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
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