British Journal of Pharmacology and Toxicology 2(3): 127-131, 2011
ISSN: 2044-2467
© Maxwell Scientific Organization, 2011
Received: April 10, 2011
Accepted: May 13, 2011
Published: August 05, 2011
Simultaneous Treatment of Cancer Cells Lines with the Anticancer
Drug Cisplatin and the Antioxidant Fucoxanthin
Takeshi Mise and Takeshi Yasumoto
Okinawa Science and Technology Promotion Center, Marine Bioindustry Division,
Okinawa, Japan
Abstract: The anti-oxidative properties and the other health benefits of fucoxanthin (Fx) make it a good
candidate for a health food supplement. However, the use of antioxidant supplements in patients undergoing
chemotherapy has been widely debated because of concerns that the antioxidants may interfere with the
mechanisms of the anticancer drugs. To investigate this concern, we studied the effect of Fx on the anticancer
drug cisplatin. Fucoxanthinol (Fxol), which is a deacetylated product produced during intestinal absorption,
and other antioxidants (fucoxanthin acetate, astaxanthin, and "-tocopherol) were also included in the
experiments. Both Fx and Fxol suppressed the proliferation of cultured cancer cells (Caco-2, Hep G2, and
Neuro2a) in a dose-dependent manner, whereas the other antioxidants showed no evident effect on their growth.
Moreover, when added to a medium containing cisplatin, none of the studied antioxidants mitigated the
inhibitory growth effects of cisplatin. Our results suggested that intake of Fx may help to prevent cancer and
that it may not interfere with the mechanisms of anticancer drugs during chemotherapy. However, further
studies, using animal models in particular, are required to elucidate these interactions.
Key words: Anticancer agent, carotenoid, fucoxanthinol
INTRODUCTION
Cisplatin is one of the most potent anticancer drugs
used for cancer therapy (Boulikas and Vougiouka, 2003)
and has been shown to induce nephrotoxicity through
oxidative stress (Yao et al., 2007). For investigating the
effects of Fx on chemotherapy, the interaction of cisplatin
and Fx was investigated. Fucoxanthinol (Fxol), which is
a de-acetylated product generated from the hydrolysis of
Fx during intestinal absorption, was also investigated for
its interaction with cisplatin. Additional antioxidants
(fucoxanthin acetate, astaxanthin, and "-tocopherol) were
also investigated in order to compare their effects with Fx
and Fxol.
Our cultured cell (Caco-2, Hep G2, and Neuro2a)
studies showed that Fx and Fxol increased the level of
cytotoxicity in a dose-dependent manner. However,
fucoxanthin acetate (Fxac), astaxanthin, and "-tocopherol
had no evident cytotoxic effects. In addition, although the
antioxidants were potent, they did not interfere with the
mechanism of action of cisplatin. Further studies will be
necessary to clarify the effect of Fx during chemotherapy,
especially in animal experiments.
Fucoxanthin (Fx) is a major marine carotenoid found
in brown seaweeds (Chandini et al., 2008). Recently, an
Fx-based functional food has been developed (Mise et al.,
2011). Fx exhibits anti-carcinogenic, anti-obesity, antiinflammatory, anti-angiogenic, and anti-oxidative
activities (Okuzumi et al., 1993; Maeda et al., 2005;
Shiratori et al., 2005; Sugawara et al., 2006; Heo et al.,
2008). Moreover, orally administered Fx is safe in terms
of mutagenic potential (Beppu et al., 2009). Though Fx is
a useful marine carotenoid, no studies have described the
use of the antioxidant Fx in patients undergoing
chemotherapy. A great deal of debate has focused on the
use of antioxidant supplements in patients undergoing
chemotherapy because of the concerns that the
antioxidants may interfere with the mechanisms of
anticancer drugs and subsequently decrease their efficacy.
Some cancer patients have understood from a general
perspective that "supplements protect the body from
oxidative damage, and the mechanism for protection
against chemotherapy is well understood." However,
Gabriella and Andrea (2005) have reported that multiple
lines of evidence from extensive human studies are
needed before patients are advised to take antioxidants
during cytotoxic therapy. Nonetheless, the evidence from
different studies has suggested that antioxidant
supplements may reduce the toxic side effects of ROSgenerating chemotherapies (Block et al., 2008).
MATERIALS AND METHODS
Fucoxanthin, fucoxanthinol, and fucoxanthin acetate:
Fucoxanthin (Fx) was extracted from Undaria pinnatifida,
and fucoxanthinol (Fxol) and fucoxanthin acetate (Fxac)
were synthesized in laboratory (Fig. 1). Fx was isolated
from brown seaweed Undaria pinnatifida in May 2009, as
Corresponding Author: Takeshi Mise, Okinawa Polytechnic College, Microcomputer Control Technology Department,
Okinawa, Japan
127
Br. J. Pharmacol. Toxicol., 2(3): 127-131, 2011
Fig. 1: (a) A fucoxanthin crystal as observed by a light microscope. The scale bar represents 20 :m. (b) An Fx crystal as observed
by a stereomicroscope. The scale bar represents 500 :m. (c) The chemical structures of Fxol, Fx, and Fxac
:g/mL streptomycin (SIGMA). Neuro2a cells were
maintained in MEM Earle’s liquid (GIBCO) containing
10% heat-inactivated FBS, 100 units/mL penicillin, and
100 :g/mL streptomycin. The cells were cultured in a
humidified atmosphere with 5% CO2 at 37ºC. This
experiment was conducted in January-March 2011.
previously reported (Kadekaru et al., 2008). Fxol was
prepared by hydrolysing Fx with lipase in September
2009, as described previously (Mise et al., 2011). Fxac
was prepared from Fx by acetylation in June 2010: Fx
(12.8 mg) was dissolved in pyridine (300 :L), mixed with
acetic anhydride (180 :L), and incubated at 32ºC for 3 h.
The reaction mixture was partitioned between diethyl
ether and water. The ether layer was evaporated, and the
residue was dissolved in methanol (MeOH). The purity of
Fx, Fxol, and Fxac was determined by HPLC and
monitored at 440 nm. The structures of these compounds
were identified by NMR.
Cell viability: For all experiments, cells were seeded at a
density of approximately 1×104 cells per well containing
100 :L of culture medium in 96-well plates. After 24 h
incubation, the medium was replaced with fresh medium
containing an antioxidant (Fx, Fxol, Fxac, astaxanthin, or
"-tocopherol) and/or the anticancer drug cisplatin. The
cells were incubated for an additional 72 h. All
antioxidants were dissolved in DMSO and added to the
culture medium (2-8 :M). "-Tocopherol was purchased
from Wako Pure Chemical Industries, Ltd., and
astaxanthin was purchased from Santa Cruz
Biotechnology. The final concentration of DMSO was
0.5% (v/v), and the control culture received only DMSO.
The anticancer drug cisplatin was purchased (Wako) and
dissolved in water (2.5 :g/mL). Cell viability was
determined by Cell Counting Kit-8 (Dojindo). After 72 h,
the medium in each well of a 96-well plate was replaced
Cell culture: The human colonic cancer Caco-2,
hepatocarcinoma Hep G2, and rat neuroblastoma Neuro2a
cell lines were purchased from the RIKEN BRC Cell
Bank. Caco-2 cells were maintained in D-MEM (high
glucose) with L-glutamine and phenol red (Wako)
containing 10% heat-inactivated FBS, 100 units/mL
penicillin, and 100 :g/mL streptomycin. Hep G2 cells
were maintained in Dulbecco's modiWed Eagle's medium
(D-MEM; low glucose) with L-glutamine and phenol red
(Wako) containing 10% heat-inactivated foetal bovine
serum (FBS, GIBCO), 100 units/mL penicillin, and 100
128
Br. J. Pharmacol. Toxicol., 2(3): 127-131, 2011
Fig. 2: Cell viability (% of control) of Caco-2, Hep G2, and Neuro2a cells treated with Fx (2-8 :M) and/or cisplatin (2.5 :g/mL)
*: DMSO-free
Fig. 3: Cell viability (% of control) of Caco-2, Hep G2, and Neuro2a cells treated with Fxol (2-8 :M) and/or cisplatin (2.5 :g/mL)
*: DMSO-free
Fig. 4: Cell viability (% of control) of Hep G2 treated with Fxac (2-8 :M), astaxanthin (2-8 :M), or "-tocopherol (2-8 :M) and/or
cisplatin (2.5 :g/mL), *: DMSO-free
with Phosphate-Buffered Saline (PBS). After a 1-min
wash, the PBS was replaced with a solution of 5 :L of
WST-8 reagent and 95 :L of cultured medium, and the
cells were then incubated for 1 h at 37ºC. The absorbance
at 440 nm was measured using a microplate
spectrophotometer (TECAN GENios). This experiment
was conducted in January-March 2011.
RESULTS AND DISCUSSION
Cytotoxicity of the antioxidants: The culture media of
Caco-2, Hep G2, and Neuro2a cells were supplemented
with Fx, Fxol, and the other antioxidants to investigate
their cytotoxic effects on cancer cells (Fig. 2-4). Dosedependent effects on the viability of Caco-2, Hep G2, and
129
Br. J. Pharmacol. Toxicol., 2(3): 127-131, 2011
Neuro2a cells were shown for Fx. At 4 :M, Fx reduced
cell viability to 59% for Caco-2 and to 85% for Hep G2.
At 8 :M, Fx reduced cell viability to 30% for Caco-2, 5%
for Hep G2, and 69% for Neuro2a (Fig. 2). Figure 3
shows the dose-dependent effects of Fxol on the viability
of Caco-2, Hep G2, and Neuro2a cells. At 4 :M, Fxol
reduced cell viability to 46% for Caco-2, 85% for Hep
G2, and 60% for Neuro2a. At 8 :M, Fxol reduced cell
viability to 24% for Caco-2, 6% for Hep G2, and 7% for
Neuro2a. Based on these results, Fx and Fxol have
cytotoxic effects and are able to suppress the proliferation
of cancer cells in the absence of chemotherapeutic agents.
In contrast, fucoxanthin acetate, astaxanthin, and "tocopherol had no effect on the cell viability of Hep G2 at
2-8 :M (Fig. 4).
REFERENCES
Beppu, F., Y. Niwano, E. Sato, M. Kohno, T. Tsukui,
M. Hosokawa and K. Miyashita, 2009. In vitro and in
vivo evaluation of mutagenicity of fucoxanthin (FX)
and its metabolite fucoxanthinol (FXOH). J. Toxicol.
Sci., 34(6): 693-698.
Block, K.I., A.C. Koch, M.N. Mead, P.K. Tothy,
R.A. Newman and C. Gyllenhaal, 2008. Impact of
antioxidant supplementation on chemotherapeutic
toxicity: A systematic review of the evidence from
randomized controlled trials. Int. J. Cancer, 123:
1227-1239.
Boulikas, T. and M. Vougiouka, 2003, Cisplatin and
platinum drugs at the molecular level (Review).
Oncol. Rep., 10: 1663-1682.
Chandini, S.K., P. Ganesan, P.V. Suresh and N. Bhaskar,
2008. Seaweeds as a source of nutritionally beneficial
compounds-A review. J. Food Sci. Tehnol., 45(1):
1-13.
Gabriella, M. and D. Andrea, 2005. Use of antioxidants
during chemotherapy and radiotherapy should be
avoided. CA Cancer J. Clin., 55(5): 319-321.
Heo, S.J., S.C. Ko, S.M. Kang, H.S. Kang, J.P. Kim,
S.H. Kim, K.W. Lee, M.G. Cho and Y.J. Jeon, 2008.
Cytoprotective effect of fucoxanthin isolated from
brown algae Sargassum siliquastrum against H2O2induced cell damage. Eur. Food Res. Technol., 228:
145-151.
Kadekaru, T., H. Toyama and T. Yasumoto, 2008. Safety
Evaluation of Fucoxanthin Purified from Undaria
pinnatifida. Nippon Shokuhin Kagaku Kogaku
Kaishi, 55(6): 304-308.
Maeda, H., M. Hosokawa, T. Sashima, K. Funayama and
K. Miyashita, 2005. Fucoxanthin from edible
seaweed, Undaria pinnatifida, shows antiobesity
effect through UCP1 expression in white adipose
tissues. Biochem. Biophys. Res. Commun., 332:
392-397.
Mise, T., M. Ueda and T. Yasumoto, 2011. Production of
Fucoxanthin-Rich Powder from Cladosiphon
okamuranus. Adv. J. Food Sci. Technol., 3(1): 73-76.
Okuzumi, J., T. Takahashi, T. Yamane, Y. Kitao,
M. Inagake, K. Ohya, H. Nishino and Y. Tanaka,
1993. Inhibitory effects of fucoxanthin, a natural
carotenoid, on N-ethyl-N'-nitro-N-nitrosoguanidineinduced mouse duodenal carcinogenesis. Cancer
Lett., 68: 159-168.
Shiratori, K., K. Ohgami, I. Ilieva, X.H. Jin, Y. Koyama,
K. Miyashita, K. Yoshida, S. Kase and S. Ohno,
2005. Effects of fucoxanthin on lipopolysaccharideinduced inflammation in vitro and in vivo. Exp. Eye
Res., 81: 422-428.
Effect of antioxidants on cisplatin: To investigate the
effects of Fx on chemotherapy, we studied the interaction
of cisplatin and Fx (Fig. 2). Cisplatin reduced cell
viability to 33% for Caco-2, 24% for Hep G2, and 25%
for Neuro2a. DMSO mitigated the growth inhibition
effect of cisplatin and recovered the cell viability to 47%
for Caco-2, 52% for Hep G2, and 65% for Neuro2a.
However, Fx did not affect the growth inhibition effect of
cisplatin (at 2 :M Fx: 42% for Caco-2, 51% for Hep G2,
and 69% for Neuro2a). The anti-oxidative potential of Fx
did not influence cisplatin cytotoxicity. The anti-oxidative
potential of Fxol and other antioxidants also did not
influence the growth inhibition effect of cisplatin
(Fig. 3, 4). It was shown that DMSO decreased the effect
of cisplatin, while the antioxidants had no influence on
the effect of the chemotherapy agent. When cisplatin is
added to the culture medium, one should dissolve the
carotenoids by using other solvents.
CONCLUSION
Our cell culture studies showed that Fx and Fxol
were able to suppress the proliferation of cancer cells in
a dose-dependent manner. Moreover, none of the
antioxidants used in this study affected the growth
inhibition potency of cisplatin. We suggest that Fx and
Fxol have the potential for cancer prevention and will not
interfere with anticancer drugs during chemotherapy.
When 5Fu was used instead of cisplatin, it showed a
comparable result (data not shown). Further studies are
necessary to clarify the effect of Fx during chemotherapy.
ACKNOWLEDGMENT
This study was supported by Okinawa prefecture and
the Ministry of Education, Culture, Sports, Science and
Technology.
130
Br. J. Pharmacol. Toxicol., 2(3): 127-131, 2011
Yao, X., K. Panichpisal, N. Kurtzman and K. Nugent,
2007. Cisplatin nephrotoxicity: A review. Am. J.
Med. Sci., 334(2), 115-124.
Sugawara, T., K. Matsubara, R. Akagi, M. Mori and
T. Hirata, 2006. Antiangiogenic activity of brown
algae fucoxanthin and its deacetylated product,
fucoxanthinol. J. Agric. Food Chem., 54: 9805-9810.
131