そらたね祭

These results indicated for the first time that fucoxanthin
reduces the viability of human colon cancer cells by
inducing apoptosis. A similar induction of apoptosis in
colon cancer cells has been reported for h-carotene and
cantaxanthin [14–16]. However, in this study, h-carotene
and astaxanthin did not reduce cell viability or induce
apoptosis in Caco-2, even after 48 h of incubation at a
concentration of 7.6 AM, whereas fucoxanthin, at the same
concentration, significantly increased DNA fragmentation.
One possible reason for this might be the concentrations of
Fig. 6. Expression of Bcl-2 protein in Caco-2 cells treated with fucoxanthin.
Caco-2 cells were incubated in culture medium containing 22.6 AM
fucoxanthin for 48 or 72 h. Cellular protein was extracted, and levels of
Bcl-2 protein were detected using Western blot analysis.
Fig. 7. Combined effect of fucoxanthin and troglitazone on viability of Caco-2 cells. All values presented are calculated considering control as 100% survival.
(A) Caco-2 cells were incubated in culture medium containing 100 AM troglitazone for 24 or 48 h. Cell viability was measured by WST-1 assay. Values are
meansFS.D., n=3. The asterisk indicates a value significantly different from the control value ( Pb0.01) (Student’s t test). (B) Caco-2 cells were incubated in
culture medium containing 3.8 AM fucoxanthin for 24 h and then troglitazone was added into culture medium. After additional 24 or 48 h of incubation, cell
viability was measured by WST-1 assay. Values are meansFS.D., n=3. In each incubation time, the values with different letters were significantly different
from each other. Pb0.01 (Scheffe’s F-test).
M. Hosokawa et al. / Biochimica et Biophysica Acta 1675 (2004) 113–119 117
the two carotenoids (h-carotene and canthaxanthin) used in
the current study, which were too low to induce apoptosis
in Caco-2 cells. In addition, fucoxanthin is converted to
fucoxanthinol during the uptake by Caco-2 cells [29].
Since fucoxanthinol exhibits stronger growth inhibition
than fucoxanthin, the antiproliferative effect of fucoxanthin
may be attributable to the action of its metabolites
[30].
Caspases are known to play an important role in
inducing apoptosis [31]. DNA fragmentation in Caco-2
cells treated with fucoxanthin was significantly diminished
by a general caspase inhibitor, Z-VAD-fmk. However, since
Z-VAD-fmk diminished DNA fragmentation by only 40%,
it is suggested that the apoptosis signaling in Caco-2 cell by
fucoxanthin is mediated by both caspase-dependent and
-independent pathways. Caspase-independent apoptosis has
been reported by other investigators [32]. In addition,
exposure to fucoxanthin decreased the level of the apoptosis-
suppressing protein, Bcl-2. This indicates that the downregulation
of Bcl-2 protein may contribute to fucoxanthininduced
apoptosis in Caco-2 cells.
Fucoxanthin has a unique structure including an unusual
allenic bond and 5,6-monoepoxide in its molecule (Fig. 1).
Epoxy-h-carotene, neoxanthin, halocynthiaxanthin and
fucoxanthin containing epoxide in their molecules induced
a remarkable reduction in the growth of leukemia and
prostate cancer cells [10–12,33]. The structure of carotenoids
may be of importance in the reduction of growth and
in apoptosis induction in cancer cells. Previous studies have
reported the antioxidant activity of fucoxanthin [34–36]. In
contrast, the pro-oxidant action of carotenoids is shown to
induce apoptosis through the production of reactive oxygen
species [37]. This suggests that carotenoids act either as an
antioxidant or as a pro-oxidant, depending on their environment.
Fucoxanthin may also regulate the redox signals, and
then facilitate the progression of apoptosis through Bcl-2
protein suppression and the caspase-dependent and -independent
pathway.
Our study demonstrates the combination effect of
fucoxanthin and troglitazone on the reduction of Caco-2
cell viability. Pre-incubation of Caco-2 cells with fucoxanthin
remarkably enhanced the effect of troglitazone. Troglitazone
is known to inhibit cell growth and induce apoptosis
through the activation of PPARg [20–24]. Recently, oral
administration of troglitazone has been reported to inhibit the
early stage of colon tumorigenesis [25,26]. The combined
action of PPARg ligand and fucoxanthin may provide
new perspectives in developing novel chemopreventive
approaches.
In conclusion, fucoxanthin decreased cell viability and
induced apoptosis in the human colon cancer cell line, Caco-
2. Since the expression level of Bcl-2 protein was decreased
in Caco-2 cells treated with fucoxanthin, its down-regulation
may contribute to fucoxanthin-induced apoptosis. Further,
fucoxanthin enhanced the antiproliferative effect of a PPARg
ligand, troglitazone. Our findings indicate the possibility of a
chemopreventive or chemotherapeutic effect of fucoxanthin,
with or without troglitazone, on colon cancer.
Acknowledgements
This work was partly supported by PROBRAIN Project
from Bio-oriented Technology Research Advancement
Institution and carried under the Core University Program
on Fisheries Sciences between Japan and Korea (FiSCUP),
with the support of JSPS and KOSEF.
References

mg/ml phenylmethylsulfonyl fluoride, 50 Ag/ml aprotinin
and 1 mM Na3VO4. Further, cell lysates were centrifuged at
4 8C, 15,000 rpm for 20 min. The supernatants (40 Ag
protein/lane) were then separated by 10% SDS-polyacrylamide
gel electrophoresis. Proteins were transferred to
polyvinylidene difluoride membrane, and membrane was
then blocked with TBS-T (20 mM Tris–HCl (pH 7.6), 137
mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry
milk for 1 h at room temperature. The membrane was
incubated with anti-human Bcl-2 antibody for 1 h. After
washing, the membranes were incubated with a secondary
antibody, anti-mouse IgG-HRP (Santa Cruz Biotechnology),
for 1 h at room temperature. Finally, the membrane was
treated with the reagents in the chemiluminescence detection
kit (ECL system, Amersham Pharmacia Biotech,
Piscataway, NJ, USA) according to the manufacturer’s
instructions. Actin was used as the control with human
actin antibody (Santa Cruz Biotechnology).
2.7. Combined treatment with fucoxanthin and troglitazone
Caco-2 cells (2_103 cells/well) were cultured in 96-well
microplate with 100-Al medium per well for 24 h.
Fucoxanthin was added into culture medium as described
in cell viability assay and incubated for an additional 24 h.
Then, 10 AM troglitazone was added into culture medium as
DMSO solution, followed by incubation for 24–48 h. Cell
viability was measured by WST-1 assay as described above.
2.8. Statistical analysis
Data are expressed as meansFS.D. Statistic analyses
between multiple groups were determined by ANOVA.
Statistical comparisons were made by Scheffe’s F-test or
Tukey’s test. Analysis between two groups was determined
using the unpaired Student t test. Differences with Pb0.01
or Pb0.05 were considered significant.
3. Results
3.1. Effect of fucoxanthin on the viability of human colon
cancer cells
Fucoxanthin remarkably decreased the viability of Caco-2
cells. After 72 h of incubation with 7.6 AM fucoxanthin, the
number of viable cells decreased by 39% compared to the
control (Fig. 2). On the other hand, incubation with 7.6 AM
h-carotene and astaxanthin, which are major carotenoids in
natural products, did not alter the viability of Caco-2 cells.
We conducted a detailed investigation of the effects of
fucoxanthin on colon cancer cells. Fucoxanthin reduced the
cell viability of three human colon cancer cell lines, Caco-2,
DLD-1 and HT-29 cells, in a dose- and time-dependent
manner (Table 1). After 72 h of incubation, 15.2 AM
fucoxanthin significantly reduced the viability of Caco-2,
DLD-1 and HT-29 cells to 14.8%, 29.4% and 50.8%,
respectively, in relation to the control. The viability of
DLD1 and HT-29 cells was not altered by incubation with
7.6 AM fucoxanthin for 72 h, while the viability of Caco-2
cells was reduced to 36.8% compared to control. The level
of sensitivity against fucoxanthin of the cells tested was as
follows: Caco-2NDLD-1NHT-29.
3.2. Induction of apoptosis by fucoxanthin treatment
The morphological evidence in Caco-2 cells treated with
11.6 AM fucoxanthin for 72 h indicated a diminished size
Fig. 2. Comparison of viability of Caco-2 cells incubated with fucoxanthin,
astaxanthin or h-carotene. Caco-2 cells were incubated with 7.6 AM each
carotenoid for 72 h. Cell viability was measured by WST-1 assay. Values
are meansFS.D., n=3–4. The asterisk indicates a value significantly
different from the control value ( Pb0.01) (Student’s t test).
Table 1
Effect of fucoxanthin on cell viability of human colon cancer cell lines
Colon cancer
cell lines
24-h incubation 72-h incubation
7.6 AM 15.2 AM 7.6 AM 15.2 AM
Caco-2 79.6F2.0a,c 56.1F4.9b 36.8F4.3a 14.8F0.4c
HT-29 93.1F2.9a 82.2F0.5a,c 93.7F1.1b 50.8F0.6d
DLD-1 88.5F1.5a 70.9F4.4c 103.8F5.2b 29.4F2.3a
Colon cancer cells were incubated with 7.6 or 15.2 AM fucoxanthin.
Cell growth was measured by WST-1 assay.
Values are meanFS.D. (n=3).
The interaction of concentration and cell line was significant for both 24-h
incubation and 72-h incubation.
The values with different letters were significantly different from each
other, Pb0.01 (Tukey’s test).
M. Hosokawa et al. / Biochimica et Biophysica Acta 1675 (2004) 113–119 115
and rounded shape (data not shown). Further, the cell
membrane had shrunk with a condensed cytoplasm. The
morphological appearance of Caco-2 cells treated with
fucoxanthin has the properties observed in apoptotic cells.
To prove that apoptosis induction occurred by means of
fucoxanthin, DNA fragmentation in Caco-2 cells was
measured as an indicator of apoptosis with quantitative
sandwich ELISA using an anti-histone antibody and an anti-
DNA antibody. Fucoxanthin induced a dose-dependent
increase in cellular DNA fragmentation (Fig. 3). When
Caco-2 cells were incubated in culture medium with 22.6 AM
fucoxanthin for 24 h, a 10-fold increase in DNA fragmentation
over that of control cells was observed. Conversely, hcarotene
and astaxanthin did not induce DNA fragmentation
in Caco-2 cells during 48 h of incubation at a concentration of
7.6 AM, whereas fucoxanthin increased DNA fragmentation
even at a concentration of 7.6 AM (Fig. 4).
3.3. Involvement of caspases and Bcl-2 protein in apoptosis
induced by fucoxanthin
To investigate the involvement of the caspase pathway,
Caco-2 cells were treated with a broad spectrum caspase
inhibitor, Z-VAD-fmk, together with fucoxanthin for 24 h.
As shown in Fig. 5, DNA fragmentation induced by
fucoxanthin was significantly diminished by Z-VAD-fmk.
However, the suppression of DNA fragmentation by ZVAD-
fmk was only 40%.
We also evaluated the expression of Bcl-2 protein, which
suppresses programmed cell death as a survival factor
[27,28]. The Bcl-2 protein level in Caco-2 cells was
decreased remarkably by treatment with 22.6 AM fucoxanthin
(Fig. 6). After 72 h of incubation, the relative
expression level of Bcl-2 protein vs. actin was less than 20%
that of the control cells.
Fig. 3. DNA fragmentation in Caco-2 cells treated with fucoxanthin. Caco-2 cells were incubated in culture medium containing fucoxanthin. DNA
fragmentation was measured by a sandwich enzyme immunoassay using anti-histone antibody and anti-DNA antibody. Values are meansFS.D., n=3. In each
incubation time, the values with different letters were significantly different from each other. Pb0.01 (Scheffe’s F-test).
Fig. 4. Comparison of DNA fragmentation in Caco-2 cells incubated with
fucoxanthin, astaxanthin or h-carotene. Caco-2 cells were incubated in
culture medium containing 7.6 AM each carotenoid for 48 h. DNA
fragmentation was measured by the sandwich enzyme immunoassay using
anti-histone antibody and anti-DNA antibody. Values are meansFS.D.,
n=3. The values with different letters were significantly different from each
other. Pb0.01 (Scheffe’s F-test).
Fig. 5. Effect of caspase inhibitor, Z-VAD-fmk, on apoptosis in Caco-2 cells
induced by fucoxanthin. Caco-2 cells were incubated in culture medium
containing 22.6 AM fucoxanthin and/or 10 AM Z-VAD-fmk for 24 h. DNA
fragmentation was measured by the sandwich enzyme immunoassay. Values
are meansFS.D., n=3. The values with different letters were significantly
different from each other. Pb0.05 (Scheffe’s F-test).
M. Hosokawa et al. / Biochimica et Biophysica Acta 1675 (2004) 113–119 116
3.4. Combined effect of fucoxanthin and troglitazone on the
viability of Caco-2 cells
We determined the combined effect of fucoxanthin and
troglitazone on the viability of Caco-2 cells. In previous
studies, troglitazone was reported to reduce cell growth
and to induce apoptosis in colon cancer cells [23].
However, in the previous study, the concentration of
troglitazone that was effective in reducing cell growth in
the presence of 10% FBS was more than 100 AM. In Fig.
7, 100 AM troglitazone caused a decrease in cell viability
to less than 50% after 48 h of incubation. However, a
concentration of troglitazone lower than 10 AM troglitazone
did not affect cell viability at all. In contrast, a
combined treatment with 3.8 AM fucoxanthin and 10 AM
troglitazone resulted in a notable reduction of the viability of
Caco-2 cells, whereas 3.8 AM fucoxanthin alone did not
affect cell viability (Fig. 7).
4. Discussion
In the current study, we demonstrated for the first time
that fucoxanthin induces apoptosis in human colon cancer
cells. Moreover, the complementary relation between
fucoxanthin and troglitazone in reducing the viability of
colon cancer cells has been demonstrated.
Fucoxanthin remarkably reduced the viability of human
colon cancer Caco-2 cells. Further, fucoxanthin inhibited
the viability of other human colon cancer cell lines, HT-29
and DLD-1, although the sensitivity for fucoxanthin
among the three was different. In addition, dose-dependent
DNA fragmentation was observed in Caco-2 cells treated
with fucoxanthin. These effects were correspondingly
reflected in the reduction of cell viability. Internucleosomal
degradation of DNA, due to the activation of endogenous
endonuclease, is one feature that occurs during apoptosis.

Fucoxanthin induces apoptosis and enhances the antiproliferative effect
of the PPARg ligand, troglitazone, on colon cancer cells
Masashi Hosokawaa,*, Masahiro Kudoa, Hayato Maedaa, Hiroyuki Kohnob,
Takuji Tanakab, Kazuo Miyashitaa
aGraduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hakodate, Hokkaido 041-8611, Japan
bDepartment of Pathology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
Received 14 January 2004; received in revised form 10 August 2004; accepted 26 August 2004
Available online 11 September 2004
Abstract
The effect of fucoxanthin, from the edible seaweed Undaria pinnatifida on viability of colon cancer cells and induction of apoptosis was
investigated. Fucoxanthin remarkably reduced the viability of human colon cancer cell lines, Caco-2, HT-29 and DLD-1. Furthermore,
treatment with fucoxanthin induced DNA fragmentation, indicating apoptosis. The DNA fragmentation in Caco-2 cells treated with 22.6 AM
fucoxanthin for 24 h was 10-fold higher than in the control. Fucoxanthin suppressed the level of Bcl-2 protein. Also, DNA fragmentation
induced by fucoxanthin was partially inhibited by a caspase inhibitor Z-VAD-fmk. Moreover, combined treatment with 3.8 AM fucoxanthin
and 10 AM troglitazone, which is a specific ligand for peroxisome proliferator-activated receptor (PPAR) g, effectively decreased the viability
of Caco-2 cells. However, separate treatments with these same concentrations of fucoxanthin nor troglitazone did not affect cell viability.
These findings indicate that fucoxanthin may act as a chemopreventive and/or chemotherapeutic carotenoid in colon cancer cells by
modulating cell viability in combination with troglitazone.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Fucoxanthin; Colon cancer cell; Antiproliferative effect; Apoptosis; PPARg
1. Introduction
Colon cancer is one of the most malignant neoplasia in the
world [1]. Colon carcinogenesis is considered to be linked
with dietary habits like high animal fat intake [2]. In contrast,
a number of studies have suggested that high consumption of
fruit and vegetables decreases the risk of colon cancer [3,4].
Among various plant constituents, carotenoids such as hcarotene
and lycopene have been extensively studied and
implicated as cancer preventive agents [5,6]. Fucoxanthin is
found in edible brown algae such as Undaria pinnatifida
and, along with h-carotene, is one of the most abundant
carotenoids found in nature [7]. Recently, fucoxanthin has
also been focused on as an anticancer carotenoid [8].
Previous studies have reported that fucoxanthin causes cell
growth inhibition of human neuroblastoma GOTO cells,
human leukemia cells, and prostate cancer cells [9–12].
Additionally, a chemopreventive effect of fucoxanthin on the
development of aberrant crypt foci in the colons of mice has
also been reported [13]. The antiproliferative effect of
fucoxanthin is stronger than the effects of h-carotene and
lycopene. However, the mechanism by which fucoxanthin
suppresses colon carcinogenesis is not fully understood.
It was reported that cantaxanthin and h-carotene inhibit
the growth of human colon cancer cell lines by inducing
apoptosis [14–16]. We and others have already reported that
fucoxanthin induces apoptosis in human leukemia and
prostate cancer cells [11,12]. However, there is as yet no
report of fucoxanthin inducing apoptosis in colon cancer
cells. An understanding of the underlying mechanism of the
induction of apoptosis by fucoxanthin will benefit the
development of chemopreventives and/or chemotherapeutics
for colon cancer.
0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2004.08.012
* Corresponding author. Tel.: +81 138 40 5530; fax: +81 138 40 5530.
E-mail address: hoso@fish.hokudai.ac.jp (M. Hosokawa).
Biochimica et Biophysica Acta 1675 (2004) 113– 119
http://www.elsevier.com/locate/bba
Recently, we have focused extensively on the ability of
peroxisome proliferator-activated receptor (PPAR) g to
suppress colon carcinogenesis [17–19]. Troglitazone has
been reported to induce growth inhibition and apoptosis in
colon cancer cells [20–24]. Furthermore, we reported that
PPAR ligands, including troglitazone, inhibit the early
stages of colon tumorigenesis, which is induced by azoxymethane
and dextran sodium sulfate [25,26]. To enhance the
effects of PPARg ligands and to reduce their toxic side
effects on normal cells, a combination of lower doses of
fucoxanthin and PPARg ligand may be effective.
In the present study, we demonstrate for the first time that
fucoxanthin, not h-carotene or astaxanthin, reduces the
viability of colon cancer cells and induces apoptosis in
colon cancer cells. Fucoxanthin reduced the expression of
Bcl-2 protein in Caco-2 cells. In addition, combined
treatment with fucoxanthin and troglitazone resulted in a
strong reduction of Caco-2 cell viability.
2. Methods and materials
2.1. Materials
h-Carotene and astaxanthin were purchased from Sigma
Chemical (St. Louis, MO, USA). Troglitazone was a kind
gift from Sankyo Co. (Tokyo, Japan). Dulbecco’s modified
Eagle’s medium, RPMI 1640 medium and antibiotics were
purchased from Gibco (Grand island, NY, USA). Fetal
bovine serum (FBS) was obtained from ThermoTrace
(Melbourne, Australia). A caspase inhibitor, Z-VAD-fmk,
was purchased from Clontech Laboratories, Inc. (Palo Aito,
CA, USA). Monoclonal antibodies against Bcl-2 was
obtained from Santa Cruz Biotechnology (Santa Cruz,
CA, USA).
2.2. Cell lines and cell culture
Colon cancer cell lines, Caco-2 (ATCC HTB-37), HT-29
(ATCC HTB-38) and DLD-1 (ATCC CCL-21), were
obtained from the American Type Culture Collection
(Rockville, CT, USA). Caco-2 cells were cultured in
minimum essential medium (MEM) supplemented with
10% fetal bovine serum (FBS), 1% nonessential amino
acid, 100 U/ml penicillin and 100 Ag/ml streptomycin. HT-
29 and DLD-1 were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) and RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 U/
ml penicillin and 100 Ag/ml streptomycin, respectively. Cell
cultures were maintained in a humidified atmosphere of
95% air and 5% CO2 at 37 8C.
2.3. Preparation of fucoxanthin
Fucoxanthin(3V-acetoxy-5,6-epoxy-3,5V-dihydroxy-6V7Vdidehydro-
5,6,7,8, 5V,6V-hexahydro-hh-caroten-8-on) was
extracted from U. pinnatifida as previously reported [11].
Purification of fucoxanthin was finally carried out by lowpressure
liquid chromatography equipped with RP-8 column
with methanol/H2O (17:3, v/v). The purity of isolated
fucoxanthin was more than 98% by HPLC analysis
equipped with ODS column. The chemical structure of
carotenoids used in the present study is shown in Fig. 1.
2.4. Cell viability assay
Human colon cancer cells (2_103 cells/well) were
cultured in a 96-well microplate with 100-Al medium per
well for 24 h. Fucoxanthin was dissolved in ethanol and
then 5% ethanol solution was prepared using medium. Ten
microliters of carotenoid solution (5% ethanol solution) was
added into culture medium. h-Carotene and astaxanthin
were added into the culture medium as described above
using tetrahydrofuran or dimethyl sulfoxide (DMSO)
instead of ethanol. Cell viability was assessed with WST-1
regent (Wako Pure Chemical, Osaka, Japan). This assay is
based on cleavage of the tetrazolium salt WST-1 by
mitochondrial dehydrogenase of viable cells to formazan
dye. A number of viable cells were measured colorimetrically
and expressed as percentage of viability in relation to
control cultures.
2.5. Measurement of DNA fragmentation
Level of fragmented DNA was measured as an indicator
of apoptotic cell death. This was performed using a
commercial kit (Cell Death Detection ELISA, Roche
Diagnostics GmbH, Mannheim, Germany) according to
the manufacturer’s instructions. The assay is based on a
quantitative sandwich enzyme immunoassay to detect the
histone-associated DNA fragments produced during apoptosis.
Cell culture conditions were the same as in WST-1
assay.
Fig. 1. Structure of the carotenoids used in the current study.
M. Hosokawa et al. / Biochimica et Biophysica Acta 1675 (2004) 113–119 114
2.6. Western blot analysis
Caco-2 cells (1.5_106 cells) were cultivated in 150-mm
tissue cultured dish for 24 h and fucoxanthin was then added
into culture medium as ethanol solution. The final ethanol
concentration was below 0.1% (v/v). After incubation,
adherent cells were trypsinized and washed three times
with phosphate buffered saline (PBS). Pellet was then
scraped in a cold RIPA buffer (pH 7.4) containing 20 mM
Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1