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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46

Available online at www.sciencedirect.com

jo u r n al hom ep age: www.elsev ier .com/ locate /e tap

nhibition of tumor growth in vitro and in vivo byucoxanthin against melanoma B16F10 cells

il-Nam Kima, Ginnae Ahnb, Soo-Jin Heoc, Sung-Myung Kangd, Min-Cheol Kangd,ye-Mi Yanga, Daekyung Kima, Seong Woon Roha, Se-Kwon Kime, Byong-Tae Jeonf,yo-Jam Parkg, Won-Kyo Jungh, You-Jin Jeonb,∗

Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of KoreaLaboratory of Veterinary Molecular Pathology and Therapeutics, Tokyo University of Agricultural and Technology, 3-5-8 Saiwaicho,uchu, Tokyo 183-8509, JapanGlobal Bioresources Research Center, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of KoreaDepartment of Marine Life Science, Jeju National University, Jeju 690-756, Republic of KoreaDepartment of Chemistry, Pukyong National University, Busan 608-737, Republic of KoreaKorean Nokyong Research Center, College of Medicine Konkuk University, Chungju 380-701, Republic of KoreaDepartment of Biotechnology, Konkuk University, Chungju 380-701, Republic of KoreaDepartment of Marine Life Science, Chosun University, Gwangju 501-759, Republic of Korea

r t i c l e i n f o

rticle history:

eceived 27 April 2012

eceived in revised form

October 2012

ccepted 5 October 2012

vailable online 13 October 2012

eywords:

ucoxanthin

a b s t r a c t

The present study was designed to evaluate the molecular mechanisms of fucoxanthin

against melanoma cell lines (B16F10 cells). Fucoxanthin reduced the proliferation of B16F10

cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during

the G0/G1 phase and apoptosis. Fucoxanthin-induced G0/G1 arrest was associated with a

marked decrease in the protein expressions of phosphorylated-Rb (retinoblastoma protein),

cyclin D (1 and 2) and cyclin-dependent kinase (CDK) 4 and up-regulation of the protein levels

of p15INK4B and p27Kip1. Fucoxanthin-induced apoptosis was accompanied with the down-

regulation of the protein levels of Bcl-xL, an inhibitor of apoptosis proteins (IAPs), resulting

in a sequential activation of caspase-9, caspase-3, and PARP. Furthermore, the anti-tumor

elanoma cell lines (B16F10 cells)

ell arrest

poptosis

27Kip1

cl-xL

effect of fucoxanthin was assessed in vivo in Balb/c mice. Intraperitoneal administration

of fucoxanthin significantly inhibited the growth of tumor mass in B16F10 cells implanted

mice.

© 2012 Elsevier B.V. All rights reserved.

Metastatic melanoma presents a serious clinical challenge,

. Introduction

alignant melanoma is a life-threatening skin cancer thatriginates from melanocytes, the specialized pigmented cellsound in the epidermis (Gray-Schopfer et al., 2007). Its

∗ Corresponding author. Tel.: +82 64 754 3475; fax: +82 64 756 3493.E-mail address: youjinj@jejunu.ac.kr (Y.-J. Jeon).

382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2012.10.002

incidence is steadily increasing worldwide, resulting in a grow-ing public health problem (Ferlay et al., 2004; Jemal et al., 2008).

because it is resistant to systemic therapy and has an aggres-sive nature after dissemination (Amiri et al., 2004). Its onlyknown environmental risk factor is exposure to ultraviolet

n d p

40 e n v i r o n m e n t a l t o x i c o l o g y a

radiation via sunlight (Hacker et al., 2006). At present, thereis no proven effective therapy to improve the survival ofpatients with metastatic melanoma (Jiang et al., 2007). There-fore, novel therapeutic agents and strategies are urgentlyneeded to decrease morbidity and enhance survival.

Cell cycle control mechanisms serve major regulatoryfunctions for cell growth. Many cytotoxic agents and DNA-damaging agents arrest the cell cycle at the G0/G1, S, or G2/Mphase, and then induce apoptotic cell death (Kaina, 2003;Nakanishi et al., 2006). In fact, the anti-cancer properties ofmany anti-cancer agents act through the induction of cellcycle arrest and/or apoptotic cell death. Cyclin D/CDK4, cyclinD/CDK6, or cyclin E/CDK2 complex phosphorylates retinoblas-toma protein (Rb), and by doing so, allows cell cycle G1–Stransition (Sherr, 1993, 1994; Weinberg, 1995). Therefore, theinhibitors of CDK4 and CDK6 can control the G1 restriction. Theinhibitors of CDK4 (INK4) family (p15INK4B, p16INK4A, p18INK4C,and p19INK4D) competes with cyclin D to bind with CDK4,CDK6, and kinase inhibitor protein (KIP) family (p21WAF1/Cip1,p27Kip1, and p57Kip2) to form associations with a wider rangeof cyclin/CDK complexes, including CDK4, CDK6, and CDK2(Sherr and Roberts, 1995; Harper and Elledge, 1996). Apoptosiscan occur as part of the normal physiological process or in thepathological deletion of cells to regulate the balance betweencell proliferation and cell death. Apoptosis is characterized bydistinct morphological changes, some of which are membraneblebbing, cytoplasmic shrinkage, dissipation of mitochon-drial membrane potential, nuclear condensation, and DNAfragmentation (Steller, 1995; Hengartner, 2000). Apoptosis iscontrolled by the Bcl-2 family of proteins and by caspases,which is a family of cysteine proteases (Hockenbery et al.,1993; Korsmeyer, 1999; Ham et al., 2012). Apoptosis inducedby these molecules can prevent carcinogenesis by eliminat-ing damaged cells or inhibiting abnormal cell development(Hengartner, 2000). Therefore, the induction of cell-cycle arrestand apoptosis in cancer cells is the basis of anticancer therapy.

Several recent reports have indicated that fucoxanthin,a carotenoid found in seaweed, inhibits tumor cell growthby modulating the expression of cell cycle-regulatory andapoptosis-related genes (Kim et al., 2010a; Yu et al., 2011;Satomi and Nishino, 2009; Das et al., 2008; Nakazawa et al.,2009). However, information concerning its ability to inducecell-cycle arrest and apoptosis in melanoma is lacking. In thisstudy, we aimed to investigate the molecular mechanisms offucoxanthin-induced cell-cycle arrest and apoptosis in mousemelanoma cell line (B16F10 cells).

2. Materials and methods

2.1. Reagents

Fucoxanthin was isolated from marine alga Ishige oka-murae (Specimen No. Jeju-B-18). The details of theisolation have been published recently (Kim et al.,2010b). Dulbecco’s modified Eagle’s medium (DMEM),

fetal bovine serum (FBS), penicillin–streptomycin, andtrypsin–ethylenediaminetetraacetic acid (EDTA) were pur-chased from Gibco BRL (Life Technologies, Burlington,ON, Canada). RNase A, 3-(4,5-dimethylthiazol-2-yl)-2,

h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46

5-diphenyltetrazolium bromide (MTT), propidium iodide(PI), dimethyl sulfoxide (DMSO), and Hoechst 33342 werepurchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-bodies against phospho-Rb (p-Rb), CDK4, cyclin D1 and D2,p15INK4B, p27Kip1, B-cell lymphoma 2-associated X (Bax),B-cell lymphoma-extra large (Bcl-xL), cleaved caspase-3 and-9, poly(ADP-ribose) polymerase (PARP), cellular inhibitor ofapoptosis 1 and 2 (c-IAP-1 and -2), X-linked inhibitor of apo-ptosis (XIAP), and �-actin were purchased from Cell SignalingTechnology (Danvers, MA, USA). The other chemicals andreagents used were of analytical grade.

2.2. Cell culture

B16F10 cells were grown in DMEM supplemented with 10%(v/v) heat-inactivated FBS, penicillin (100 U/ml), and strepto-mycin (100 �g/ml). Cultures were maintained at 37 ◦C in a 5%CO2 incubator.

2.3. Cytotoxicity assay

The cytotoxicity of fucoxanthin was determined by a col-orimetric MTT assay. B16F10 cells were seeded in a 96-wellplate at a concentration of 2 × 104 cells/ml. After 16 h, the cellswere treated with various concentrations (12, 25, 50, 100, and200 �M) of fucoxanthin and incubated for 72 h at 37 ◦C. MTTstock solution (50 �l; 2 mg/ml in phosphate-buffered saline[PBS]) was then added to each well to obtain a total reac-tion volume of 250 �l. After 4 h of incubation, the plate wascentrifuged at 2000 rpm for 10 min, and the supernatant wasaspirated. The formazan crystals in each well were dissolvedin DMSO. The amount of purple formazan was determined bymeasuring the absorbance at 540 nm.

2.4. Nuclear double staining

The nuclear morphology of B16F10 cells was studied byusing cell-permeable DNA dyes Hoechst 33342 and PI. B16F10cells were seeded in a 24-well plate at a concentration of1 × 105 cells/ml. After 16 h, the cells were treated with vari-ous concentrations (50, 100, and 200 �M) of fucoxanthin andincubated for 24 h. Then, Hoechst 33342 and PI were added tothe culture medium at a final concentration of 10 and 5 �g/ml,respectively, and the plate was incubated for another 10 minat 37 ◦C. The stained cells were observed under a fluorescencemicroscope equipped with a CoolSNAP-Pro color digital cam-era to examine the degree of nuclear condensation. Cells withhomogeneously stained nuclei were considered to be viable,whereas the presence of chromatin condensation and/or frag-mentation was considered indicative of apoptosis (Gschwindand Huber, 1995; Lizard et al., 1995).

2.5. Cell-cycle analysis

Cell-cycle analysis was performed to determine the proportionof apoptotic sub-G1 hypodiploid cells (Nicoletti et al., 1991).

B16F10 cells were seeded in a 6-well plate at a concentrationof 1.0 × 105 cells/ml. After 16 h, the cells were treated with vari-ous concentrations (50, 100, and 200 �M) of fucoxanthin. After24 h of incubation, the cells were harvested at the indicated

d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46 41

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Fig. 1 – Inhibitory effect of fucoxanthin against the growthof B16F10. Each point represents the mean ± S.D. of threeindependent experiments. Means with different letters

e n v i r o n m e n t a l t o x i c o l o g y a n

ime and fixed in 1 ml of 70% ethanol for 30 min at 4 ◦C. Theyere then washed twice with PBS and incubated in the dark in

ml of PBS containing 100 �g PI and 100 �g RNase A for 30 mint 37 ◦C. Flow cytometric analysis was performed with a FAC-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).he effect of fucoxanthin on the cell cycle was determinedy changes in the percentage of cells in each phase of theell cycle and assessed with histograms generated by softwarerograms CellQuest and ModFit (Wang et al., 1999).

.6. Western blot analysis

16F10 cells (2 × 105 cells/ml) were treated with various con-entrations (50, 100, and 200 �M) of fucoxanthin. Harvestedells were lysed in a lysis buffer (50 mM Tris–HCl [pH 7.5],50 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM ethyl-ne glycol tetraacetic acid [EGTA], 1 mM NaVO3, 10 mM NaF,

mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluo-ide [PMSF], 25 �g/ml aprotinin, and 25 �g/ml leupeptin] and

aintained on ice for 30 min. The cell lysates were washedy centrifugation, and protein concentrations were deter-ined by using a BCATM protein assay kit (Pierce, Thermo

isher Scientific). Aliquots of the lysates (30–50 �g of protein)ere separated on a 12% sodium dodecyl sulfate (SDS)-olyacrylamide gel and transferred onto a polyvinylideneuoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) with

glycine transfer buffer (192 mM glycine, 25 mM Tris–HClpH 8.8], and 20% MeOH [v/v]). After the nonspecific siteas blocked with 1% bovine serum albumin (BSA), theembrane was incubated with diluted specific primary anti-

odies (1:1000 or 1:500) in 10 ml dilution buffer (5% milk,.05% Tween 20, 50 mM Tris, 150 mM NaCl) at 4 ◦C overnight.he membrane was further incubated for 60 min with aeroxidase-conjugated secondary antibody (1:5000) at roomemperature. The immunoreactive proteins were detected bysing an enhanced chemiluminescence (ECL) western blottingetection kit.

.7. Induction of tumor in mice

alb/c mice weighing 20–22 g (6 weeks old) were purchasedrom Orientbio, Inc. (Sungnam, Korea) and housed in con-entional animal facilities with an NIH-07-approved diet andater ad libitum at a constant temperature (23 ± 1 ◦C) according

o the guidelines for the Care and Use of Laboratory Animals ofhe Institutional Ethical Committee of Jeju National University.

The mice were randomly separated into three groups (5ice per a group): a sham B16F10 cells-implanted group,

B16F10 melanoma cells-implanted control group, and aucoxanthin plus B16F10 cells-implanted group. For thexperimental induction of the B16F10 melanoma tumor, anssessment was made by intradermal (i.d.) inoculation ofumor cells (5 × 105 cells/100 �l/mouse) into mice of both the16F10 cells-implanted control group. For the fucoxanthinlus B16F10 cells-implanted group, fucoxanthin dissolved inaline (pH 7.4) (300 �g/100 �l/mouse) was applied to mice by

ntraperitoneal injection at 24 h and 2 h before the implan-ation of B16F10 cells. Then, after the induction of the16F10 melanoma tumor, fucoxanthin (300 �g/100 �l/mouse)as applied into the mice once every 5 days by intraperitoneal

within a column are significantly different (* p < 0.05).

injection. Additionally, as naïve control group, mice were i.p.injected with saline instead of fucoxanthin or B16F10 cells.The mice were analyzed at 20 days after the induction ofB16F10 melanoma tumor.Statistical analysisAll data are pre-sented as the mean ± SD of at least three replicates. Significantdifferences among the groups were determined by using theunpaired Student’s t-test. p < 0.05 was considered statisticallysignificant.

3. Results

3.1. Cytotoxic effect of fucoxanthin

As shown in Fig. 1, cell growth was markedly inhibited 72 hafter exposure to fucoxanthin in a dose-dependent man-ner. B16F10 cell proliferation was reduced by 87% upon 72 hexposure to 200 �M fucoxanthin. In addition, observationunder an inverted microscope showed that numerous mor-phological changes occurred in cells treated with fucoxanthin(Fig. 2A).

3.2. Apoptosis induced by fucoxanthin

Apoptosis was confirmed by the presence of apoptotic bod-ies and nuclear condensation detected with Hoechst 33342.Costaining of the cells with PI allowed the discrimination ofdead cells from apoptotic ones. The control, cultured withoutfucoxanthin, showed a clear image and no DNA damage. How-ever, fucoxanthin treated cells exhibited significant apoptoticbody and nuclear condensation, destruction characteristic ofapoptosis (bright blue color), and cell death (red color). Inaddition, the amounts of nuclear condensation and apoptoticbodies dramatically increased with increasing concentrationsof fucoxanthin (Fig. 2B).

3.3. Cell-cycle arrest induced by fucoxanthin

The induction of apoptosis and cell-cycle arrest is consid-ered the major cause of antiproliferation (Hsiao et al., 2007).

42 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46

Fig. 2 – Fucoxanthin-induced cell death in B16F10 cells. B16F10 cells were stimulated with the indicated concentrations (50,100, and 200 �M) of fucoxanthin for 24 h at 37 ◦C. The cells were double stained with Hoechst 33342 and PI solution, andthen observed under an inverted microscope (A) and a fluorescent microscope (B). Exposure to fucoxanthin resulted inapoptosis and cell death in a concentration-dependant manner, as indicated by the bright blue (indicated by arrow) andred-stained nuclei, respectively. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of the article.)

Table 1 – Effect of fucoxanthin on the cell cycle pattern and the apoptotic portion of B16F10 cells by flow cytometricanalysis.

Concentrations (�M) % of cells

Sub-G1 G0/G1 S G2/M

0 0.9 ± 0.9 58.5 ± 2.2 29.5 ± 3.5 11.1 ± 1.950 3.0 ± 2.3 74.9 ± 3.8* 15.0 ± 2.5* 7.1 ± 2.8100 8.5 ± 4.9 77.8 ± 4.5* 7.2 ± 2.7* 6.5 ± 1.2*

200 14.5 ± 4.1* 74.3 ± 5.2* 5.2 ± 1.2* 6.0 ± 1.5*

B16F10 cells were seeded at 1 × 105 cells/ml and treated with the different concentrations (50, 100, and 200 �M) of fucoxanthin for 24 h. The cellsprese.

were stained with PI and analyzed by flow cytometry. Each point redifferent letters within a column are significantly different (* p < 0.05)

Table 1 shows representative histograms of the relative per-centage of B16F10 cells in each phase of the cell cycle afterincubation in the absence or presence (50, 100, and 200 �M) offucoxanthin for 24 h. Fucoxanthin treatment for 24 h caused

an increase in the percentage of cells in the G0/G1 phase,which was accompanied by a corresponding reduction in thepercentages of cells in the S and G2/M phases (Fig. 3). In addi-tion, a distinct sub-G1 peak (14.5%) was observed in the cells

Fig. 3 – Effect of fucoxanthin on the cell cycle pattern and the apB16F10 cells were stimulated with the indicated concentrations (cells were stained with PI and analyzed by flow cytometry.

nts the mean ± S.D. of three independent experiments. Means with

treated with 200 �M fucoxanthin, suggesting the induction ofapoptosis.

3.4. Effects of fucoxanthin on cell cycle-regulatory

protein levels

Because fucoxanthin induced cell-cycle arrest of B16F10cells in the G0/G1 phase, its effects on cell cycle-regulatory

optotic portion of B16F10 cells by flow cytometric analysis.50, 100, and 200 �M) of fucoxanthin for 24 h at 37 ◦C. The

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46 43

Fig. 4 – Effect of fucoxanthin on protein expression relatedcell cycle arrest of B16F10 cells. The cells were treated with50 and 100 �M fucoxanthin for 24 h. Equal amounts of celllysates were resolved by SDS-PAGE, transferred tonitrocellulose, and probed with specific antibodies(anti-p15, anti-p27, anti-CDK4, anti-Cyclin D1, D2, anda

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Fig. 5 – Fucoxanthin treatment results in modulation ofanti- and pro-apoptotic proteins in B16F10 cells. The cellswere treated with 50, 100 and 200 �M or 200 �Mfucoxanthin for 24 h or difference time. Equal amounts ofcell lysates were resolved by SDS-PAGE, transferred tonitrocellulose, and probed with specific antibodies (A:anti-Bax, -Bcl-xL, -pro-caspase-9, -cleaved caspase-3, and-PARP; B: anti-c-IAP-1, -2, and -XIAP).

nti-p-Rb).

olecules involved in the G0/G1 phase were investigated. P-b, p15INK4B, and p27Kip1 play a critical role in the transitionrom the G1 phase to the S phase (Bai et al., 2003; Lloydt al., 1999; Puri et al., 1999). Fucoxanthin treatment obviouslyecreased the p-Rb level but markedly increased the p15INK4B

nd p27Kip1 levels in a dose-dependent manner (Fig. 4). Fur-her, CDKs and cyclins play crucial roles in the regulation ofhe cell cycle (Morgan, 1995). Fucoxanthin treatment caused aose-dependent decrease in cyclin D1 and D2 levels (Fig. 4),ccompanied by a reduction in the CDK4 level. CDK4 wasarely detectable in these cells, especially at 100 �M fucoxan-hin.

.5. Effects of fucoxanthin on apoptosis-relatedrotein expression levels

o clarify the mechanism of fucoxanthin-induced apopto-is, the expression levels of Bax, Bcl-xL, cleaved caspase-3nd -9, and PARP were evaluated by western blot analy-is. The expression level of Bcl-xL, an antiapoptotic protein,ecreased gradually with increasingly fucoxanthin concentra-ion, but that of Bax, a proapoptotic protein, did not changeFig. 5A). The expression levels of cleaved caspase-3 and -9ncreased upon treatment with 200 �M fucoxanthin, and PARPas cleaved at 200 �M fucoxanthin (Fig. 5A). IAP family pro-

eins bind to caspases and induce caspase inactivation for anntiapoptotic effect in eukaryotic cells (Deveraux and Reed,999). Therefore, the expression levels of XIAP, cIAP-1, andIAP-2 in 200 �M fucoxanthin-treated cells were evaluated.

ucoxanthin treatment dramatically decreased the expressionevels of these IAP family members in a time-dependent man-er (Fig. 5B).

3.6. Effect of fucoxanthin on tumor growth in vivo

As shown in Fig. 6, the melanoma tumor mass was markedlyformed in B16F10 cells-injected mice group and its meanweight amounted to 124 mg in comparison with normal micegroup. In contrast, the application of fucoxanthin markedlydecreased the weight of melanoma tumor mass up to 27 mg(5-fold reduction) by retarding the formation of tumor mass,compared with the B16F10 cells-injected mice group. Theseresults suggest that fucoxanthin has anti-tumor effect asinhibiting the melanoma tumor growth in vivo.

4. Discussion

Fucoxanthin induces apoptosis in human gastric adenocarci-noma MGC-803 cells (Yu et al., 2011) and leukemia cells (Kimet al., 2010a). In addition, it causes cell-cycle arrest in DU145cells (Satomi and Nishino, 2009) and human hepatoma HepG2

cells (Das et al., 2008). Here, we have demonstrated that fucox-anthin inhibits B16F10 cell growth through cell-cycle arrestin the G0/G1 phase and the apoptotic pathway. Furthermore,

44 e n v i r o n m e n t a l t o x i c o l o g y a n d p

Fig. 6 – Effect of fucoxnathin on the inhibition of tumor(B16F10) growth in Balb/c mice. Results are expressed asthe mean ± S.D. Means with different letters within acolumn are significantly different (* p < 0.05).

this research has also been the first to show that fucoxanthinsuppressed in vivo growth of B16F10 melanoma in Balb/c mice.

We examined the antiproliferative effect of fucoxanthin onB16F10 cells by using concentrations ranging from 12 �M to200 �M for 72 h. Fucoxanthin significantly decreased the pro-liferation of these cells in a dose-dependent manner. Moreimportantly, the fucoxanthin also exhibited effective anti-cancer properties in Balb/c mice. This finding is consistentwith previous reports that fucoxanthin inhibits the growthof human gastric adenocarcinoma, leukemia, neuroblastoma,and hepatoma cells (Yu et al., 2011; Kim et al., 2010a; Das et al.,2008). Suppression of growth by fucoxanthin can be partiallyexplained by the occurrence of an arrest during the cell cycle.Cell cycle analysis using flow cytometry showed that treat-ment with 100 �M fucoxanthin induced G0/G1 phase arrestof the cell cycle at 24 h. However, this concentration did notinduce a similar level of apoptosis, as evidenced by the rel-ative percentage of sub-G1 cells. On the other hand, 200 �Mfucoxanthin induced both cell-cycle arrest and apoptosis.

Rb is the key regulator of the cell cycle, and its continualphosphorylation parallels the transition of cells through the G1

and S phases (Weinberg, 1995). Most invasive and metastaticmelanoma specimens and cell lines express Rb (Albino et al.,2000). In its hypophosphorylated state, the Rb family of pro-

teins associates with and inhibits the activity of the E2Ffamily of transcription factors, which are involved in the tran-scription of cell-cycle regulators. Upon growth stimulation,the G1-specific CDKs/cyclins phosphorylate Rb on multiple

h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 39–46

residues, resulting in the release of E2F-related transcriptionfactors (Roy et al., 2007). We found that fucoxanthin causes adose-dependent decrease in the level of p-Rb.

Many studies have shown that cyclins and CDKs controlthe G1–S transition in the cell cycle. Therefore, the regulationof their activity is the most productive strategy for design-ing anticancer agents targeting the cell cycle (Wu et al., 2006).Further, Weinstein (2000) reported that CKIs play a major rolein cell-cycle regulation. CDKs in the G1 phase are inactivatedby 2 families of CKIs: the KIP family, including p21WAF1/Cip1,p27Kip1, and p57Kip2, and the INK4 family, including p15INK4B,p16INK4A, p18INK4C, and p19INK4D (Bai et al., 2003; Lloyd et al.,1999; Pei and Xiong, 2005). Accordingly, we found that fucox-anthin decreased the expression levels of cyclin D1 and D2,which correlated with the decrease in the expression level ofCDK4. Concomitantly, the expression levels of p15INK4B andp27Kip1 increased in B16F10 cells exposed to fucoxanthin.

Apoptosis is important to maintain homeostasis betweencell division and cell (Green and Reed, 1998; Kaufmann andHengartner, 2001). It is mediated by the activation of an evo-lutionary conserved intracellular pathway (Bold et al., 1997).Therefore, the induction of apoptosis in cancer cells is a usefulstrategy for developing anticancer drugs (Hu and Kavanagh,2003; Kim et al., 2012). Apoptosis is a tightly regulated pro-cess, involving changes in the expression of distinct genes.Bcl-2 family proteins (e.g., Bcl-2 and Bcl-xL) are a criticalregulator of the apoptotic pathway (Korsmeyer, 1999). Bcl-2 and Bcl-xL are upstream molecules in this pathway andpotent suppressors of apoptosis (Hockenbery et al., 1993). Wefound that fucoxanthin treatment of B16F10 cells resultedin a concentration-dependent decrease in the Bcl-xL expres-sion level. In addition, caspase activation is often regulatedby various cellular proteins, including members of the IAP(Deveraux and Reed, 1999) and Bcl-2 families (Adams andCory, 1998; Antonsson and Martinou, 2000). Our data revealthat the expression levels of c-IAP-1, c-IAP-2, and XIAP inB16F10 cells decreased upon fucoxanthin treatment. Thecleavage of caspase-3 and -9 appeared to be correlated withfucoxanthin-induced apoptosis in B16F10 cells. Caspase-3 and-9 are key components in the mitochondria-initiated pathway(Budihardjo et al., 1999). Once caspases are activated, variouscellular proteins are targeted, leading ultimately to apopto-sis. Moreover, PARP is the best known substrate of caspasesand is cleaved from the 116 kDa intact form to a 85 kDa frag-ment (Konopleva et al., 1999). This phenomenon is importantfor cells to maintain their viability; cleavage of PARP facilitatescellular disassembly and serves as a marker of cells undergo-ing apoptosis (Oliver et al., 1998). In our study, the intact andcleaved forms of PARP were detected in fucoxanthin-treatedB16F10 cells.

In conclusion, fucoxanthin has antiproliferative effects onB16F10 cells by inducing apoptosis and cell-cycle arrest. Itincreased the proportion of cells in the G1 phase of the cellcycle, which was associated with decreased cyclin D1 andD2, and CDK4 expressions and increased p15INK4B and p27Kip1

expressions. Fucoxanthin-induced apoptosis might be related

to caspase-3 and -9 activation and the downregulation of Bcl-xL and IAP expressions. Taken together, the data presented inthis in vitro and in vivo study provide important insights intothis cell-cycle-based therapeutic strategy and form a strong

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asis for the development of fucoxanthin as an anticancergent.

onflict of interest statement

he authors declare that there are no conflicts of interest.

cknowledgment

his research was supported by a grant from Marine Biopro-ess Research Center of the Marine Bio21 Project funded by theinistry of Land, Transport and Maritime, Republic of Korea.

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