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
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