were filtered 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-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 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 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
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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 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).

2.8. 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 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 by Guimarães
et al. (2009), and observed using JEOL JSM- 6360 LV SEM.

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 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, 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
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 1 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 min. 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 5 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,
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S.M.P. Biscaia et al.

Fig. 1. (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
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S.M.P. Biscaia et al.

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

1

2

3

4

5

6a
→2,6)-α-D-Galp-(1→ (Residue A)

13

C
H
C
1
H
13
C
1
H
13

C
1
H
13
C
1
H
13
C
1
H
13
C
1
H
13
C
1
H
1

→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

1

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

6

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. 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.

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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 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 1 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
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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 eects (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. 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 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.

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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.
4. 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. 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 MGPe (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).

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 2
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
0.7980
0.9862
0.8786
0.9356

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 Ttest, * p < 0.05.

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Fig. 6. (A) Relative organs weight (ratio organ-to-body weight) and (B) animals body
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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 Dittrich, and MSc. Olair Carlos
Beltrame from the UFPR Veterinary Hospital.
Appendix A. Supplementary data
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