Yonago Acta medica 2015;58:1–7

Original Article

Anti-Proliferation Activity of Fucoidan in MKN45 Gastric Cancer Cells
and Downregulation of Phosphorylated ASK1, a Cell Cycle-Regulated Kinase
Miwa Yoshimoto,* Katsumi Higaki,† Eiji Nanba† and Masahide Ikeguchi**Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504,
Japan and †Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503,
Japan

ABSTRACT

nose, xylose and uronic acid. Over the past decades, fucoidan has been extensively studied, owing to its numerous interesting biological activities, such as antioxidant,1
immunomodulatory,2 antiviral,3 antithrombotic 4 andanticoagulant effects.5 In particular, we have focused on
the anti-cancer activities of fucoidan.
Multiple studies have demonstrated antitumor activity for many polysaccharides. Fucoidans from Eisenia
bicyclis and Laminaria japonica are effective against
sarcoma 180.6, 7 Fucoidan from L. japonica can inhibit
hepatoma QGY7703 cell growth in logarithmic phase invitro, thus restraining the growth of tumors.8 Fucoidans
from L. saccharina, L. digitata, Fucus serratus, F. distichus and F. vesiculosus strongly blocked MDA-MB-231
breast carcinoma cell adhesion to platelets, an effect thatmight have critical implications in tumor metastasis.9
Among its various anti-cancer effects, we havefocused on the antiproliferation activities of fucoidan.
We hypothesized that fucoidan exerts its anti-cancer
effects through alterations of gene expression. Thus, inthis study, we performed mRNA microarray analysis to
examine the effects of fucoidan on cellular gene expression. We selected MKN45 gastric cancer cells for analysis, as this common cancer cell line is relatively easy toculture and is often used for experimental analyses.10–12

Background

Fucoidan is a high-molecular polysaccharide whose main constituent is sulfated fucose. We
specifically focused on the anti-proliferation activity of
fucoidan and examined the underlying mechanism in
MKN45 gastric cancer cells.
Methods MKN45 cell proliferation was analyzed by
BrdU assay and fucoidan cytotoxicity was examined by
LDH and clonogenic assays. The Agilent Human microarray kit was used to identify upregulated and downregulated genes in response to fucoidan, and western blot
analyses evaluated cell cycle proteins.
Results
Fucoidan impeded the MKN45 cell cycle
by approximately 50%, and inhibited cell proliferation. LDH assays showed no immediate cytotoxic effects of fucoidan at 24 h exposure, however longer timecourses revealed cell growth inhibition at 4 days in adose-dependent manner. Microarray analysis identified
MAP3K5, or ASK1 (apoptosis signal-regulating kinase),
which was upregulated by 1.38-fold upon fucoidan treatment. Fucoidan increased ASK1 protein levels, while reducing phosphorylated ASK1 levels. Reduction of ASK1
by siRNA decreased proliferation of MKN45 cells.
Conclusion Our findings show that fucoidan may
suppress cellular proliferation and DNA synthesis in
MKN45 cells by suppressing the ASK1-p38 signaling
pathway through reduction of phosphorylated ASK1 levels.

MATERIALS AND METHODS
Reagents

The human gastric adenocarcinoma cell line, MKN45
was obtained from the Riken BioResource Center(National Bio-Resource Project of the Ministry of
Education, Culture, Sports, Science and Technology,
Tsukuba, Japan) in November 20, 2012. Cells weremaintained in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS). MKN45 gastric adenocarcinoma
cells were obtained from a metastatic liver tumor in a
62-year-old Japanese female.
Dulbecco’s modified Eagle medium (DMEM),
trypsin-EDTA, and the lactate dehydrogenase (LDH)
assay kit were obtained from Wako (Tokyo, Japan).
FBS was from Biowest (Nuaillé, France). Rabbit polyclonal anti-ASK1 (H-300), anti-ERK1 (K-23), mousemonoclonal anti-phospho-ERK (E-4), and ASK1 siRNA

anti-proliferation activity; ASK1; cell
cycle; fucoidan; MKN45

Key words

Fucoidan is a high-molecular polysaccharide only foundin brown algae and its main constituent is sulfated fucose. The saccharide chain also includes galactose, manCorresponding author: Miwa Yoshimoto,m-yoshimoto@med.tottori-u.ac.jp

Received 2014 November 21
Accepted 2014 November 27
Abbreviations: ASK-1, apoptosis signal-regulating kinase one;
BrdU, bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle
medium; IgG, immunoglobulin; LDH, lactate dehydrogenase;
MAPK, mitogen-activated protein kinase; MTT, methyl thiazolyl
tetrazorium; RPMI, Roswell Park Memorial Institute1

M. Yoshimoto et al.

0.7

30

0.6

25

0.5

20

0.4

15

0.3

10

0.2
0.1

5

0

0

Fig. 1. Effect of fucoidan on MKN45 cells.
A: Fucoidan impedes proliferation of MKN45 cells. The rate of MKN45 cell proliferation inhibition in the presence of increasing
amounts of fucoidan was examined using BrdU incorporation assay. Over 5 mg/mL of fucoidan resulted in decreased BrdU incorporation. Inhibition rate was approximately 50%.
B: Cytotoxic effects of fucoidan in MKN45 cells. Cytotoxic effects of increasing amounts of fucoidan in MKN45 cells were examinedby LDH assay. Cells were cultured with fucoidan as indicated for 24 h. Increasing fucoidan concentrations did not affect the rate ofcell death, even at high doses.
AU, arbitrary unit.

were purchased from Santa Cruz Biotech (Santa Cruz,
CA). Rabbit polyclonal anti-phospho-ASK1 (Thr845),
rabbit monoclonal anti-p38 MAPK (D13E1) and antiphospho-p38 MAPK (Thr180/Tyr182) (D3F9) were from
Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin(IgG), anti-mouse IgG, and the ECL plus detection kit
were from GE Healthcare (Buckinghamshire, United
Kingdom). Alexa Fluor 488-conjugated anti-rabbit IgG
was from Invitrogen (Carlsbad, CA).

evaluated by an enzymatic reaction that results in formazan products, which were measured with a spectrophotometer (Tecan Sunrise Rainbow, Tecan Japan, Tokyo)
at 600 nm.
siRNA transfection

Transfection of siRNA was performed with Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s protocol. Briefly, ASK1 siRNA (sc-20748;
Santa Cruz Biotech) was mixed with Lipofectamine
RNAiMAX reagent in serum-free DMEM. Suspended
cells were added to the mixture and the cell mixture wasplated and cultured for 24 h before experimental analysis.

Preparation of fucoidan

High molecular weight fucoidan was obtained from
Marine Products Kimuraya (Sakaiminato, Japan) as asulfated polysaccharide, extracted from the brown seaweed Cladosiphon okamuranus (Okinawa Mozuku) as
previously described.13, 14 The mean molecular weightwas 4 × 105 and the active ingredient contained 14.2%
sulfate (SO42−). The material has been previously used instudies on biological activity and with respect to safetyof human use.15–17

Microarray expression analysis

Total RNA was collected from MKN45 cells using the
RNeasy mini kit (Qiagen, Hilden, Germany). Microarray expression analyses were performed using theAgilent SurePrint G3 Human GE v2 8×60K Microarray(Design ID: 039494) (Agilent Technologies, Palo Alto,CA).

Cell culture, cell growth and cell death assay
BrdU assay

Cells were cultured in DMEM with 10% FBS. For fucoidan treatment, culture medium was replaced with fresh
medium mixed with indicated concentrations of fucoidan, and cells were cultured until the assay. Cell growth
assay was performed by counting cell numbers usinga hemocytometer. Cytotoxic effects were evaluated by
the LDH assay following the manufacturer’s protocol.
Briefly, extracellular LDH activity in the medium was

BrdU assay was performed using the BrdU Cell Proliferation assay kit (Merck, Marmstadt, Germany) following
the manufacturer’s protocol. Briefly, cells in a 96-wellplate were cultured in medium with BrdU for 12 h. BrdU
incorporation was detected with anti-BrdU. Signals were
measured by spectrophotometer analysis at 450/540 nm.

2Anti-proliferation activity of fucoidan

Fig. 2. Fucoidan inhibition of MKN45 cells.
A: Fucoidan inhibition of MKN45 cell growth. Clonogenic assays were performed by culturing MKN45 cells for 6 days in the presenceof increasing amounts of fucoidan as indicated (F0-F20), and counting cells with a hemocytometer. Starting at day 4, the numbers ofcells were clearly reduced with higher doses of fucoidan.
B: Fucoidan inhibits colony formation of MKN45 cells in a dose-dependent manner. Colony formation assay of MKN45 cells incubatedwith increasing amounts of fucoidan. Cell density was lower with increasing concentrations of fucoidan.

Western blot

RESULTS

Cells were lysed by sonication in 10 mM Tris-HCl(pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
and a protease inhibitor cocktail (Roche Diagnostics,
Mannheim, Germany) at 4 °C. Protein concentrations
were determined with a protein assay rapid kit (Wako,
Tokyo). Cell lysates (10 μg of protein) were separated by
SDS-PAGE and transferred to a PVDF membrane. The
membrane was incubated with primary antibodies at
1:1000 dilution with TBS-Tween 20 buffer, followed byincubation with HRP-conjugated secondary antibody at
1:5000 dilution. The membranes were processed using
the ECL detection kit and images were obtained using a
LAS-4000 image analyzer (Fujifilm, Tokyo).

Fucoidan impedes the cell cycle

We first examined the effects of fucoidan on proliferation of MKN45 cells using the BrdU incorporation assay. Low doses of fucoidan had a minimal impact on
MKN45 cell proliferation, while high doses of fucoidan,
over 5 mg/mL, dramatically decreased BrdU incorporation. The rate of MKN45 cell proliferation inhibition byfucoidan was approximately 50% (Fig. 1A).
Cytotoxicity of fucoidan by LDH assay

The cytotoxic effects of treatment of MKN45 cells withincreasing amounts of fucoidan for 24 h were examinedby LDH assay (Fig. 1B). The results showed that 24 h
fucoidan treatment did not induce cell death, even athigh doses.

Immunofluorescence staining

Cells were plated on coverslips and fixed with 3.7%
formaldehyde in PBS for 30 min, followed by permeabilization in 0.1% Triton X-100 in PBS for 15 min. Cells
were then incubated with anti-phospho-p38 antibody(1:200 in 0.1% BSA/PBS) for 1 h, followed by incubation
with Alexa Fluor 488-conjugated anti-rabbit IgG for 1
h. The coverslips were mounted with mounting medium
containing DAPI (ProLong Gold Antifade Mounting
with DAPI; Invitrogen), and fluorescence images wereobtained with confocal microscopy (Leica TCS-SP2,
Wetzler, Germany).

Clonogenic assay reveals fucoidan cytotoxic effects

To gain fuller insight into the potential cytotoxic effects of fucoidan, we performed clonogenic assays andextended the incubation time to 6 days (Fig. 2A). Starting at day 4, the numbers of cells were clearly reducedwith larger doses of fucoidan. Colony formation assays
showed similar results (Fig. 2B). We observed lower celldensity of MNK45 cells cultured with high doses of fucoidan, and this reduction occurred in a dose-dependent
manner.
3

M. Yoshimoto et al.
Table 1. The differentially expressed G0 categories andgene lists in fucoidan treated MKN45 cells
Downregulated
Upregulated
Human insulin signaling (7)
SORBS1FLOT2PSCD3
MAP4K1
MAP3K5FOS
EGR1
GPCRs, class a rhodopsin-like (5)
HTR6
OR3A3
P2RY4
ADRA2A
OR5V1
T-cell receptor signaling pathway (5)MAP4K1RASGRP2FOSCABIN1
CARD11
Monoamine GPCRs (2)HTR6
ADRA2A
MAPK signaling pathway (5)HSPA8MAP4K1
MAP3K5FOS
MAPT

Irinotecan pathway(Homo sapiens) (2)
CES1
ABCC2
Blood clotting cascade (2)SERPINE1
F10
Fatty acid omega oxidation (2)CYP1A2
CYP1A1
Focal adhesion (9)TNC
EGFR
CAV1 (A_24_P12626)
CAV3
CAV1 (A_23_P134454)
RAPGEF1
COMP
MAPK8
LAMC2
Complement activation,
classical pathway (3)CFH
SERPINE1
F10Genes that showed significant (P < 0.05) changes in upregulated or
downregulated expression in MKN45 cells in the presence of fucoidan.

Microarray analyses of fucoidan-treated cells

To identify potential genes involved in mediating thecellular effects of fucoidan, total RNA was collectedfrom MKN45 cells and microarray expression
analyses were performed using the Agilent Human
microarray kit. A total of 20278 sequences were examined at DNA Chip Research (Kanagawa, Japan),and 496 genes were found to be upregulated and 774
genes were downregulated by fucoidan. Table 1 lists
the genes in MKN45 cells with significant changes
in expression in response to fucoidan (P < 0.05).
Among these genes, we observed that MAP3K5,
also known as ASK1 (apoptosis signal-regulating
kinase), was upregulated by 1.38-fold in response to
fucoidan. Microarray analyses accession number:
GSE58774.
Fucoidan downregulates cell cycle promoting
proteins

From the results of the BrdU incorporation assay
and mRNA microarray analysis, we hypothesizedthat fucoidan affected the expressions of cell cyclerelated genes. Western blot analysis revealed thattreatment of MNK45 cells with over 5 mg/mL of
fucoidan resulted in decreased levels of phosphorylated ASK1 and phosphorylated p38 MAPK. In contrast, the expression of ASK1 was slightly increased
in a dose-dependent manner. The expressions of p38
MAPK, ERK, and phosphorylated ERK were unchanged (Fig. 3A).

Fig. 3. Effects of fucoidan on phosphorylation.
A: Fucoidan downregulates cell-cycle promoting proteins. Western blot analysis revealed that over 5 mg/mL of fucoidan deceased thelevels of phosphorylated ASK1 and phosphorylated p38 MAPK. However, the expression of ASK1 was slightly increased in a dosedependent manner. The expressions of p38 MAPK, ERK, and phosphorylated ERK were not changed.
B: Fucoidan reduces phosphorylated p38 in the nucleus. MKN45 cells were incubated with fucoidan as indicated and analyzed by immunofluorescence for phosphorylated p38. Treatment with fucoidan at 5 and 10 mg/mL clearly reduced the levels of phosphorylated
p38 protein in the nucleus of MKN45 cells, which was consistent with the western blot results.

4Anti-proliferation activity of fucoidan

tory molecules in cancer therapy. The anti-proliferative
activity of fucoidan may be a result of its interference incell cycle progression.
Previous studies demonstrated that fucoidan caused
a cell cycle arrest at the G0/G1 phase in hepatocarcinoma cell lines18 and suppressed cell proliferation andarrested the cell cycle in hepatocellular carcinoma celllines.19 Our BrdU results showed that cell proliferation
was suppressed at a dose of fucoidan of over 5 mg/mL
in 24 h, which impeded DNA synthesis and cell proliferation during S phase in MKN45 cells. This result
demonstrated that fucoidan induced cell cycle arrest in
MKN45 cells.
We explored the cytotoxic effects of fucoidan in
MKN45 cells by LDH assay. After 24 h of incubation, fucoidan-treated MKN45 cells did not exhibit any elevationin LDH release compared with the control group, even athigh doses (Fig. 1B). This result was consistent with previous studies.18, 19 However, other studies demonstrated
cytotoxicity of fucoidan in different cancer cells.19, 20
These discrepancies in cytotoxicity among fucoidans are likely due to variations in fucoidan polymer
structure and function, as fucoidans originate fromvarious species, anatomical regions, and under different
growing conditions of brown seaweeds. Fucoidans arealso subject to specific extraction and purification procedures. Furthermore, fucoidans may also exhibit celltype-specific effects, with different functions in different
cancer cell lines. Additionally, even though cell proliferation was repressed by fucoidan in previous reports,
the repression was not evident at 24 h by MTT assay.19
We also did not observe cytotoxic effects of fucoidan at
24 h. We speculated that a longer time course may better
reveal fucoidan effects under long exposure conditions,and subsequently extended the incubation time to 6 days.
Under longer treatment conditions, we observed both amarked decrease in cell number and reduction of celldensity in a dose-dependent manner.
We performed mRNA microarray analysis to identify potential gene targets of fucoidan in mediating its
cell cycle effects. MAP3K5 or ASK1 that was identified
as a MAPK kinase kinase, was upregulated 1.38-foldin response to fucoidan. ASK1 is involved in regulating
the cell cycle and is phosphorylated at the beginning of
S phase.21 This suggested that phosphorylation might
facilitate ASK1’s function in the spindle-kinetochoreinteraction, which presumably occurs during the S phase
after the duplication of kinetochores. This also implies
that decreasing phosphorylated ASK1 would impair thecell cycle during S phase.
In the MAPK pathway, p38 MAPK functions as adownstream kinase of ASK1. p38 MAPK was reported

We also performed fluorescence immunostainingof MNK45 cells with phosphorylated p38 antibody andfound that 5 and 10 mg/mL fucoidan treatment clearly
reduced phosphorylated p38 protein levels in the nucleus(Fig. 3B), which was consistent with the western blot
results. These results suggested the possibility that fucoidan inhibited proliferation of MKN45 cells by suppressing the ASK1-p38 signaling pathway.
ASK1 knockdown reduces proliferation of MKN45
cells

To examine the effect of ASK1 suppression on MKN45
cell proliferation, ASK1 siRNA was transfected into
MKN45 cells. Western blot analyses confirmed that
ASK1 siRNA reduced ASK1 protein levels comparedwith control cells (Fig. 4A). BrdU assay revealed thatgenetic inhibition of ASK1 repressed MKN45 cell proliferation (Fig. 4B).
DISCUSSION
Recent studies have shown that marine bioactive compounds can either modulate signaling pathways leading
to cell cycle regulation or directly alter cell cycle regula-Fig. 4. ASK1 knockdown decreases proliferation of MKN45 cells.
A: Western blot analysis confirmed that transfection with ASK1
siRNA reduced the expression level of ASK1 protein compared with control cells. Beta-tubulin was used as an internal
control.
B: To examine the effect of ASK1 suppression on proliferationof MKN45 cells, ASK1 siRNA was transfected into MKN45
cells and Brdu assay was performed. Genetic inhibition of
ASK1 repressed the proliferative activity of MKN45 cells.
AU, arbitrary unit.

5M. Yoshimoto et al.

to promote cancer cell growth and survival.22 In a study
investigating the effect of fucoidan on lung carcinoma,fucoidan treatment resulted in decreased phospho-p38
levels,23 which demonstrated anti-cancer effect due tofucoidan.
Another MAPK, ERKs are known to promote
cell survival, differentiation, cell proliferation 24 andcell death.25 A previous study showed fucoidan clearlydecreased the phosphorylation of ERK in HS-Sultan
cells.26
In evaluating the results of the mRNA microarray analysis, we therefore focused on genes involved incell cycle regulation to examine potential mediators of
fucoidan. Based on the BrdU incorporation results, wehypothesized that the protein levels of ASK1 and its
related proteins would be altered by fucoidan, and we
performed western blot analysis of ASK1, p38 MAPK,
and ERK steady state and phosphorylated forms to determine the effects of fucoidan.
Treatment with over 5 mg/mL fucoidan effectively
deceased phosphorylated ASK1 and phosphorylated
p38 MAPK levels, while ERK expression remained
unchanged. The western blot results furthered the BrdU
incorporation findings, suggesting that fucoidan caused
reduction of phosphorylated ASK1 expression, arresting
the cell cycle in S phase and eventually impairing DNA
synthesis. This results in damaging proliferation of cancer cells.
Consistent with the microarray analysis, fucoidan
increased ASK1 protein levels. Increased ASK1 protein
could possibly result from a reduction of phosphorylated
ASK1 by fucoidan. As a consequence of reduced phosphorylated ASK1, the mRNA levels were compensatory
amplified, resulting in increased expression of ASK1.
A previous study demonstrated elevation of ASK1 inhuman gastric cancer and showed that overexpression of
ASK1 promoted gastric cancer cell proliferation.27 Another report found that ASK1 inhibition prevents gastriccancer cell growth in vitro and in vivo.28 This mechanism involved cyclin D, a known regulator of the G1 to
S phase transition.29 However, this mechanism seemed
specific to gastric cancer cells, as ASK1 knockdownin other cell lines did not inhibit proliferation.28 The
previous study supports the results of the current study,
demonstrating that genetic inhibition of ASK1 repressed
MKN45 cell proliferation.
To elucidate the mechanisms underlying the anticancer activities of fucoidan, determining its binding
partners and targets would help clarify its activities. One
study reported that fucoidan acts as a polysaccharide
ligand of P-selectin, with nanomolar affinity.30 P-selectin
is an adhesion molecule expressed at the surface of en-

dothelial cells and platelets on activation. P-selectin promotes platelet, endothelium, and leukocyte interactions,and was reported to cause cardiovascular events.31
Fucoidan exhibited the highest affinity for Pselectin32 and repressed its function in platelet aggregation.31 This implied that fucoidan targets the surfaceof endothelial cells, with potential consequences on
downstream signal transduction pathways. Our findings
show that fucoidan induces alterations of gene expression, which may possibly involve its function at the cellsurface.
In conclusion, our study indicated that fucoidan may
suppress cellular proliferation and DNA synthesis in
MKN45 cells by suppressing the ASK1-p38 signaling
pathway through a reduction in phosphorylated ASK1
levels. Our findings strongly suggest fucoidan as a potential therapeutic agent for cancer therapies. Further
investigations would provide new approaches to the development of effective chemotherapy regimens.
Acknowledgments: This work was supported by Marine Products
Kimuraya Co., Ltd. (Sakaiminato, Japan).
The authors declare no conflict of interest.
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