Abstract. Fucoidan, the general term for sulfated polysaccha-
rides, is reported to engage in various biological activities
having anti-tumor, anti-coagulation and anti-viral effects.
Though it has been investigated, the mechanism of its anti-
tumor effects remains elusive. The current study examined the
anti-tumor effects of fucoidan extracted from Okinawa mozuku
on 15 human cancer cell lines (6 hepatocellular carcinomas,
1 cholangiocarcinoma, 1 gallbladder cancer, 2 ovarian cancers,
1 hepatoblastoma, 1 neuroblastoma and 3 renal cancers) using
an MTT assay. Changes in apoptosis and the cell cycle were
analyzed by flow cytometry. The results revealed that cell
proliferation was suppressed in 13 cell lines in a time- and/or
dose-dependent manner; this suppression was marked in the
hepatocellular carcinoma, cholangiocarcinoma and gallbladder
carcinoma cell lines. In contrast, proliferation of the neurob-
lastoma and 1 of the 2 ovarian carcinoma cell lines was not
affected. The ratio of apoptotic cells significantly increased in
5 of the 6 hepatocellular carcinoma cell lines, and the ratio of
G2/M cells increased in the 3 hepatocellular cell lines exam-
ined. These observations indicate that fucoidan is a potential
anti-tumor agent for the treatment of bile duct cancers, such as
hepatocellular carcinoma, cholangiocarcinoma and gall-bladder
Malignant tumors are a major cause of death in humans
and, though treatment methods for cancer have markedly
progressed, curative regimens and protocols are still under
investigation. At present, chemotherapy is regarded as the
most effective treatment for solid tumors.
Recent studies have shown that the natural substances
extracted from green tea (1) and marine products, among
others, have favorable preventive effects against cancers.
‘Mozuku’ (brown seaweed) is a marine plant which grows off-
shore around Okinawa Island. It has attracted the attention of
scientists as well as the general public, and is considered a
food which has various beneficial effects on the body.
Fucoidan, the major component of ‘mozuku’, is the general
term used for sulfated polysaccharides, which are also found
in other seaweeds such as ‘wakame’, ‘hijiki’, ‘mekabu’.
Sulfated polysaccharides are reported to engage in various
biological activities having anti-tumor effects (2-6), anti-
thrombin activity (7,8), anti-coagulant activity (9) and anti-
viral effects (10,11). Their anti-tumor effects have been
investigated using various methods, but the mechanism of
their action has remained elusive.
In order to increase the understanding of these anti-tumor
effects, this in vitro study was conducted using 15 cell lines
from 5 types of human cancers (liver cancer, cholangiocarci-
noma, ovarian cancer, hepatoblastoma and neuroblastoma)
along with fucoidan extracted from ‘Okinawa mozuku’.
Materials and methods
Preparation of culture medium with fucoidan. Fucoidan
solution was kindly provided by FCC Horiuchi & Co.
(Kurume, Japan). The fucoidan was extracted from mozuku
(Cladosiphon Okamuranus Tokida) collected from the shores
of Okinawa Island in Japan, sold as a sea product (containing
fucose 22.1 mg/100 mg) with government approval, and used
as raw material for a health drink. The structure of the
polysaccharide from C. okamuranus has been investigated
The basal medium for cell culture was Dulbecco's modified
Eagle's medium (Nissui Seiyaku Co., Tokyo, Japan) supple-
mented with 5% fetal bovine serum (Whittaker Bioproducts
Inc., Walkersville, MD), 100 units/ml penicillin, and 100 μg/ml
streptomycin (Gibco, Chagrin Falls, OH). Fucoidan was
diluted with this medium and prepared in 10 concentrations
(0.35, 0.70, 1.40, 2.82, 5.63, 11.25, 22.50, 45, 90 and 180 μg/
ml). The osmotic pressures and pH values of the cultures
with or without fucoidan were within normal physiological
MOLECULAR MEDICINE REPORTS 1: 537-542, 2008
Fucoidan, a major component of brown seaweed, prohibits
the growth of human cancer cell lines in vitro
SUGURU FUKAHORI1-3, HIROHISA YANO1,3, JUN AKIBA1,3, SACHIKO OGASAWARA1,3,
SEIYA MOMOSAKI1,3, SAKIKO SANADA1,3, KEITARO KURATOMI1,3, YASUHIRO ISHIZAKI1,3,
FUKUKO MORIYA1,3, MINORU YAGI2,3and MASAMICHI KOJIRO1,3
1Departments of Pathology, and 2Pediatric Surgery, Kurume University School of Medicine; 3Research Center of Innovative
Cancer Therapy of the 21st Century COE Program for Medical Science, Kurume University, Kurume 830-0011, Japan
Received March 7, 2008; Accepted April 30, 2008
Correspondence to: Dr Suguru Fukahori, Department of Pathology
and Pediatric Surgery, Kurume University School of Medicine,
67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan
Key words: fucoidan, apoptosis, cell cycle, anti-tumor
Cell lines and cell culture. Fifteen cell lines were used. These
included 6 human hepatocellular carcinoma (HCC) cell lines
[KIM-1 (12,14), KYN-1 (15), KYN-2 (16), KYN-3 (17),
HAK-1A, HAK-1 (18)], one cholangiocarcinoma cell line
(KMC-1) (19) and one gallbladder carcinoma cell line
(KMG-C) which were originally established in our laboratory.
Two human ovarian clear cell carcinoma cell lines (KOC-5C
and KOC-7C) were established as described elsewhere (20,21),
as were 3 human renal cell carcinoma cell lines (KURU II,
KURM and OSRC2) (22). The human neuroblastoma cell
line (SK-N-SE) was a generous gift from Dr K. Ueda of the
Department of Pediatrics and Child Health of Kurume
University. The human hepatoblastoma cell line (HuH-6)
was purchased from the Japan Health Sciences Foundation
Observation of morphological changes. For light microscopic
observations, the cells were seeded on Lab-Tek Tissue Culture
Chamber Slides (Nunc Inc., Roskilde, Denmark), cultured
with or without fucoidan (2.82, 22.5 or 90 μg/ml) for 72 h,
fixed in Carnoy's solution for 10 min, then stained with hema-
toxylin and eosin (H&E) and observed under a microscope
(Olympus BH-2, Olympus Optical, Tokyo, Japan).
Effect of fucoidan on the proliferation of each cell line. The
effect of fucoidan on cell proliferation was investigated with
colorimetric assays using MTT [3-(4,5-dimethylthiazol-2yl-yl)-
2,5-diphenyltetrazolium bromide] cell growth assay kits
(Chemicon International Inc., Temecula, CA) as previously
described (23). Briefly, cells were seeded on 96-well plates
(Falcon, Becton Dickinson Labware, Tokyo, Japan) and
cultured for 24 h. The medium was then replaced with 100 μl
of fresh medium alone or containing the diluted fucoidan
solution (0.35, 0.70, 1.40, 2.82, 5.63, 11.25, 22.50, 45, 90 or
180 μg/ml). After 24, 48, 72 or 96 h, the number of viable
cells was counted. Each experiment was repeated at least
twice. The 50% inhibitory concentration (IC50) was defined as
the fucoidan concentration (μg) that caused a 50% reduction
FUKAHORI et al: FUCOIDAN PROHIBITS CANCER CELL GROWTH
Figure 1. Photomicrograph of HAK-1B (A and B), KIM-1 (C and D) and KMC-1 (E and F) cells cultured for 72 h on a Lab-Tek Chamber slide. (A, C and E)
No fucoidan in culture medium. Some mitotic figures were noted. (B, D and F) With 22.5 μg of fucoidan in culture medium. Apoptotic cells were character-
ized by cytoplasmic shrinkage, chromatic condensation and nuclear fragmentation (H&E staining, x200).
in cellular viability. The IC50value was calculated and used as
a parameter in the comparison of the relative cytotoxicity of
each cell line.
Quantitative analysis of fucoidan-induced apoptosis. Six HCC
cell lines were cultured with or without fucoidan (22.5 or
90 μg/ml) for 72 h and then stained with Annexin V-EGFP
(enhanced green fluorescent protein) using Apoptosis Detection
Kits (Medical & Biological Laboratories, Nagoya, Japan)
according to the manufacturer's protocol. After staining, the
cells were analyzed using a FACScan (Becton Dickinson
Immunocytometry Systems, San Jose, CA), and the percentage
of Annexin V-EGFP-positive cells was determined.
Cell cycle analysis. Three HCC cell lines (HAK-1A, KYN-2,
KYN-3) were cultured with or without fucoidan (22.5 μg/ml)
for 24, 48 or 72 h, labeled with 10 μmol/l BrdUrd for 30 min,
fixed in 70% cold ethanol at 4˚C overnight, and stained with
anti-BrdUrd antibody and propidium iodide (Sigma Chemical
Co., St. Louis, MO) using a previously described technique
(23). The stained cells were analyzed by FACScan using the
CellQuest software program (ver. 3.3, Becton Dickinson). The
distribution of cells in the G0/G1, S, or G2/M phase of the cell
cycle was calculated and shown as a percentage of each phase.
Statistical analysis. All data were expressed as the means ± SD.
For data analysis, the Student's t-test was used. A P-value
<0.05 was considered to be statistically significant.
Effect of fucoidan on morphological changes. Cell morphology
72 h after the addition of fucoidan solution was observed using
H&E staining. The cell density in culture decreased dose-
dependently (except for neuroblastoma) in order from HCC
(KIM-1, HAK-1A, HAK-1B, KYN-1, KYN-2, KYN-3),
cholangiocarcinoma (KMC-1), gallbladder carcinoma
(KMG-C), renal cell carcinoma (KURU II, KURM, OSRC2),
to ovarian cancer (KOC-5C, KOC-7C). The cell density of
the neuroblastoma cell line (SK-N-SE) did not decrease at
any concentration of fucoidan. As shown in Fig. 1, 3 HCC
cell lines (HAK-1B, KIM-1 and KMC-1) presented dose-
dependent apoptotic changes such as nuclear condensation,
cell shrinkage and nuclear fragmentation.
Effects of fucoidan on cell proliferation. MTT assay revealed
chronological and dose-dependent suppression of the prolifer-
ation of 4 cell lines (HAK-1B, KIM-1, KMG-C and KMC-1),
chronological suppression in 9 cell lines (KYN-1, KYN-2,
KYN-3, HAK-1A, KOC-5C, HuH-6, KURM, OSRC2,
KURU II), and no suppression in 2 cell lines (KOC-7C, SK-
N-SE) (Fig. 2).
The IC50values at 72 h of culture ranged from 18.71 to
299.20 μg/ml. Levels at 72 h could not be obtained for
KMG-C, KMC-1, HAK-1B and KYN-2 because >50% sup-
pression occurred; however, at 48 h the IC50was 13.33 μg for
MOLECULAR MEDICINE REPORTS 1: 537-542, 2008
Figure 2. Antiproliferative effect of fucoidan (A-C) 72 h after 0.35, 0.70, 1.40, 2.82, 5.63, 11.25, 22.50 or 45 μg were added. Cell proliferation was suppressed in
a dose-dependent manner in the 4 cell lines (KIM-1, HAK-1B, KMG-C, KMC-1), but not in the other 11 cell lines. The values represent the means ± SE of the
experiments. The experiment was repeated at least twice for each cell line. (D-F) Chronological changes in the relative viable cell number (% of the control) after
adding 22.5 μg of fucoidan. A time-dependent growth inhibition was observed in 13 cell lines with the exception of KOC-7C, and growth was suppressed over
time to varying degrees.
KMC-1, 50.64 μg for HAK-1B, 52.32 μg for KYN-2 and
76.25 μg for KMG-C. According to tumor type, the IC50was
lowest in HCC and cholangiocarcinoma (Fig. 3).
Effect of fucoidan on apoptosis. Apoptosis was examined at
72 h of culture in the 6 cell lines that presented pronounced
growth suppression in the MTT assay. The number of apop-
totic cells increased significantly in the 5 HCC cell lines with
the exception of KYN-1, indicating that fucoidan induced
apoptosis (Fig. 4).
Effect of fucoidan on the cell cycle. Flow cytometry of the 3
HCC cell lines (HAK-1A, KYN-2, KYN-3) revealed an
increased number of cells in the G2/M phase at 72 h after the
addition of the fucoidan solution (22.5 μg/ml) to the culture
The current study examined the anti-tumor effect of Okinawa
mozuku fucoidan on 15 human cancer cell lines. Chronological
and/or dose-dependent suppression of cell proliferation was
observed in 13 of the 15 lines (87%). Previous studies have
reported direct anti-tumor effects of fucoidan on HTLV-1-
infected T cell lines and primary ATL cells (24), lymphoma
cells (25) and a bronchopulmonary carcinoma cell line
(NSCLC-N6) (4). The various possible mechanisms of
fucoidan have also been explored, such as its anti-tumor
effect induced by anti-angiogenesis (5), growth suppression
due to immunopotentiation (26), and the suppression of
metastasis (27,28), but only on one cell line. This current
study investigated the effects had by fucoidan from the same
source at the same time on multiple cell lines.
The major components of fucoidan are L-fucose and sulfate
content. Previous studies used fucoidan extracted from Fucus
vesiculosus (25), Ascophyllum nodosum (4,7), Sargassum
kjellmanianum (29), Sargassum thunbergii (26) or Cladosiphon
okamuranus Tokida (24), in which the percentage of L-fucose
ranged from 12.6 to 36.0%, and the percentage of sulfate
content from 8 to 25%. Sulfate content was also reported to be
a factor with growth suppression effects (4,29). The fucoidan
solution used in the current study contained these 2 substances
within the above-mentioned ranges.
In this study, the suppression of cell proliferation was
more apparent in the cell lines of HCC, cholangiocarcinoma
and gallbladder carcinoma than in those of neuroblastoma,
hepatoblastoma, ovarian carcinoma and renal carcinoma. The
growth suppression effects also varied among the cell lines of
the same tumor type. The IC50values of the HAK-1B (HCC)
and KMC-1 (cholangiocarcinoma) cell lines were additionally
markedly lower in comparison to previously reported data
(4,25). These findings indicate that the anti-tumor effects of
fucoidan vary according to tumor type and, along with previous
findings, demonstrate the anti-tumor effects of fucoidan on
colon cancer but not on mammary tumors (30). This indicates
that the supression of cell proliferation does not occur in all
cancer cell lines. The mechanism behind the more potent
growth suppression observed in the HCC and cholangiocarci-
noma cell lines should therefore be explored in future studies.
FUKAHORI et al: FUCOIDAN PROHIBITS CANCER CELL GROWTH
Figure 3. The IC50of 15 cell lines treated with fucoidan for 72 h. *The IC50
values of KMG-C, KMC-1, HAK-1B and KYN-2 cell lines at 72 h could not
be analyzed because the viable cell number was suppressed to <50% for all
concentration levels of fucoidan.
Figure 4. An analysis of apoptosis in 6 hepatocellular carcinoma cell lines
treated with or without fucoidan for 72 h. HAK-1A, HAK-1B, KIM-1, KYN-1,
KYN-2 and KYN-3 cell lines were treated with or without fucoidan (22.5 or
90 μg) for 72 h. The cells were harvested, labeled with Annexin-V-FITC and
then analyzed by flow cytometry. (A) Percentage of apoptotic cells in the
KYN-3 cell line. (B) Data represent the average (± SE) percentage of apoptotic
cells. *P<0.001 vs. untreated cells.
Haneji et al (24) reported that apoptosis is induced by the
activation of the caspase pathway, and Aisa et al (25) demon-
strated anti-tumor effects accompanied by the activation of
the caspase pathway and the down-regulation of the ERK
pathway. In the current study, 5 of the 6 HCC cell lines pre-
sented a significant dose-dependent increase in apoptosis. The
activation of caspase-3 and -9 in HAK-1B, which presented
marked apoptosis, was therefore investigated. However, no
clear activation was observed (data not shown), indicating that
the anti-tumor effects of fucoidan on HCC cell lines could be
associated with a different pathway.
With regard to cell cycle effect, Haneji et al (24) reported
that fucoidan induced the accumulation of cells in the G1/S
phase, and Riou et al (4) observed that anti-tumor effects
were accompanied by a G1phase block. On the other hand,
Aisa et al (25) reported no effect on the cell cycle. The current
findings regarding the cell cycle demonstrate for the first
time an increase in cells in the G2/M phase.
In the current study, fucoidan suppressed cell proliferation
in a time- and dose-dependent manner at various degrees, and its
effects were particularly pronounced in the HCC, cholangio-
carcinoma and gallbladder carcinoma cell lines. The results
indicate that the mechanisms of fucoidan action include the
induction of apoptosis and the inhibition of the cell cycle.
At present, the clinical results of treatment for advanced
HCC and cholangiocarcinoma are unsatisfactory. Fucoidan
could be a potential anti-tumor remedy for specific cancers,
such as HCC or cholangiocarcinoma. More detailed informa-
tion on the anti-tumor effects of fucoidan should therefore be
obtained in future animal studies.
We thank Ms. Akemi Fujiyoshi for her valuable assistance
with our experiments.
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MOLECULAR MEDICINE REPORTS 1: 537-542, 2008
Table I. Flow cytometric analysis of the effect of fucoidan (22.5 μg/ml) on the cell cycle of hepatocellular carcinoma cell lines
at 24, 48 and 72 h of culture.
Control, cells cultured without fucoidan. Fucoidan, cells cultured with fucoidan (22.5 μg/ml). Values represent the percentage of cells at each
phase of the cell cycle.
48 h 72 h
ControlCell cycle Control FucoidanFucoidanFucoidan
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FUKAHORI et al: FUCOIDAN PROHIBITS CANCER CELL GROWTH