Green does not always mean go: A sulfated galactan from Codium isthmocladum green seaweed reduc

Carbohydrate Polymers 250 (2020) 116869

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Green does not always mean go: A sulfated galactan from Codium
isthmocladum green seaweed reduces melanoma metastasis through direct
regulation of malignancy features

T

D.L. Bellana, S.M.P. Biscaiaa, G.R. Rossia, A.M. Cristala, J.P. Gonỗalvesa, C.C. Oliveiraa,
F.F. Simasa, D.A. Sabryb, H.A.O. Rochab, C.R.C. Francoa, R. Chammasc, R.J. Gilliesd,
E.S. Trindadea,*
a

Cell Biology Department, Universidade Federal do Paraná, Curitiba, Paraná, Brazil
Biochemistry Department, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil
Center for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo, São Paulo, São Paulo, Brazil
d
Cancer Physiology Department, Moffitt Cancer Center, Tampa, FL, USA
b
c

A R T I C LE I N FO

A B S T R A C T

Keywords:

Cancer
Melanoma
Galactan
Seaweed
Polysaccharide

Melanoma is the most lethal form of skin cancer, with a worldwide increase in incidence. Despite the increased
overall survival of metastatic melanoma patients given recent advances in targeted and immunotherapy, it still
has a poor prognosis and available treatment options carry diverse severe side effects. Polysaccharides from
seaweed have been shown to exert antitumor activities. Here we show in vitro and in vivo antitumor activities of a
sulfated homogalactan (named 3G4S) from Codium isthmocladum seaweed in the B16-F10 murine melanoma cell
line. 3G4S did not induce cytotoxicity or proliferation changes; however, it was able to reduce solid tumor
growth and metastasis, while not inducing side effects in mice. B16-F10 cells traits related to the metastatic
cascade were also impaired by 3G4S, reducing cell invasion, colony-forming capacity and membrane glycoconjugates. Therefore, 3G4S shows promising antitumor activities without the commonly associated drawbacks
of cancer treatments and can be further explored.

1. Introduction
Cancer, comprising at least 200 distinct diseases, is one of the
leading causes of mortality worldwide with more than 9.6 million
deaths in 2018 (Bray et al., 2018). The shadows of a terminal illness are
cast when malignant cells from the primary tumor are able to complete
the multi-step process known as metastasis, which is responsible for
more than 90% of cancer related deaths (Lambert, Pattabiraman, &
Weinberg, 2016).
Because of its high metastatic capacity, melanoma is the most lethal
form of skin cancer when diagnosed at later stages, being one of the few
types of cancer with an increasing incidence rate over the last decades
(Ward & Farma, 2017). Melanoma’s high metastatic capacity arises
from its high mutation rate and epigenetic alterations, resulting in
protein expression and glycosylation patterns associated with migratory

and invasive traits, rapidly enabling the metastatic process over its
progression (Moran, Silva, Perry, & Gallagher, 2017). Recent advances
in metastatic melanoma treatment increased patient’s overall survival,

⁎

thanks to the development of targeted therapies such as BRAF-mutant
inhibitors (vemurafenib and drabafenib) and immunotherapies such as
the immune checkpoints inhibitors anti-CTLA-4 (ipilimumab) and antiPD-1 (nivolumab), overcoming the limited benefits of chemotherapies
such as dacarbazine (Domingues, Lopes, Soares, & Populo, 2018).
Despite the improvement in treatment, metastatic melanoma still
poses as a major clinical challenge. Targeted and immunotherapies are
still limited in their efficacy and application to different patients given
the heterogeneity of the tumor genetic landscape and the development
of resistance, while patient’s quality of life is severely compromised by
side effects induced by the available treatments (Kroschinsky et al.,
2017; Melis, Rogiers, Bechter, & van den Oord, 2017). These obstacles
engender the search for anti-tumor and specifically anti-metastatic
compounds that can provide new treatment strategies with reduced
toxicity.
Polysaccharides have shown to induce selective cytotoxicity besides
promoting cell cycle arrest, affect cell migration and invasion and reduce solid tumors and metastatic progression (Khan, Date, Chawda, &

Corresponding author.
E-mail address: (E.S. Trindade).

/>Received 5 May 2020; Received in revised form 10 July 2020; Accepted 30 July 2020
Available online 13 August 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 250 (2020) 116869

D.L. Bellan, et al.

Patel, 2019), reaching even clinical trials (Zhang et al., 2018). Seaweeds are an important font of sulfated polysaccharides, being galactans one of them (Jiao, Yu, Zhang, & Ewart, 2011; Manlusoc et al.,
2019).
Sulfated galactans are mainly found in red seaweeds, being also
present in minor amounts in green seaweed, especially in Codium sp
(Pomin & Mourão, 2008). They are composed of α-L- and/or α-D- or βD-Galp units and usually have a molecular mass higher than 100 KDa
(Pomin, 2010).
Codium sp seaweeds have a wide global distribution and there are
already described methods of their artificial cultivation (de OliveiraCarvalho, Oliveira, Pereira, & Verbruggen, 2012; Hwang, Baek, & Park,
2008). Interesting biological activities from polysaccharides obtained
from Codium species are already reported, such as anticoagulant (Li
et al., 2015), immunostimulant (Lee, Ohta, Hayashi, & Hayashi, 2010)
and liver-kidney protection against induced obesity (Kolsi et al., 2017).
The green seaweed Codium isthmocladum biosynthesize a highly
sulfated homogalactan composed mainly of β-D-Galp 3-O-linked and 4O-sulfated units (named here as 3G4S, and SG1 in the original description paper), with Mw of 14 KDa (Farias et al., 2008). This galactanrich fraction has antioxidant and anticoagulant activity, as well as antiproliferative capacity when exposed to HeLa cell line (Costa et al.,
2010), but none is known of its activity against melanoma.
Based on the relevance of sulfated polysaccharides with anti-cancer
activities and the promising structure features and origin of 3G4S our
hypothesis is that 3G4S acts directly on cancer cells modulating traits
related to cancer progression. Hence, the aim of this study was to test
the anti-tumor and anti-metastatic activities of this compound against
the highly metastatic B16-F10 cell line.

12657029, Waltham, Massachusetts, USA) and sterilized in 0.22 μm
membranes (Millipore, Cat. SLGV033RS, Kenilworth, New Jersey,
USA), and DMEM without FBS was used as control. For in vivo experiments 3G4S was dissolved in phosphate buffer saline (PBS), and PBS

alone was used as control.

2. Materials and methods

2.4. Cell morphology

2.1. Purification, characterization and preparation of C. isthmocladum
polysaccharide

B16-F10 cells were treated with 100 μg/mL 3G4S for 72 h and then
cell morphology was analyzed by confocal and scanning electronic
microscopy (SEM). Cytoskeleton was labeled with ActinGreen
ReadyProbes (Invitrogen, Cat.R37110, Waltham, Massachusetts, USA)
and cells nuclei with DAPI (Invitrogen, Cat.D1306, Waltham,
Massachusetts, USA), and imaged with A1R MP + confocal microscope
(Nikon Instruments Inc, Tokyo, Japan). Cells were fixed in Karnovsky
solution (glutaraldehyde 2 %, paraformaldehyde 4 %, CaCl2 1 mM in
sodium cacodylate buffer 0.1 M), washed and post-fixed in 1 % osmium
tetroxide (in sodium cacodylate buffer 0.1 M) for 1 h, and then dehydrated using increasing ethanol concentrations. Samples were dried to
critical point and metallized using gold. Images were acquired by a
JEOL JSM 6360 –LV (Tokyo, Japan) SEM microscope.

2.2. Cell lines
B16-F10 murine melanoma cell line, obtained from Banco de Células
do Rio de Janeiro (Rio de Janeiro, Brazil), was cultivated in DMEM,
supplemented with 10 % FBS, 0.25 μg/mL of penicillin/streptomycin
(Thermo Fisher, Cat. 15140122, Waltham, Massachusetts, USA) and
1.57 g/L sodium bicarbonate (Merck, Cat. 36486, Kenilworth, New
Jersey, USA). Luciferase expressing B16-F10-luc-G5 murine melanoma
cell line was purchased from Caliper LifeSciences (Hopkinton, USA).

Cells were cultivated in DMEM/F-12 (Thermo Fisher, Cat. 11320033,
Waltham, Massachusetts, USA), supplemented with 5 % FBS and 0.25
μg/mL of penicillin/streptomycin.
2.3. Cytotoxicity and proliferation assays
B16-F10 cells (500 cells/well) were exposed to 10, 100 or 1000 μg/
mL of 3G4S for 72 h. Cytotoxicity and proliferation were measured
using MTT (Mosmann, 1983) and Crystal Violet (Gillies, Didier, &
Denton, 1986) assays, respectively. Apoptosis and cell cycle analyses
were performed after 72 h treatment with 100 μg/mL 3G4S, using FITC
Annexin V Apoptosis Detection Kit (BD Biosciences, Cat. 556447,
Franklin Lakes, New Jersey, USA) and BD PI/RNAse kit (BD Biosciences, Cat. 550825, San Jose, California, USA), respectively.

Specimens of C. isthmocladum (Vickers) were collected from
Pirambúzios beach, (Rio Grande do Norte, Brazil - 5°59′01″S/
35°07'20"W) with agreement of the Brazilian National System of
Management of Genetic Heritage and Associated Traditional
Knowledge (SISGEN; protocols A8C31A3 and A72AD2B). The seaweed
was identified according to its morphology (Wynne, 1986) and a voucher specimen was deposited in the Herbarium of the Biosciences Institute, Universidade Federal do Rio Grande do Norte (UFRN; registration code UFRN25933).
3G4S was isolated and purified as previously described (Farias et al.,
2008) (Supplementary material). 3G4S molecular weight was estimated
by reference to a calibration curve made by dextran sulfate standards
(10, 40, 70, 147 and 500 KDa) (Sigma-Aldrich®, Cat. 75027, St. Louis,
Missouri, USA).
Monosaccharide composition was analyzed after total acid hydrolysis (4 M HCl, 100 °C, 6 h) using a LaChrom Elite® HPLC system (VWRHitachi, Radnor, Pennsylvania, USA) coupled to a LichroCART® 250-4
column (250 mm × 40 mm) (Merck, Cat. MC1508330001, Kenilworth,
New Jersey, USA) packed with Lichrospher® 100 NH2 (Merck,
Kenilworth, New Jersey, USA) and equipped with a refractive index
detector (L-2490) (VWR-Hitachi, Radnor, Pennsylvania, USA). A 2DNMR heteronuclear (1H–13C) HSQCed (Edited Heteronuclear Single
Quantum Coherence) spectrum was obtained using Bruker Avance III
Ascend 600 MHz (14.1 T) spectrometer (Billerica, Massachusetts, USA)

equipped with a 5 mm inverse probe. The chemical shift of 1H and 13C
were expressed in δ (ppm) relative to TMSP (trimethylilsilylpropionate)
(Cambridge Isotope Laboratories, Tewksbury, Massachusetts, USA) as
an internal standard (δ =0 ppm).
For in vitro experiments 3G4S was dissolved in Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco, Cat. 12800-017, Waltham,
Massachusetts, USA) without fetal bovine serum (FBS) (Gibco, Cat.

2.5. Solid tumor and experimental metastasis mouse models
C57BL/6 mice (8–12 week old) were maintained and treated in
accordance with animal use ethical principles. Procedures were previously approved by Ethics Committee on Animal Experimentation
(Universidade Federal do Paraná: certificate #1025; Faculdade de
Medicina de São Paulo: process 049/17; Moffitt Cancer Center: IACUC
#R IS00003462).
B16-F10 cells (5 × 105 cells in 100 μL of PBS) were subcutaneously
inoculated in the right flank of male mice. After 5 days, mice started
receiving daily intraperitoneal (I.P.) doses of 3G4S (50 mg/kg) diluted
in PBS or PBS alone (control group) for 10 days, an experimental design
based on previous results from our group (Biscaia et al., 2017). Tumors
were daily measured using a digital caliper, and tumor volume was
calculated using the formula “V = dxdxDx0.52” (“d” = smaller tumor
dimension, “D” = bigger dimension).
3G4S antimetastatic effect was analyzed by the experimental metastasis model. A 24 h polysaccharide pre-treatment regimen was established in order to simulate an intervention to prevent and/or reduce
metastasis progression after a diagnosed primary melanoma. Male and
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Carbohydrate Polymers 250 (2020) 116869

D.L. Bellan, et al.

Fig. 1. HSQC spectrum of 3G4S from C. isthmocladum.
→3)-β-D-Galp4S-(1→ units and →2)-3,4-Pyruvylated-β-D-Galp-(1→ (amplified). Numbers refer to the position of each 1H/13C correlation.

paraformaldehyde, and incubated for 30 min with ActinGreen™
ReadyProbes. With a humidified cotton-swab, non-invasive cells were
gently removed from Transwells top. Inserts membranes were assembled into a glass microscope slide, using Fluoromount-G™ mounting
medium with DAPI (Electron Microscopy Sciences, Cat. 17984-24,
Haltfield, Pennsylvania, USA). Slides were scanned in the VSlide Carl
Zeiss and Metasystems, using 20x objective to capture the entire
membrane. The number of cells that invaded Matrigel was counted by
detection of DAPI stained cells nuclei using ImageJ Fiji Software.

female mice were I.P. pre-treated with 3G4S (50 mg/kg) diluted in PBS
or PBS alone 24 h before B16-F10 cells (5 × 105 cells in 100 μL of PBS)
intravenous inoculation. Post-cell inoculation, treatment was carried
out daily for 9 days, following ex-vivo lungs imaging for metastasis foci
counting, or for 20 days, following ex-vivo lungs imaging for analysis of
colonized area in relation to total lung area, using ImageJ Fiji Software
(Schindelin et al., 2012) and staining with hematoxylin and eosin (H&
E). Entire lobe images were obtained on a histological slide scanner
VSlide Carl Zeiss and Metasystems (Oberkochen, Germany) using a 20x
objective.
Bioluminescence imaging of experimental metastasis progression
was performed using In vivo Imaging System (IVIS). Male and female
mice were pre-treated with 3G4S (50 mg/kg) I.P. for 24 h, following
B16-F10-luc-G5 cells intravenous inoculation (5 × 105 cells).
Treatment was carried out daily for 15 days. After 2 h or 9 days postcell inoculation, mice were I.P. injected with 150 μg/mL XenoLight Dluciferin (PerkinElmer, Cat. 122799, Walthman, Massachusetts, USA),
and subsequently bioluminescence was captured in a Xenogen IVIS 200
(Xenogen Corporation, Hopkinton, USA). Ex vivo bioluminescence

imaging of mice organs (lungs, kidneys, liver, pancreas and spleen) was
performed.

2.8. Glycoconjugates labeling
B16-F10 cells (1.2 × 104 cells/well) were exposed to 100 μg/mL
3G4S for 72 h. Cell labeling was performed with WGA lectin Alexa
Fluor 488 conjugate (Invitrogen, Cat. W11261, Waltham,
Massachusetts) that specifically binds to N-acetylglucosanime and Nacetylneuraminic acid (sialic acid). Samples were acquired using BD
FacsVerse flow cytometer (Missouri, USA).

2.9. Colony formation assay
2.6. Treatment side effects assessment
Anchorage-independent colony formation assay was performed
using AlgiMatrix ™ 3D Culture System (ThermoFisher, Cat. 12684023,
Walthmam, Massachusetts, USA). 3G4S pre-treated B16-F10 cells (100
μg/mL of 3G4S for 72 h) were plated in the reconstituted AlgiMatrix
hydrogel in new DMEM medium with 10 % FBS without the treatment.
DMEM was replaced after three days of incubation and kept for more
three days. Colonies-containing alginate matrix were fixed, stained with
CV and counted using ImageJ Fiji Software.

Body weight differences from before and after treatment were recorded. After animal anesthesia, cava vein blood was collected and
stored in EDTA-containing tubes. Blood cell count and biochemical
parameters analyses were performed using a chemistry analyzer
Mindray BS-200 (Shenzhen, China). Organs were harvested and
weighed.
2.7. Invasion assay

2.10. Statistical analysis

B16-F10 cells (1.2 × 104 cells/well) were exposed to 100 μg/mL
3G4S for 72 h. Cells were detached using a scraper and plated in DMEM
(FBS free) on top of Matrigel™ (2.6 mg/mL, 35 μL/well) (BD
Biosciences, Cat. 356234, San Jose, California, USA) pre-coated
Transwells (Millipore, Cat. MCEP24 h48, Massachusetts, USA). DMEM
with 10 % FBS at the wells bottom was used as chemoattractant, followed by 72 h incubation. Transwells were fixed for 1 h with 2 %

Significant differences between experimental and control groups
were determined by Mann Whitney t-test and by Two-Way ANOVA for
tumor volume over time, using GraphPad Prism 6 software. Data present
as median ± interquartile range.

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D.L. Bellan, et al.

Table 1
Chemical shift assignments of the HSQCspectrum of 3G4S from C. isthmocladum.
Unit

Structure

A

→2)-3,4-Pyruvylated-β-D-Galpb-(1→

A6S

→2)-3,4-Pyruvylated-β-D-Galp6S-(1→b

B

→3)-β-D-Galp4S-(1→

a
b

Chemical shifts, δ (ppm)a
H1
C1

H2
C2

H3
C3

H4
C4

H5
C5

H6
C6

4.73

103.5
4.73
103.5
4.56
103.0

3.57
73.4
3.82
74.3
3.72
70.3

3.84
82.4
4.24
78.6
3.80
70.7

4.30
70.8
4.12
74.9
4.86
77.4

3.72
75.0
4.06

73.0
3.93
73.1

3.80
61.1
4.34
67.1
3.80
61.1

Chemical shifts are referred to internal standard trimethylsilyl propionic acid (δ =0.00 ppm). Assignments based on (Farias et al., 2008).
Signals at 1.62/23.3 ppm correspond to C2 and C3/H3 of pyruvic acid ketal linked to O-3 and O-4 of galactose units.

3. Results

post-cell inoculation. Ex vivo lungs imaging showed 77.03 % reduction
in metastasis colonization after 3G4S treatment (Fig. 5C-D). Besides,
surface intratissue colonization was also reduced by 3G4S (Fig. 5E).
After 10 days of treatment, treated mice spleens were heavier than
control mice spleens, relative to body weight. After 21 days of treatment, control mice lost weight over the experiment course, while 3G4S
mice gained corporal weight. 3G4S treated mice spleens and livers were
heavier than those from control mice, however biochemical parameters
and blood cell count did not show any indication of hepatotoxicity nor
nephrotoxicity (as verified by AST, ALT and urea measurements)
(Table 2).
Data represent the mean ± SD of at least 4 animals per group for 10
days of treatment, and of at least 6 animals per group for 21 days of
treatment.

3.1. Purification and characterization of 3G4S
The characterization analysis performed (Supplementary data)
confirmed that 3G4S was highly similar to SG1 previously described by
Farias et al. (2008).
3G4S was composed of galactose with 1.2 ° of sulfation (DS), and Mw
of 14.1 KDa.
HSQC spectrum also confirms 3G4S identity (Fig. 1). Two major
spin systems are evident from the HSQC spectrum, named unit A and
unit B, which have anomeric hydrogen and carbon signals at δ 4.73/
103.5 and 4.56/103.0, respectively. All units consist of β-D-Galp residues, and their respective chemical shifts are showed in Table 1.
3.2. 3G4S induces low cytotoxicity without inducing apoptosis, proliferation
or morphology changes

3.5. 3G4S alters metastatic melanoma dynamics
3G4S treatment of B16-F10 cell line reduced glycoconjugates present in cell membrane by 33.47 %, as shown by WGA labeling (Fig. 6B).
Anchorage-independent colony formation capacity in a tridimensional
scaffold was reduced by 31.41 % (Fig. 6E). B16-F10 invasion capacity
was also reduced by 27.95 % (Fig. 6G).

MTT (Fig. 2A) and CV (Fig. 2B) assays did not show reduction in
mitochondrial activity or in cell proliferation, respectively, in any
tested concentration. Based on previous results from our group (Bellan
et al., 2020; Biscaia et al., 2017), as well as in recent literature (Khan
et al., 2019) the concentration of 100 μg/mL was chosen to the subsequent in vitro assays.
3G4S treatment did not induce apoptosis (Fig. 2C) or cell cycle alterations (Fig. 2D-E).
Representative areas of confocal microscopy (Fig. 2 F a–d) and SEM
(Fig. 2F e–h) did not show cell morphology differences between control
and 3G4S treated cells.

4. Discussion

Currently melanoma treatments collateral effects are a significant
drawback given their non-selective characteristics, so we first sought to
determine 3G4S possible cytotoxic activity. However, 3G4S, sulfated
galactan from Codium isthmocladum did not induce cytotoxicity or
proliferation changes (Figs. 1 and 2). Polysaccharides exerting direct
cytotoxic and antiproliferative effects on cancer cells, inducing cell
apoptosis and cell cycle arrest, are commonly found (Khan et al., 2019;
Zong, Cao, & Wang, 2012) including sulfated polysaccharides from
seaweeds (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer, 2011; Kim
et al., 2007; Sae-Lao, Tohtong, Bates, & Wongprasert, 2017). Nonetheless, some non-cytotoxic polysaccharides already showed antimelanoma activities as a sulfated heterorhamnan from the green seaweed Gayralia brasiliensis (Bellan et al., 2020) and the partially 3-Omethylated mannogalactans from Pleurotus eryngii (Biscaia et al., 2017).
Given the high number of collateral effects commonly associated with
direct cytotoxic treatments, a non-cytotoxic compound still able to induce antitumor activities, as 3G4S, is of significant relevance.
Daily treatment with 50 mg/Kg 3G4S, a similar dose with satisfactory effects as used in other antitumoral polysaccharide studies (Jiang
et al., 2014; Jin et al., 2007), significantly reduced tumor growth over
time, final tumor volume as well as tumor weight (Fig. 3), which is a
desirable clinical effect in advanced stages of unresectable melanoma
(Nixon et al., 2018; Perez et al., 2019).
Based on the promising antitumor activity induced by 3G4S and the
threat posed by metastatic melanoma, we sought to investigate its

3.3. Melanoma solid tumor progression is reduced by 3G4S
Daily 3G4S treatment resulted in tumor volume reduction over time
when compared to control group since 7th day of treatment (40.47 %;
Fig. 3B). Final tumor volume (Fig. 3D) and tumor weight (Fig. 3E) were
also reduced (59.10 % and 37.79 % respectively).
3.4. 3G4S shows antimetastatic effect
Melanoma cells distribution in control and treated mice was observed 2 h after cell inoculation by IVIS (Fig. 4B and C). Nine days postcell inoculation, 3G4S treated group showed reduced metastatic cells
presence (Fig. 4B and C). IVIS ex vivo imaging of organs showed a reduction in metastatic colonization in all tissues analyzed for female and
male mice (Fig. 4D and E respectively). Female mice spleens and pancreas and male mice kidneys showed a statistically significant reduction
in metastatic colonization (Fig. 4F and G).

Metastasis foci count 9 days post-cell inoculation was also reduced
in 3G4S treated mice, resulting in 74.79 % less foci (Fig. 5A and B).
The experimental metastasis model was also carried out for 20 days
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Fig. 2. 3G4S cell cytotoxicity, proliferation and morphology.
(A) MTT. (B) Cell proliferation. (C) Annexin V and 7 AAD. (D) Cell cycle. (E) Cell cycle distribution histogram. These results represent the set of at least three
biologically independent experiments for A, C, D and E, and two for B. Control represented as a dotted line for A and B. Data normalized for A and B (F) Confocal
microscopy (a,b – Control; c,d – 3G4S). (F) SEM (e,f – control; g,h – 3G4S).

containing N-acetylglucosamine and sialic acid (Fig. 6). Tumor progression and metastasis are accompanied by a series of glycosylation
modifications, promoting sustained proliferative signals, resistance to
cell death, immune evasion, migration and invasion amongst other
tumor promoting effects (Peixoto, Relvas-Santos, Azevedo, Lara Santos,
& Ferreira, 2019). One of the mechanisms responsible for increasing
cancer cells migration and invasion through glycosylation modulation
is sialylation, the addition of syalic acid in N-glycans, promoting cancer
cell detachment through physical disruption of cell adhesion (Schultz,
Swindall, & Bellis, 2012). Thus the observed reduction in glycans
containing N-acetylglucosamine and sialic acid observed post 3G4S
treatment corroborates with a less invasive phenotype, as shown in the
invasion assay and in the experimental metastasis results.
Anchorage-independent colony formation capacity of B16-F10 cell
line was reduced after 3G4S treatment (Fig. 6F), in a similar manner

antimetastatic potential. IVIS bioluminescence capture and ex vivo
images of lungs presented a reduction in 3G4S treated mice metastatic
progression in all experiments endpoints (Fig. 4). Histology images also
show a visual reduction in tumors inside the lungs (Fig. 5). In order to
understand how 3G4S modulates metastasis progression, we analyzed
its effects in cell malignancy related traits that corroborate to metastatic
dissemination.
In vitro 3G4S treatment reduced B16-F10 cell line invasive activity
(Fig. 6). Polysaccharides are able to affect different cell dynamics associated with metastatic capacity, reducing MMPs production and activity as well as modulating cancer cell membrane surface receptors,
reducing migratory and invasive capacities (Khan et al., 2019; Zong
et al., 2013), modulations that can be associated with experimental
metastasis reduction in vivo (Yu et al., 2018).
3G4S reduced glycoconjugates labeling, specifically glycans

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Fig. 3. Solid tumor progression is impaired with 3G4S.
(A) Experimental design. (B) Tumor volume over time. *p = 0.0141; ****p < 0.0001. (C) Solid tumors (a,b – Control; c,d – 3G4S). Representative tumors images
from the same experiment based on median tumor weight. (D) Final tumor volume. (E) Tumor weight. These results represent the set of four biologically
independent experiments (Control N = 25; 3G4S N = 24; male mice).

capacity to sustain proliferation, as well as making them more prone to
cell death induced by immune system cells; and the B16-F10 3G4Streated diminished anchorage independent colonization capacity may
be affecting the subsequent proliferation and lung colonization necessary for the metastatic progression. Hence, the results described here
point to the modulation of some of the most important cellular dynamics to the successful of the early stages of the metastasis cascade extravasation, tissue remodeling, immune evasion and colonization

(Lambert et al., 2016).
3G4S treated mice gained corporal weight when compared to
treatment start, while control mice lost weight (Table 2). This result
could be an indicative of a protective effect from 3G4S against cancer
cachexia, a disease adverse effect leading to skeleton muscle mass loss
and progressive functional impairment (Fearon et al., 2011). Similar
protective effects can be found exerted by other polysaccharides already described (Chen et al., 2018; Fitton, Stringer, Park, & Karpiniec,
2019). 3G4S treated mice also presented heavier spleens and liver when
compared to control mice (Table 2). The increase in spleen weight
should be further investigated as a possible collateral effect or an immune modulation activity, as shown by other polysaccharides in the
form of spleenocyte proliferation and T cell activation (Ramberg,

some polysaccharides are able to interfere in colony formation in the
same concentration (100 μg/mL) and cell line (B16-F10) (Oliveira et al.,
2019; Varghese, Joseph, Aravind, Unnikrishnan, & Sreelekha, 2017).
After tissue extravasation the initial seeding of metastatic cells depends
on its capacity to survive and colonize the new microenvironment,
normally assisted by growth factors and inflammatory proteins released
by the primary tumor, generating a premetastatic niche (Pachmayr,
Treese, & Stein, 2017). To further generate micro and macrometastasis
these cells need to enable sustained proliferative capacity, a trait simulated in the colony formation assay (Franken, Rodermond, Stap,
Haveman, & van Bree, 2006). Cells treated with 3G4S presented a long
lasting reduction in their colony formation capacity even after treatment removal, indicating a modulation in another metastatic cascade
initial step (Fig. 5D and E).
Although the exact mechanism underlying 3G4S antimetastatic activity is still unclear, the modulation of metastatic related features demonstrated in vitro could be strongly correlated to the significant impairment of B16-F10 metastasis progression. 3G4S may be affecting
B16-F10 extravasation and lung’s tissue colonization through a reduction in cells invasive and tissue remodeling capacities; the reduction in
glycoconjugates surrounding metastatic cells may be reducing their
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Fig. 4. Metastasis progression impairment by 3G4S observed by IVIS.
(A) Experimental design. (B) Control and (C) 3G4S Bioluminescence capture 2 h and 9 days post-cell inoculation (Control N = 5, 3G4S N = 4; female mice). (D–E)
Ex vivo organ bioluminescence. (D- female mice; Control N = 5, 3G4S N = 4; E- male mice; Control N = 5, 3G4S N = 4). *p = 0.0286. (F–G) Incidence of
metastasis in each mouse. Presence of metastasis in the specified organ accounted for each mouse (F- female mice; Control N = 5, 3G4S N = 4; G- male mice;
Control N = 5, 3G4S N = 4).

The combination of structure features, namely molecular weight,
sulfate content, monosaccharide composition and type of glycosidic
linkage is closely related to the extent of polysaccharide activity.
However, the highly structural diversity of polysaccharides makes a
direct relationship between structure and biological effects a complex
subject (Jiao et al., 2011; Xu, Huang, & Cheong, 2017). Nonetheless,
when comparing 3G4S structure and activities with some of the most
common polysaccharides obtained from seaweeds, we can find interesting similarities and differences regarding structure and activity.

Nelson, & Sinnott, 2010) including in galactan-containing polysaccharides (Awadasseid et al., 2017; Zheng et al., 2016). Although the
increase in 3G4S treated mice liver weight, biochemical analyses did
not show hepatotoxicity (Table 2). Cancer treatment commonly induces
severe patient’s quality life loss through a myriad of collateral effects,
even reaching the point of treatment withdrawal by patients (Clarke,
Johnston, Corrie, Kuhn, & Barclay, 2015), therefore 3G4S absence of
toxicity results indicates another promising component of this polysaccharide.
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Fig. 5. Metastasis progression impairment over time.
(A) Experimental metastasis end point 9 days post cell inoculation. (a–d) Control. (e–h) 3G4S. Lungs ventral view of 4 animals. (B) Metastasis foci count.
(Control N = 4; 3G4S N = 5; female mice). *p = 0.0317. (C) Experimental metastasis end point 20 days post cell inoculation. (a–d) Control. (e–h) 3G4S.
Ventral view of lungs. (D) Total metastasis colonization area. Total lung area / metastatic area. (Control N = 12; 3G4S N = 8; female mice). ***p = 0.0002. (E) H
&E from mice lungs. Tumor colonies indicated by arrowheads (intratissue) and arrows (superficial) (a–d) Control. (e–h) 3G4S. Images corresponding to left lung
lobe from the same lungs and in the same order as (C).

activities of fucoidans, preponderantly inducing cell cycle arrest and
selective cytotoxicity in cancer cells (Ale et al., 2011; Atashrazm,
Lowenthal, Woods, Holloway, & Dickinson, 2015; Zhang, Teruya, Eto,
& Shirahata, 2011). Differently from fucoidans, 3G4S presents

Fucoidans
are
sulfated
L-fucose-rich
branched
heteropolysaccharides obtained from brown seaweeds. They present chains of
α-(1→3)-L-Fuc and/or alternated α-(1→3)- and α-(1→4)-L-Fuc units
(Li, Lu, Wei, & Zhao, 2008). A variety of studies present antitumor
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D.L. Bellan, et al.

Table 2
Analysis of physiological, biochemical and hematological parameters.
Parameter
Treatment regimen: 10 days
Corporal weight variation
Lung weight/body weight
Spleen weight/body weight
Liver weight/body weight
Kidney weight/body weight
Biochemical parameters
Alkaline phosphatase
Creatinine
Gamma GT
Treatment regimen: 21 days
Corporal weight variation
Lung weight/body weight
Spleen weight/body weight
Liver weight/body weight
Kidney weight/body weight
Biochemical parameters
Alanine transaminase
Aspartate aminotransferase
Creatinine
Urea
Total cholesterol
Triglycerides
VLDL
Procalcitonin
Complete blood count
White blood cells

Lymphocytes
Monocytes
Granulocytes
Red blood cells parameters
Red blood cells
Mean corpuscular volume
Mean corpuscular hemoglobin
Red cell distribution width
Platelets parameters
Platelets
Mean platelet volume
Platelet distribution width

Unit

Control

3G4S

P value

%
%
%
%
%

8.05 ± 2.644
0.7799 ± 0.1211
0.4915 ± 0.04467

5.611 ± 0.4878
1.216 ± 0.02170

11.37 ± 2.538
0.7758 ± 0.1354
0.6153 ± 0.04842 **
6.031 ± 0.4204
1.233 ± 0.1568

0.0952
0.8016
0.0079
0.3095
0.6667

U/L
mg/dL
U/L

95.88 ± 8.115
0.425 ± 0.05
4.85 ± 2.068

60.4 ± 5.662
0.35 ± 0.05774
5.833 ± 2.04

0.0286
0.2857
0.5714

%
%
%
%
%

−1.298 ± 2.027
2.224 ± 1.135
0.421 ± 0.09374
5.215 ± 0.4865
1.527 ± 0.09001

+ 1.375 ± 0.6455 ***
0.9443 ± 0.1482 **
0.5601 ± 0.05745 **
6.424 ± 0.4598 ****
1.512 ± 0.04374

0.0005
0.0011
0.0022
< 0.0001
0.7102

U/L
U/L
mg/dL
mg/dL
mg/dL

mg/dL
mg/dL
%

45.84 ± 11.27
130.2 ± 53.05
0.375 ± 0.04629
59.88 ± 5.802
81.49 ± 4.041
100.9 ± 15.06
20.19 ± 2.998
0.2443 ± 0.0336

54.53 ± 7.751
179.6 ± 48.84
0.3667 ± 0.05164
51.95 ± 5.685
75.85 ± 6.95
90.53 ± 23.36
18.1 ± 4.704
0.2378 ± 0.03207

0.1419
0.1079
> 0.9999
0.0593
0.1512
0.3983
0.3983
0.7193

6.088 ± 1.454
4.475 ± 0.9498
0.125 ± 0.04629
1.488 ± 0.7019

7.783 ± 1.534
5.75 ± 1.343
0.15 ± 0.05477
1.883 ± 0.5636

0.1325
0.0593
0.5884
0.2278

x106 / μL
fL
pg
%

8.237 ± 0.4923
41.3 ± 1.223
12.09 ± 0.2673
15.79 ± 1.145

7.775 ± 0.4598
40.67 ± 1.472
11.8 ± 0.3162
16.02 ± 0.5672

0.0734
0.5589
0.1282
0.3817

x103 / μL
fL
%

421.5 ± 53.71
5.8 ± 0.1309
14.74 ± 0.1847

395.5 ± 52.76
6.033 ± 0.3141
14.9 ± 0.2757

0.4715
0.1275
0.3124

x103
x103
x103
x103

/
/
/

/

μL
μL
μL
μL

Data represent the Mean ± SD of at least 4 animals per group for 10 days of treatment, and of at least 6 animals per group for 21 days of treatment.

modification associated with a higher degree of biological effects in
seaweed polysaccharides (Patel, 2012) and that has also been linked to
a higher biological and antitumor activity in polysaccharides in general
(Xie et al., 2020).
Antitumor and antimetastatic combined activities of 3G4S and its
apparent absence of side effects described here represents a new step on
melanoma treatment. This promising compound could be administered
over a long period of time since the first diagnosis of a primary tumor,
leading to tumor growth rate decrease and possible reduction and inhibition of metastatic progression.

antitumor activities without interfering in cell proliferation and viability. This could be related, at least in part, to its differences in monosaccharide composition and glycosidic linkage types: 3G4S is a homogalactan mainly composed of β-(1→3)- linkage, whereas fucoidans are
α-(1→3) and α-(1→4) linked and contain fucose units. Interestingly,
3G4S has a Mw around 14 KDa, which is a value near of sulfated fucoidans molar mass that showed better antitumor activities (Choi &
Kim, 2013; Kasai, Arafuka, Koshiba, Takahashi, & Toshima, 2015).
Another example of abundant seaweed sulfated polysaccharides are
carrageenans which are highly sulfated homogalactans. They are
composed of alternating units of D-Galp β-(1→3)-linked and D-Galp α(1→4)-linked, Galactose α-(1→4)-linked that can be replaced by 3,6anhydrogalactose units (Necas & Bartosikova, 2013). Many reports
demonstrate carrageenans anticancer activities, especially through cell
cycle arrest (Ling, 2012; Prasedya, Miyake, Kobayashi, & Hazama,
2016) and cell cytotoxicity, being those depolymerized carrageenans
(with lower Mw) highly cytotoxic (Calvo et al., 2019; Z. Jin, Han, &

Han, 2013; Liu et al., 2019). Although monosaccharide composition
and the relative low Mw of non-cytotoxic 3G4S approximate it to cytotoxic carrageenans, some structural features are different. Carrageenans can be 2-O-, 4-O-, and 6-O-sulfated while 3G4S is preponderantly
4-O-sulfated (Campo et al., 2009). Moreover, 3G4S does not present
alternated β-(1→3) and α-(1→4) glycosidic linkages nor 3,6-anhydrogalactose units, a structural moiety of carrageenans already linked
to cell cytotoxic effects (Alves et al., 2012).
Additionally, part of 3G4S antitumor activity could be associated to
its high sulfate content, a structural characteristic and chemical

5. Conclusion
The polysaccharide 3G4S modulated in vitro malignancy features
and reduced solid tumor and lung’s metastasis progression of melanoma
without side effects. This is the first report of a galactan from green
seaweed with antitumor activities both in solid tumor model and experimental metastasis induced by the highly aggressive B16-F10 melanoma cell line, revealing it as a promising compound to further studies.
CRediT authorship contribution statement
D.L. Bellan: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Validation, Writing - review & editing.
S.M.P. Biscaia: Data curation, Formal analysis, Investigation,
Methodology. G.R. Rossi: Data curation, Formal analysis,
9


Carbohydrate Polymers 250 (2020) 116869

D.L. Bellan, et al.

Fig. 6. 3G4S modulates B16-F10 malignancy related traits.
(A) Glycoconjugate labeling representative histogram. (B) Glycoconjugate labeling analysis. ***p = 0.0007. (C–E) Colony formation anchorage-independent assay. (C) Control. (D) 3G4S. (E) Colony formation analysis. *p = 0.0286. (F) Invasion assay. Representative image (a) Control (b) 3G4S. (G)
Invasion assay analysis. **p = 0.0095. These results represent the set of at least three biologically independent experiments. Data normalized.

Acknowledgements

Investigation, Methodology. A.M. Cristal: Formal analysis,
Investigation, Methodology. J.P. Gonỗalves: Data curation, Formal
analysis, Investigation, Methodology. C.C. Oliveira: Conceptualization,
Funding
acquisition,
Validation,
Writing.
F.F.
Simas:
Conceptualization, Funding acquisition, Validation, Writing. D.A.
Sabry: Formal analysis, Investigation, Methodology. H.A.O. Rocha:
Funding acquisition, Validation, Methodology. C.R.C. Franco: Funding
acquisition, Validation, Supervision. R. Chammas: Conceptualization,
Funding
acquisition,
Validation,
Writing.
R.J.
Gillies:
Conceptualization, Funding acquisition, Validation. E.S. Trindade:
Conceptualization, Funding acquisition, Validation, Supervision,
Writing, Project administration.

The authors would like to thank the Brazilian funding agencies
CAPES (Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nớvel Superior)
and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)
for financial support (Grant numbers: CAPES- 001-CIMAR 1985/2014
and PROCAD 2965/2014; CNPq - 309260/2015-9). We also would like
to thank the UFPR Multi-user Confocal Microscopy Center, UFPR

Electron Microscopy Center, Prof Dra. Rosangela Locatelli Dittrich and
MSc. Olair Carlos Beltrame from the UFPR Veterinary Hospital.

10


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D.L. Bellan, et al.

Appendix A. Supplementary data

from Ascophyllum nodosum on the growth of sarcoma-180 solid tumor in mice.
Anticancer Research, 34(4), 1663–1672.
Jiao, G., Yu, G., Zhang, J., & Ewart, H. S. (2011). Chemical structures and bioactivities of
sulfated polysaccharides from marine algae. Marine Drugs, 9(2), 196–233. https://
doi.org/10.3390/md9020196.
Jin, L. Q., Zheng, Z. J., Peng, Y., Li, W. X., Chen, X. M., & Lu, J. X. (2007). Opposite effects
on tumor growth depending on dose of Achyranthes bidentata polysaccharides in
C57BL/6 mice. International Immunopharmacology, 7(5), 568–577. />10.1016/j.intimp.2006.12.009.
Jin, Z., Han, Y. X., & Han, X. R. (2013). Degraded iota-carrageenan can induce apoptosis
in human osteosarcoma cells via the wnt/β-catenin signaling pathway. Nutrition and
Cancer, 65(1), 126–131. />Kasai, A., Arafuka, S., Koshiba, N., Takahashi, D., & Toshima, K. (2015). Systematic
synthesis of low-molecular weight fucoidan derivatives and their effect on cancer
cells. Organic & Biomolecular Chemistry, 13(42), 10556–10568. />1039/C5OB01634G.
Khan, T., Date, A., Chawda, H., & Patel, K. (2019). Polysaccharides as potential anticancer
agents—A review of their progress. Carbohydrate Polymers, 210(January), 412–428.
/>Kim, J. Y., Yoon, M. Y., Cha, M. R., Hwang, J. H., Park, E., Choi, S. U., ... Hwang, Y. I.
(2007). Methanolic extracts of plocamium telfairiae induce cytotoxicity and caspasedependent apoptosis in HT-29 human colon carcinoma cells. Journal of Medicinal
Food, 10(4), 587–593. />Kolsi, R. B. A., Jardak, N., Hajkacem, F., Chaaben, R., Jribi, I., Feki, A. E.l., ... Belghith, K.

(2017). Anti-obesity effect and protection of liver-kidney functions by Codium fragile
sulphated polysaccharide on high fat diet induced obese rats. International Journal of
Biological Macromolecules, 102, 119–129. />04.017.
Kroschinsky, F., Stölzel, F., von Bonin, S., Beutel, G., Kochanek, M., Kiehl, M., ...
Schellongowski, P. (2017). New drugs, new toxicities: Severe side effects of modern
targeted and immunotherapy of cancer and their management. Critical Care, 21(1),
1–11. />Lambert, A. W., Pattabiraman, D. R., & Weinberg, R. A. (2016). Emerging biological
principles of metastasis. Cell, 168(4), 670–691. />11.037.
Lee, J., Ohta, Y., Hayashi, K., & Hayashi, T. (2010). Immunostimulating effects of a
sulfated galactan from Codium fragile. Carbohydrate Research, 345(10), 1452–1454.
/>Li, B., Lu, F., Wei, X., & Zhao, R. (2008). Fucoidan: Structure and bioactivity. Molecules,
13(8), 1671–1695. />Li, N., Mao, W., Yan, M., Liu, X., Xia, Z., Wang, S., ... Cao, S. (2015). Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green
alga. Codium Divaricatum, 121, 175–182. />036.
Ling, N. (2012). Growth inhibition and cell cycle arrest of kappa-selenocarrageenan and
paclitaxel on HepG2 cells. Advanced Materials Research, 343–344, 530–534. https://
doi.org/10.4028/www.scientific.net/AMR.343-344.530.
Liu, Z., Gao, T., Yang, Y., Meng, F., Zhan, F., Jiang, Q., ... Sun, X. (2019). Anti-cancer
activity of porphyran and carrageenan from red seaweeds. Molecules, 24(23), 4286.
/>Manlusoc, J. K. T., Hsieh, C. L., Hsieh, C. Y., Salac, E. S. N., Lee, Y. T., & Tsai, P. W.
(2019). Pharmacologic application potentials of sulfated polysaccharide from marine
algae. Polymers, 11(7), />Melis, C., Rogiers, A., Bechter, O., & van den Oord, J. J. (2017). Molecular genetic and
immunotherapeutic targets in metastatic melanoma. Virchows Archiv, 471(2),
281–293. />Moran, B., Silva, R., Perry, A. S., & Gallagher, W. M. (2017). Epigenetics of malignant
melanoma. Seminars in Cancer Biology, (October), 0–1. />semcancer.2017.10.006.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays. Journal of Immunological Methods,
65(1–2), 55–63. />Necas, J., & Bartosikova, L. (2013). Carrageenan: A review. Veterinární Medicína, 58(4),
187–205. />Nixon, N. A., Blais, N., Ernst, S., Kollmannsberger, C., Bebb, G., Butler, M., ... Verma, S.
(2018). Current landscape of immunotherapy in the treatment of solid tumours, with
future opportunities and challenges. Current Oncology, 25(5), e373–e384. https://doi.

org/10.3747/co.25.3840.
Oliveira, R. S., Biscaia, S. M. P., Bellan, D. L., Viana, S. R. F., Di-Medeiros Leal, M. C.,
Vasconcelos, A. F. D., ... Carbonero, E. R. (2019). Structure elucidation of a bioactive
fucomannogalactan from the edible mushroom hypsizygus marmoreus. Carbohydrate
Polymers, 225. />Pachmayr, E., Treese, C., & Stein, U. (2017). Underlying mechanisms for distant metastasis - Molecular biology. Visceral Medicine, 33(1), 11–20. />000454696.
Patel, S. (2012). Therapeutic importance of sulfated polysaccharides from seaweeds:
updating the recent findings. 3 Biotech, 2(3), 171–185. />s13205-012-0061-9.
Peixoto, A., Relvas-Santos, M., Azevedo, R., Lara Santos, L., & Ferreira, J. A. (2019).
Protein glycosylation and tumor microenvironment alterations driving cancer hallmarks. Frontiers in Oncology, 9(MAY), 1–24. />00380.
Perez, M. C., Zager, J. S., Amatruda, T., Conry, R., Ariyan, C., Desai, A., ... Raskin, L.
(2019). Observational study of talimogene laherparepvec use for melanoma in clinical practice in the United States (COSMUS-1). Melanoma Management, 6(2), https://
doi.org/10.2217/mmt-2019-0012 MMT19.

Supplementary material related to this article can be found, in the
online version, at doi: />References
Ale, M. T., Maruyama, H., Tamauchi, H., Mikkelsen, J. D., & Meyer, A. S. (2011). Fucosecontaining sulfated polysaccharides from brown seaweeds inhibit proliferation of
melanoma cells and induce apoptosis by activation of caspase-3 in vitro. Marine
Drugs, 9(12), 2605–2621. />Alves, M. G., das, C. F., Dore, C. M. P. G., Castro, A. J. G., do Nascimento, M. S., Cruz, A.
K. M., Soriano, E. M., ... Leite, E. L. (2012). Antioxidant, cytotoxic and hemolytic
effects of sulfated galactans from edible red alga Hypnea musciformis. Journal of
Applied Phycology, 24(5), 1217–1227. />Atashrazm, F., Lowenthal, R. M., Woods, G. M., Holloway, A. F., & Dickinson, J. L. (2015).
Fucoidan and cancer: A multifunctional molecule with anti-tumor potential. Marine
Drugs, 13(4), 2327–2346. />Awadasseid, A., Hou, J., Gamallat, Y., Xueqi, S., Eugene, K. D., Hago, A. M., ... Xin, Y.
(2017). Purification, characterization, and antitumor activity of a novel glucan from
the fruiting bodies of coriolus versicolor. PLoS One, 12(2), 1–15. />1371/journal.pone.0171270.
Bellan, D. L., Mazepa, E., Biscaia, S. M. P., Gonỗalves, J. P., Oliveira, C. C., Rossi, G. R., ...
Franco, C. R. C. (2020). Non-cytotoxic sulfated heterorhamnan from gayralia brasiliensis green seaweed reduces driver features of melanoma metastatic progression.
Marine Biotechnology. />Biscaia, S. M. P., Carbonero, E. R., Bellan, D. L., Borges, B. S., Costa, C. R., Rossi, G. R., ...
Trindade, E. S. (2017). Safe therapeutics of murine melanoma model using a novel
antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii.

Carbohydrate Polymers, 178(August), 95–104. />2017.08.117.
Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018).
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality
worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6),
394–424. />Calvo, G. H., Cosenza, V. A., Sáenz, D. A., Navarro, D. A., Stortz, C. A., Céspedes, M. A., ...
Di Venosa, G. M. (2019). Disaccharides obtained from carrageenans as potential
antitumor agents. Scientific Reports, 9(1), 1–13. />Campo, V. L., Kawano, D. F., da Silva, D. B., & Carvalho, I. (2009). Carrageenans:
Biological properties, chemical modifications and structural analysis - A review.
Carbohydrate Polymers, 77(2), 167–180. />020.
Chen, L., Wei, Y., Zhao, S., Zhang, M., Yan, X., Gao, X., ... Gao, Y. (2018). Antitumor and
immunomodulatory activities of total flavonoids extract from persimmon leaves in
H22 liver tumor-bearing mice. Scientific Reports, 8(1), 1–11. />1038/s41598-018-28440-8.
Choi, J. I., & Kim, H. J. (2013). Preparation of low molecular weight fucoidan by gammairradiation and its anticancer activity. Carbohydrate Polymers, 97(2), 358–362.
/>Clarke, G., Johnston, S., Corrie, P., Kuhn, I., & Barclay, S. (2015). Withdrawal of anticancer therapy in advanced disease: A systematic literature review. BMC Cancer,
15(1), 1–9. />Costa, L. S., Fidelis, G. P., Cordeiro, S. L., Oliveira, R. M., Sabry, D.a., Câmara, R. B. G., ...
Rocha, H.a. O. (2010). Biological activities of sulfated polysaccharides from tropical
seaweeds. Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie, 64(1),
21–28. />de Oliveira-Carvalho, M. de F., Oliveira, M. C., Pereira, S. M. B., & Verbruggen, H. (2012).
Phylogenetic analysis of Codium species from Brazil, with the description of the new
species C. pernambucensis (Bryopsidales, Chlorophyta). European Journal of
Phycology, 47(4), 355–365. />Domingues, B., Lopes, J., Soares, P., & Populo, H. (2018). Melanoma treatment in review.
ImmunoTargets and Therapy, 7, 35–49. />Farias, E. H. C., Pomin, V. H., Valente, A. O.-P., Nader, H. B., Rocha, H.a. O., & Mourão,
P.a. S. (2008). A preponderantly 4-sulfated, 3-linked galactan from the green alga
Codium isthmocladum. Glycobiology, 18(3), 250–259. />glycob/cwm139.
Fearon, K., Strasser, F., Anker, S. D., Bosaeus, I., Bruera, E., Fainsinger, R. L., ... Baracos,
V. E. (2011). Definition and classification of cancer cachexia: An international consensus. The Lancet Oncology, 12(5), 489–495. />Fitton, J. H., Stringer, D. N., Park, A. Y., & Karpiniec, S. S. (2019). Therapies from fucoidan: New developments. Marine Drugs, 17(10), />md17100571.
Franken, N.a. P., Rodermond, H. M., Stap, J., Haveman, J., & van Bree, C. (2006).
Clonogenic assay of cells in vitro. Nature Protocols, 1(5), 2315–2319. />10.1038/nprot.2006.339.
Gillies, R. G., Didier, N., & Denton, M. (1986). Determination of cell number in monolayer
cultures. Analytical Biochemistry, 159, 109–113.

Hwang, E. K., Baek, J. M., & Park, C. S. (2008). Cultivation of the green alga, Codium
fragile (Suringar) Hariot, by artificial seed production in Korea. Journal of Applied
Phycology, 20(5), 469–475. />Jiang, Z., Abu, R., Isaka, S., Nakazono, S., Ueno, M., Okimura, T., ... Oda, T. (2014).
Inhibitory effect of orally-administered sulfated polysaccharide ascophyllan isolated

11


Carbohydrate Polymers 250 (2020) 116869

D.L. Bellan, et al.

western Atlantic. Canadian Journal of Botany, 64(10), 2239–2281. />1139/b86-298.
Xie, L., Shen, M., Hong, Y., Ye, H., Huang, L., & Xie, J. (2020). Chemical modifications of
polysaccharides and their anti-tumor activities. Carbohydrate Polymers, 229(October
(2019)), Article 115436. />Xu, S., Huang, X., & Cheong, K. (2017). Recent advances in Marine algae polysaccharides:
Isolation, structure, and activities. Marine Drugs, 15(12), 388. />3390/md15120388.
Yu, Z., Sun, Q., Liu, J., Zhang, X., Song, G., Wang, G., ... Chen, K. (2018). Polysaccharide
from Rhizopus nigricans inhibits the invasion and metastasis of colorectal cancer.
Biomedicine and Pharmacotherapy, 103(21), 738–745. />biopha.2018.04.093.
Zhang, Y., Zhang, M., Jiang, Y., Li, X., He, Y., Zeng, P., ... Zhang, L. (2018). Lentinan as an
immunotherapeutic for treating lung cancer: A review of 12 years clinical studies in
China. Journal of Cancer Research and Clinical Oncology, 144(11), 2177–2186. https://
doi.org/10.1007/s00432-018-2718-1.
Zhang, Z., Teruya, K., Eto, H., & Shirahata, S. (2011). Fucoidan extract induces apoptosis
in MCF-7 cells via a mechanism involving the ros-dependent JNK activation and
mitochondria-mediated pathways. PLoS One, 6(11), />journal.pone.0027441.
Zheng, Y., Yang, G., Zhao, Z., Guo, T., Shi, H., Zhou, Y., ... Sun, L. (2016). Structural
analysis of ginseng polysaccharides extracted by EDTA solution. RSC Advances, 6(4),
2724–2730. />Zong, A., Cao, H., & Wang, F. (2012). Anticancer polysaccharides from natural resources:

A review of recent research. Carbohydrate Polymers, 90(4), 1395–1410. https://doi.
org/10.1016/j.carbpol.2012.07.026.
Zong, A., Zhao, T., Zhang, Y., Song, X., Shi, Y., Cao, H., ... Wang, F. (2013). Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni
ink. Carbohydrate Polymers, 91(1), 403–409. />08.050.

Pomin, V. H. (2010). Structural and functional insights into sulfated galactans: A systematic
review. 1–12. />Pomin, V. H., & Mourão, P. A. S. (2008). Structure, biology, evolution, and medical importance of sulfated fucans and galactans. Glycobiology, 18(12), 1016–1027. https://
doi.org/10.1093/glycob/cwn085.
Prasedya, E. S., Miyake, M., Kobayashi, D., & Hazama, A. (2016). Carrageenan delays cell
cycle progression in human cancer cells in vitro demonstrated by FUCCI imaging.
BMC Complementary and Alternative Medicine, 16(1), 1–9. />s12906-016-1199-5.
Ramberg, J. E., Nelson, E. D., & Sinnott, R. A. (2010). Immunomodulatory dietary
polysaccharides: A systematic review of the literature. Nutrition Journal, 9(1), 54.
/>Sae-Lao, T., Tohtong, R., Bates, D. O., & Wongprasert, K. (2017). Sulfated Galactans from
red seaweed Gracilaria fisheri target EGFR and inhibit cholangiocarcinoma cell
proliferation. American Journal of Chinese Medicine, 45(3), 615–633. />10.1142/S0192415X17500367.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ...
Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis.
Nature Methods, 9(7), 676–682. />Schultz, M. J., Swindall, A. F., & Bellis, S. L. (2012). Regulation of the metastatic cell
phenotype by sialylated glycans. Cancer and Metastasis Reviews, 31(3–4), 501–518.
/>Varghese, S., Joseph, M. M., Aravind, S. R., Unnikrishnan, B. S., & Sreelekha, T. T. (2017).
The inhibitory effect of anti- tumor polysaccharide from Punica granatum on metastasis. International Journal of Biological Macromolecules, 103, 1000–1010. https://
doi.org/10.1016/j.ijbiomac.2017.05.137.
Ward, W. H., & Farma, J. M. (2017). In W. H. Ward, & J. M. Farma (Vol. Eds.), Cutaneous
melanoma: Etiology and therapy: Vol. 6Codon Publications />codon.cutaneousmelanoma.2017.
Wynne, M. J. (1986). A checklist of benthic marine algae of the tropical and subtropical

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