Adult T-cell leukemia (ATL) is a unique malignancy of mature CD4+ T cells caused by human T-cell leukemia virus type 1 (HTLV-1).1–3 Clinically, ATL is subclassified into 4 subtypes: acute, lymphoma, chronic and smoldering. In the relatively indolent smoldering and chronic types, the median survival time is ≥2 years. However, at present, there is no accepted curative therapy for ATL and the condition often progresses to death with a median survival time of 13 months in aggressive ATL.4 Death is usually due to severe infection or hypercalcemia, often associated with resistance to intensive, combined chemotherapy. Therefore, the establishment of new therapeutic strategies for ATL is very important.
Carotenoids are a family of natural pigments with at least 600 members. They have several biological functions, including provitamin A activity, radical scavenging, singlet oxygen-quenching activity, immunomodulation and chemopreventive effects on carcinogenesis.5 Among the carotenoids, β-carotene and lycopene, which are found in terrestrial plants such as vegetables and fruits, have been extensively studied with regard to physiological functions.5 Marine organisms also contain carotenoids with unique structures. Astaxanthin is a nonprovitamin A carotenoid widely distributed in marine animals and algae, and is commonly described as an antioxidant, immune modulator and scavenger of reactive oxygen species. Fucoxanthin is a major marine carotenoid, found in brown seaweed. Its structure, which includes an allenic bond and a 5,6-monoepoxide, differs from that of common carotenoids. Fucoxanthin has been reported to demonstrate anticarcinogenic6 and antiinflammatory effects7 as well as apoptotic effects in cancer cells8, 9 and radical scavenging activity.10 Orally administered fucoxanthin is known to be metabolized to fucoxanthinol and amarouciaxanthin A.11 Because fucoxanthin is a potent inducer of apoptosis in human cancer cells, the comparison of anticancer effects of fucoxanthin and its metabolite is very interesting in biochemical studies of carotenoids.
With the objective of finding newer agents for the treatment of ATL, the present study was designed to investigate the antitumor potential of carotenoids. We first assayed the antiproliferative effects of some carotenoids. We found that both fucoxanthin and fucoxanthinol have remarkable antiproliferative effects on HTLV-1-infected T-cell lines and primary ATL cells in vitro, although the effect of fucoxanthin was less than that of fucoxanthinol. We also examined the tissue distribution of these carotenoids and the underlying mechanisms involved in their antitumor activities. The results showed that orally administered fucoxanthinol induced apoptosis in tumors inoculated subcutaneously into severe combined immunodeficiency (SCID) mice.
AP-1, activator protein-1; ATL, adult T-cell leukemia; EMSA, electrophoretic mobility shift assay; HTLV-1, human T-cell leukemia virus type 1; IL-2Rα, interleukin-2 receptor α chain; NF-κB, nuclear factor-κB; PARP, poly(ADP-ribose) polymerase; PBMCs, peripheral blood mononuclear cells; ppRb, hyperphosphorylated form of the retinoblastoma protein; SCID, severe combined immunodeficiency; sIL-2Rα, soluble IL-2Rα; TUNEL, terminal deoxynucleotidyl transferase mediated nick labeling; WST, water-soluble tetrazolium.
Material and methods
HTLV-1-infected T-cell lines, MT-2,12 MT-4,13 HUT-1021 and ED-40515(-),14 HTLV-1-uninfected T-cell line, Jurkat, and chronic myelogenous leukemia cell line, K562, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS), 50 U/ml penicillin and 50 μg/ml streptomycin. MT-2 and MT-4 are HTLV-1-transformed T-cell lines and constitutively express viral genes including Tax. ED-40515(-) is a T-cell line of leukemic cell origin that was established from a patient with ATL and does not express viral genes. HUT-102 was also established from a patient with ATL and constitutively expresses viral genes, but its clonal origin is unclear.15 HeLa cervical cancer cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin.
The diagnosis of ATL was based on clinical features, hematological findings and the presence of anti-HTLV-1 antibodies in the sera. Monoclonal HTLV-1 provirus integration into the DNA of leukemic cells was confirmed by Southern blot hybridization in all patients (data not shown). Peripheral blood mononuclear cells (PBMCs) from 3 healthy volunteers, 8 patients with acute type ATL and 4 patients with chronic type ATL were analyzed. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden) and washed with PBS. All samples were obtained after informed consent.
β-carotene and astaxanthin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Fucoxanthin was extracted from brown algae Undaria pinnatifida using acetone as solvent, and purified by column chromatography, liquid–liquid partition and recrystallization up to ≥95% purity. Further purification was performed by RP-HPLC up to ≥98% purity, for in vitro assay. Fucoxanthinol was prepared by enzymatic hydrolysis of purified fucoxanthin using porcine pancreatic lipase. For this purpose, 195 mg of fucoxanthin, 2 g of sodium taurocholate and 2 g of porcine pancreatic lipase (Type II; Sigma Chemical, St. Louis, MO) were dissolved in 30 ml of 0.1 M sodium phosphate buffer (pH 7.0). The reaction buffer was incubated at 37°C for 3 hr. Fucoxanthinol was purified by ODS column chromatography, liquid–liquid partition and recrystallization. In the experiment, we prepared 142 mg of purified fucoxanthinol (≥95% purity, 72% yield). Further purification was performed by RP-HPLC up to ≥98% purity, for in vitro assay.
Rabbit polyclonal antibodies to cyclin D2, cIAP2, survivin, IκBα, JunD, nuclear factor-κB (NF-κB) subunits p65, p50, c-Rel and p52, and activator protein-1 (AP-1) subunits c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD and mouse monoclonal antibody to GADD45α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody to Bcl-xL was purchased from BD Transduction Laboratories (San Jose, CA). Mouse monoclonal antibodies to Bcl-2, CDK4, CDK6, p53, actin and hyperphosphorylated form of the retinoblastoma protein (ppRb) (Ser780) were purchased from NeoMarkers (Fremont, CA). Mouse monoclonal antibodies to XIAP and cyclin D1 were purchased from Medical & Biological Laboratories (MBL; Nagoya, Japan). Mouse monoclonal antibody to phospho-IκBα (Ser32/36), caspase-8 and -9, rabbit monoclonal antibody to cleaved caspase-3 and rabbit polyclonal antibody to cleaved poly(ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA).
Cell viability and apoptosis assays
The effect of carotenoids on cell viability was examined by the cell proliferation reagent, water-soluble tetrazolium (WST)-8 (Wako Chemicals). Briefly, 1 × 105 cells/ml (cell lines) or 1 × 106 cells/ml (PBMCs) were incubated in a 96-well microculture plate in the absence or presence of various concentrations of carotenoids. After 24 hr of culture, WST-8 (5 μl) was added for the last 4 hr of incubation and absorbance at 450 nm was measured using an automated microplate reader. Measurement of mitochondrial dehydrogenase cleavage of WST-8 to formazan dye provides an indication of the level of cell viability. Apoptotic events in cells were detected by staining with phycoerythrin-conjugated APO2.7 monoclonal antibody (Beckman Coulter, Marseille, France)16 and analyzed by flow cytometry (Epics XL, Beckman Coulter, Fullerton, CA). For analysis of morphologic changes of nuclei, cells were stained by 10 μg/ml Hoechst 33342 (Wako Pure Chemical Industries) and photographed through an ultraviolet filter using an Olympus IX70 microscopy (Olympus, Tokyo, Japan).
Cell cycle analysis
Cell cycle analysis was performed with the CycleTEST PLUS DNA reagent kit (Becton Dickinson Immunocytometry Systems, San Jose, CA). In brief, 1 × 106 cells were washed with a buffer solution containing sodium citrate, sucrose and dimethyl sulfoxide, suspended in a solution containing RNase A and stained with 125 μg/ml propidium iodide for 10 min. After passing the cells through a nylon mesh, cell suspensions were analyzed on an Epics XL. The population of cells in each cell cycle phase was determined.
In vitro measurement of caspase activity
Cell extracts were recovered with the use of the cell lysis buffer and assessed for caspase-3, -8 and -9 activities using colorimetric probes (MBL). The colorimetric caspase assay kits are based on detection of the chromophore p-nitroanilide after cleavage from caspase-specific-labeled substrates. Colorimetric readings were performed in an automated microplate reader at an optical density of 400 nm.
Western blot analysis
Cells were lysed in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 6% 2-mercaptoethanol and 0.01% bromophenol blue. Samples were subjected to electrophoresis on SDS-polyacrylamide gels followed by transfer to a polyvinylidene difluoride membrane and probing with the specific antibodies. The bands were visualized with the enhanced chemiluminescence kit (GE Healthcare Unlimited, Buckinghamshire, UK).
Preparation of nuclear extracts and electrophoretic mobility shift assay
Cells were placed in culture and examined for inhibition of NF-κB and AP-1 after exposure to carotenoids for 12 hr. Nuclear proteins were extracted, and NF-κB and AP-1 binding activities to NF-κB and AP-1 elements were examined by electrophoretic mobility shift assay (EMSA) as described previously.17 In brief, 5 μg of nuclear extracts were preincubated in a binding buffer containing 1 μg poly-deoxy-inosinic-deoxy-cytidylic acid (GE Healthcare Biosciences), followed by the addition of [α-32P]-labeled oligonucleotide probe containing NF-κB element (∼50,000 cpm). These mixtures were incubated for 15 min at room temperature. The DNA protein complexes were separated on 4% polyacrylamide gels and visualized by autoradiography. To examine the specificity of the NF-κB element probe, unlabeled competitor oligonucleotides were preincubated with nuclear extracts for 15 min before incubation with probes. The probes or competitors used were prepared by annealing the sense and antisense synthetic oligonucleotides; a typical NF-κB element from the interleukin-2 receptor α chain (IL-2Rα) gene (5′-gatcCGGCAGGGGAATCTCCCT CTC-3′) and an AP-1 element of the IL-8 gene (5′-gatcGTGAT GACTCAGGTT-3′). The oligonucleotide 5′-gatcTGTCGAATG CAAATCACTAGAA-3′, containing the consensus sequence of the octamer binding motif, was used to identify specific binding of the transcription factor Oct-1. This transcription factor regulates transcription of a number of so-called housekeeping genes. Underlined sequences represent the NF-κB, AP-1 or Oct-1 binding site. To identify NF-κB proteins in the DNA protein complex detected by EMSA, we used antibodies specific for various NF-κB family proteins, including p65, p50, c-Rel and p52, and various AP-1 family proteins, such as c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD (Santa Cruz Biotechnology), to elicit a supershift DNA-protein complex formation. These antibodies were incubated with the nuclear extracts for 45 min at room temperature before incubation with the radiolabeled probes.
In vivo administration of fucoxanthinol
Five-week-old female C.B-17/Icr-SCID mice obtained from Ryukyu Biotec (Urasoe, Japan) were maintained in containment level 2 cabinets and provided with autoclaved food and water ad libitum. Mice were engrafted with 1 × 107 HUT-102 cells by subcutaneous injection in the postauricular region and were randomly placed into 2 cohorts of 6 mice each that received vehicle and fucoxanthinol, respectively. Treatment was initiated on the day after cell injection. Fucoxanthinol was dissolved in soybean oil at a concentration of 13.3 mg/ml, and 200 mg/kg body weight of fucoxanthinol was administered by oral gavage every day for 28 days. Control mice received the same volume of the vehicle (soybean oil) only. Body weight and tumor numbers and size were monitored once a week. All mice were sacrificed on day 28, and then the tumors were dissected out and their weight was physically measured. Thereafter, tumors were fixed for paraffin embedding and tissue sectioning. Analysis of DNA fragmentation by fluorescent terminal deoxynucleotidyl transferase mediated nick labeling (TUNEL) was performed using a commercial kit (Takara Bio, Otsu, Japan) as described in the instructions provided by the manufacturer. The efficacy of the treatment was determined by measuring the serum levels of soluble IL-2Rα (sIL-2Rα) by ELISA (BioSource, Camarillo, CA) at 28 days after injection. This experiment was performed according to the Guidelines for the Animal Experimentation of the University of the Ryukyus and was approved by the Animal Care and Use Committee of the University of the Ryukyus.
Serum extraction and analysis
A mixture of serum (50 μl) and ethanol (200 μl) was vortex mixed and centrifuged at 2,000g. The precipitate was reextracted with ethanol (200 μl) and the mixture was centrifuged. The supernatants were combined and made up to 500 μl with ethanol and the solution was used for HPLC analysis. HPLC analysis was carried out on an Agilent 1100 series HPLC system equipped with a photodiode array detector under the following conditions: injected sample volume, 10 μl; column, Hypersil ODS-5 column (4.0 mm × 125 mm); mobile phase, 70% methanol (0 min)-100% methanol (25 min) linear gradient at a flow rate of 0.4 ml/min. Fucoxanthinol was detected at 440 nm wavelength and quantified from the peak area by using the standard curve prepared using pure fucoxanthin.
Tissue extraction and analysis
Respective tissues (hearts, lungs, livers, spleens, kidneys, thymus glands and transplanted tumors) from 6 mice were combined, immersed in ethanol (5 ml for thymus, 20 ml for transplanted tumors and 10 ml for other organs) and preserved at −30°C until used for analysis (about 17 days). Ethanol solutions were separated from tissues by filtration and made up with ethanol to 10 ml (thymus) or to 25 ml (other organs). The second extraction was made by cutting the tissues into small pieces and immersing them in ethanol, in a manner similar to the first extraction. The tissues were preserved in ethanol at −80°C until analysis (about 11 days). The first and second extracts were analyzed separately under the HPLC conditions described for the serum sample.
Data were expressed as mean ± SD. Volume and weight of tumors and serum levels of sIL-2Rα in fucoxanthinol-treated mice were compared with those in the vehicle-treated controls by the Mann-Whitney U-test. A p value less than 0.05 denoted the presence of a statistically significant difference.
Fucoxanthin and fucoxanthinol inhibit cell viability of HTLV-1-infected T-cell lines and primary ATL cells
We first examined the effects of carotenoids on the cell viability of HTLV-1-infected T-cell lines. Tax protein was detected by immunoblot analysis in the 3 HTLV-1-infected T-cell lines (MT-2, HUT-102 and MT-4) but not in the ATL-derived T-cell line [ED-40515(-)]. Culture of cells with various concentrations of carotenoids for 24 hr resulted in the suppression of cell viability in a dose-dependent manner in all 4 lines tested as assessed by the WST-8 assay (Fig. 1a). Inhibition of cell viability was significantly more severe in the cells treated with fucoxanthin and fucoxanthinol than those treated with β-carotene and astaxanthin. The effect of fucoxanthin was not significant on control uninfected cell lines K562 and HeLa. Cell viability of an uninfected T-cell line, Jurkat, was also inhibited, but Jurkat was less susceptible to fucoxanthin than HTLV-1-infected T-cell lines. The cell viability inhibition activity of fucoxanthinol was more pronounced than that of fucoxanthin with IC50 values of 0.86–1.83 μM (1.24 ± 0.43 μM) and 1.20–4.46 μM (3.31 ± 1.44 μM), respectively.
We also evaluated the effects of the 2 carotenoids on the cell viability of fresh ATL cells obtained from 12 independent ATL patients. As shown in Figure 1b, fucoxanthin and fucoxanthinol inhibited the cell viability of fresh ATL cells. All patients were negative for Tax protein by immunoblot analysis (data not shown). Fresh ATL leukemic cells were more susceptible to fucoxanthin and fucoxanthinol-induced cell viability inhibition than control PBMCs from healthy individuals. Like the data in Figure 1a with T-cell lines, fucoxanthinol-induced cell viability inhibition was more pronounced than that of fucoxanthin with IC50 values of 0.46–3.84 μM (1.72 ± 0.97 μM) and 1.29–3.75 μM (2.74 ± 0.83 μM), respectively.
Fucoxanthin and fucoxanthinol induce apoptosis of HTLV-1-infected T-cell lines
To examine whether induction of apoptosis accounts for the cell viability inhibition observed in HTLV-1-infected T-cell lines, cells were treated with fucoxanthin and fucoxanthinol then probed with the APO2.7 monoclonal antibody. Both carotenoids increased the proportion of apoptotic cells in all HTLV-1-infected T-cell lines, but not in HeLa (Fig. 2a). They increased the proportion of apoptotic cells in Jurkat, but Jurkat was less susceptible to them than HTLV-1-infected T-cell lines. Furthermore, Hoechst 33342 staining of nuclei showed apoptotic changes (Fig. 2b). Taken together, these results indicate that fucoxanthin and fucoxanthinol inhibit cell viability of HTLV-1-infected T-cell lines through cell apoptosis.
Fucoxanthin- and fucoxanthinol-induced apoptosis is caspase-dependent
We next examined whether caspase activation is involved in fucoxanthin- and fucoxanthinol-induced apoptosis. We measured the levels of caspase-3, -8, -9 and PARP in HUT-102 cells after exposure to fucoxanthinol. Fucoxanthinol cleaved caspase-3, -8, -9 and PARP, indicating activation of these cystein proteases (Fig. 2c). Furthermore, both carotenoids activated caspases-3, -8 and -9 in HUT-102 and ED-40515(-) cells (Fig. 2d). These results demonstrate the involvement of caspase activation in fucoxanthin- and fucoxanthinol-induced apoptosis in HTLV-1-infected T-cell lines.
Fucoxanthin and fucoxanthinol induce cell cycle arrest
We also investigated the effect of both carotenoids on the cell cycle progression in cell lines. HUT-102 cells were incubated with fucoxanthinol (5 μM) for various time intervals (0 to 24 hr) and analyzed for cell cycle distribution by flow cytometry (Fig. 3a). Cultivation with fucoxanthinol for 12 hr increased the population of cells in the G1 phase, with a marked reduction of cells in the S phase. At 24 hr after treatment, the percentage of apoptotic cells markedly increased, suggesting that cell cycle arrest is the cause of apoptosis. Therefore, cells were incubated with fucoxanthin (10 μM) and fucoxanthinol (5 μM) for 12 hr and analyzed for cell cycle distribution by flow cytometry (Fig. 3b). Both carotenoids inhibited cell cycle progression, as evidenced by the increased proportion of cells in G1 phase and reduction of cells in the S phase, indicating G1 arrest in all cell lines. These results clearly show that both carotenoids induce G1 arrest of the cells.
Effects of fucoxanthin and fucoxanthinol on the expression of cell cycle- and apoptosis-related proteins
To clarify the molecular mechanisms of fucoxanthin and fucoxanthinol-induced inhibition of cell viability and apoptosis in HTLV-1-infected T-cell lines, we examined the expression of several intracellular regulators of cell cycle and apoptosis, including cyclin D1, cyclin D2, CDK4, CDK6, p53, GADD45α, Bax, Bcl-2, Bcl-xL, XIAP, cIAP2 and survivin by Western blot analysis. As shown in Figure 4a, fucoxanthin and fucoxanthinol did not alter p53, Bax and Bcl-xL levels. In contrast, they significantly decreased the expression of survivin, XIAP, cyclin D1, cyclin D2 and CDK4, and induced the expression of GADD45α in HUT-102 and MT-2 cells in a time-dependent manner. Both carotenoids also decreased the expression of Bcl-2 in MT-2 cells but not in HUT-102 cells. Furthermore, cIAP2 levels were decreased in fucoxanthinol-treated HUT-102 and MT-2 cells and in fucoxanthin-treated MT-2 cells. Fucoxanthinol but not fucoxanthin decreased the expression of CDK6. Comparable loading of protein was confirmed with a specific antibody for the housekeeping gene product actin. Finally, fucoxanthin and fucoxanthinol decreased the expression of XIAP and cyclin D2 in freshly isolated ATL cells (Fig. 4b).
Fucoxanthin and fucoxanthinol modulate activated NF-κB
NF-κB is a transcription factor involved in the control of apoptosis, cell cycle progression and cell differentiation.18 NF-κB is constitutively activated in Tax-expressing and HTLV-1-infected T-cell lines as well as primary ATL cells,17 and such activation correlates with leukemogenesis.19 Because NF-κB regulates the expression of survivin, XIAP, cIAP2, Bcl-2, cyclin D1, cyclin D2, CDK4 and CDK6,20–26 we examined whether fucoxanthin and fucoxanthinol inhibit the NF-κB pathway. To study the DNA-binding activity of NF-κB, we performed EMSA with radiolabeled double-stranded NF-κB oligonucleotides and nuclear extracts from untreated or fucoxanthin- or fucoxanthinol-treated HTLV-1-infected T-cell lines. NF-κB oligonucleotide probe with nuclear extracts from untreated HTLV-1-infected T-cell lines generated DNA-protein gel shift complexes (Fig. 5a, top panel). These complexes were due to specific binding of nuclear proteins to the NF-κB sequence, because these binding activities were reduced by the addition of cold probe but not by an irrespective sequence (Fig. 5a, top panel, lanes 2 and 3). Furthermore, NF-κB complexes contained p50, p65 and c-Rel (Fig. 5a, top panel, lanes 4–7). Nuclear extracts prepared from HTLV-1-infected T-cell lines treated with fucoxanthin and fucoxanthinol for 12 hr exhibited a decrease in the intensity of the NF-κB-containing gel shift complexes (Fig. 5b). This finding suggests that both carotenoids downregulate the DNA-binding activities of NF-κB. Inhibition appeared specific to NF-κB and not due to cell death, because no significant change in binding activity of Oct-1 was observed after treatment of cells with fucoxanthin and fucoxanthinol.
Degradation of IκBα and subsequent release of NF-κB requires prior phosphorylation at Ser32 and Ser36 residues.27 To investigate whether the inhibitory effects of the carotenoids were mediated through alteration of phosphorylation of IκBα, HUT-102 and MT-2 cells were treated with fucoxanthin and their protein extracts were checked for phospho-IκBα expression. Untreated HUT-102 and MT-2 cells constitutively expressed Ser32/36-phosphorylated IκBα, while fucoxanthin treatment decreased the phosphorylated IκBα in a time-dependent manner (Fig. 5c), with a concomitant rise in IκBα level, suggesting that fucoxanthin inhibits phosphorylation of IκBα followed by accumulation of this protein.
Fucoxanthin and fucoxanthinol modulate activated AP-1
Transcription factor AP-1 is also identified as a crucial mediator of both cell cycle enhancing and cell death inhibiting pathways in HTLV-1-infected T-cells.28 Therefore, we focused on AP-1 inactivation after exposure to fucoxanthin and fucoxanthinol. HUT-102 and MT-2 cells exhibited elevated constitutive AP-1 DNA-binding activity (Fig. 5a, bottom panel). Supershift analysis with antibodies indicated that the AP-1 complex in both cell lines contained JunD. Fucoxanthin and fucoxanthinol reduced AP-1 DNA-binding activity (Fig. 5b) and also decreased time-dependently the expression of JunD, which composes the increased DNA-binding AP-1 protein (Fig. 5c). These findings suggest that the 2 carotenoids deplete JunD, resulting in inactivation of AP-1.
Antitumor effects of fucoxanthinol on subcutaneous HUT-102 tumors
Finally, we examined the effects of fucoxanthinol against ATL in vivo. SCID mice (n = 12) were inoculated with HUT-102 and then divided into 2 groups: untreated mice (n = 6) and fucoxanthinol-treated mice (n = 6). Treatment commenced on the day after inoculation and the effects of treatment on tumorigenicity were assessed over 4 weeks. During treatment, all mice displayed no adverse events with respect to general appearance, body weight (Fig. 6a) and food intake. Fucoxanthinol did not affect tumor incidence but significantly slowed the growth of the transplanted tumors. After 14-day treatment, fucoxanthinol significantly decreased tumor volume compared with vehicle-treated mice (p < 0.01). Statistically similar differences were found in tumor weights at necropsy (p < 0.05) (Fig. 6a). The efficacy of treatment was reflected by a decrease in serum levels of the surrogate marker sIL-2Rα (p < 0.05). On the other hand, TUNEL assay showed few apoptotic cells in tumors from untreated mice, while abundant apoptotic cells were noted in tumors from fucoxanthinol-treated mice (Fig. 6c). At necropsy, gross and histopathological examinations showed no apparent pathological findings, neoplastic lesions or metastatic tumors in the lungs, liver, pancreas, kidneys, spleen or large bowel in all mice.
We examined the tissue distribution of fucoxanthinol in mice treated orally for 28 days. Fucoxanthinol showed extensive distribution into all sampled tissues (serum, 0.51 μg/ml; heart, 10.48 μg/g; lung, 18.48 μg/g; thymus, 24.70 μg/g; liver, 15.32 μg/g; spleen, 33.02 μg/g), with highest concentrations in the kidney (33.37 μg/g). Tumor fucoxanthinol levels were 3.51 μg/g. This concentration is equivalent to 5.70 μM, suggesting that therapeutically effective concentrations of fucoxanthinol may be easy to achieve in vivo. These results suggest that fucoxanthinol is therapeutically beneficial in mice with ATL.
ATL follows an invariably fatal clinical course in spite of the introduction of various chemotherapeutic agents. Although many ATL patients initially respond to chemotherapy, drug-resistance eventually develops, which prevents curative treatment. Although allogenic hematopoietic stem cell transplantation produces better results, it often causes serious side effects and entails the risk of graft versus host-disease.29 In addition, its application is limited because of the advanced age of patients with ATL. Therefore, a novel therapeutic approach based on new insights into the pathogenesis of ATL is strongly desired. Advances in molecular biology have provided many new insights into the biology and treatment options for ATL. Recently, the strategy of targeting the molecules critical for maintenance and growth of the tumor cells was considered important for the development of more effective treatment with less undesirable effects.30 This strategy intensifies the specificity of treatment to tumor cells and minimizes undesirable effects to normal cells.
In our study, we examined the inhibitory effects of 4 carotenoids on the cell viability of HTLV-1-infected T-cell lines. The inhibitory activities of fucoxanthin and its deacetylated derivative, fucoxanthinol, were stronger than those of β-carotene and astaxanthin. Because dietary fucoxanthin is hydrolyzed into fucoxanthinol in the gastrointestinal tract before absorption in the intestine and the inhibitory effect of fucoxanthinol was higher than that of fucoxanthin, fucoxanthinol may be more effective than fucoxanthin in vivo. Furthermore, we showed that HTLV-1-infected T-cell lines and primary ATL cells are more susceptible to cell viability inhibition induced by treatment with fucoxanthin and fucoxanthinol than normal PBMCs and uninfected cell lines. These data therefore demonstrate that both carotenoids are cytotoxic to HTLV-1-infected cells but not to normal cells.
Our results showed that the cell viability-inhibitory potential of fucoxanthin and fucoxanthinol on HTLV-1-infected T-cell lines was mainly due to the induction of G1 cell cycle arrest and apoptosis. Such cell cycle arrest in G1 phase was associated with downregulation of expression of proteins in G1/S transition such as cyclin D1, cyclin D2, CDK4 and CDK6, and upregulation of expression of GADD45α, which inhibits entry of cells into S phase. In agreement with our findings, Satomi and Nishino showed that fucoxanthin induced G1 cell cycle arrest and GADD45 gene expression in human cancer cells.31 GADD45α is a known transcriptional downstream target of p53; however, this cannot solely explain fucoxanthin-induced GADD45α. Fucoxanthin and fucoxanthinol did not increase p53 levels, suggesting that upregulation of GADD45α is independent of p53.
Both carotenoids induced apoptosis of HTLV-1-infected T-cell lines, which was associated with activation of caspase-3, -8 and -9, as well as downregulation of expression of antiapoptotic proteins, XIAP, cIAP2, Bcl-2 and survivin. Bcl-2 is the principal regulator of the mitochondrial-dependent pathway for apoptosis,32 and its downregulation is involved in caspase-9-dependent apoptosis. Because XIAP and cIAP2 inhibit caspase-3 and -9 activities,33 it appears that the 2 carotenoids stimulate caspase-3- and -9-dependent apoptosis by downregulating XIAP and cIAP2 expression. In our study, the expression of survivin, another member of the IAP family, was also downregulated by fucoxanthin and fucoxanthinol. Because caspase-3 can be inhibited by survivin,34 it is possible that downregulation of survivin by the 2 carotenoids could lead to activation of caspase-3. Caspase-8 is activated by death receptors, such as CD95, tumor necrosis factor receptor and tumor necrosis factor-related apoptosis-inducing ligand receptor, which are expressed on HTLV-1-infected T cells. These death receptors may trigger signaling pathways in cells treated with fucoxanthin and its metabolite.
Detailed mapping of intracellular molecules and signaling pathways might provide more efficient and less toxic treatment opportunities in which cellular components, critical for survival of the tumor, can be selectively targeted. We found that both carotenoids possessed anti-NF-κB activity. They inhibited IκBα phosphorylation and NF-κB DNA-binding activity. Activation of NF-κB plays an important role in cell proliferation and prevention of apoptosis due to upregulation of several NF-κB-inducible molecules. We found that suppression of NF-κB by fucoxanthin and fucoxanthinol correlated with downregulation of the expression of several gene products regulated by NF-κB.20–26 Thus, both carotenoids reduced the expression levels of XIAP, cIAP2, survivin, Bcl-2, cyclin D1, cyclin D2, CDK4 and CDK6. Interestingly, various target genes showed different susceptibility to the inhibitory effects of fucoxanthin and fucoxanthinol. NF-κB participates in the transcription of over 150 target genes, but not all are activated when NF-κB is induced. It is possible that several different mechanisms confer selectivity on the NF-κB inhibitory response to fucoxanthin and fucoxanthinol. A decrease in NF-κB activity may be at least in part responsible for induction of cell cycle arrest and apoptosis by both carotenoids in HTLV-1-infected T-cell lines.
The AP-1 is known to regulate cell proliferation, differentiation and apoptosis in various cell lines.35 In HTLV-1-infected T-cell lines, fucoxanthin and fucoxanthinol inhibited JunD expression, resulting in the suppression of AP-1 DNA-binding. AP-1 is required for proliferation of HTLV-1-infected T cells.28 Although cyclin D1 expression is regulated by NF-κB,24 AP-1 proteins also bind directly to the cyclin D1 promoter and activate it.36 Furthermore, the cyclin D2 promoter contains NF-κB and AP-1 sites.37 It is therefore likely that NF-κB and AP-1, in concert, support proliferation of HTLV-1-infected T cells by activating cyclin D1 and cyclin D2. We speculate that fucoxanthin and fucoxanthinol inhibit cyclin D1 and cyclin D2 expression through the suppression of both NF-κB and AP-1, resulting in the induction of cell cycle arrest at the G1 phase. Based on these findings, it is likely that the 2 carotenoids exert their anti-ATL effects through the suppression of HTLV-1-induced activation of NF-κB and AP-1.
In HTLV-1-expressing cells, Tax, the virus-encoded regulatory protein, plays a critical role in the growth and survival of infected T cells by perturbing normal regulatory mechanisms, including transcription, signal transduction and cell cycle progression, resulting in uncontrolled cell growth.38 Tax activates NF-κB by stimulating the activity of the IκB kinase, which in turn leads to phosphorylation and degradation of IκBα.19 Tax also activates AP-1.28, 39 However, primary ATL cells are known to have very low or no expression of Tax and therefore, growth of ATL cells in vivo is believed to be Tax-independent.40 Fucoxanthin and fucoxanthinol induced cell death of Tax-negative HTLV-1-infected T-cell line, ED-40515(-), and primary ATL cells. In addition, both carotenoids did not inhibit the level of Tax expression in HTLV-1-infected T-cell lines (data not shown). Therefore, the effects of fucoxanthin and fucoxanthinol on HTLV-1-infected T-cell lines and primary ATL cells appear to be mediated through a Tax-independent pathway.
The potent and selective apoptotic effects of fucoxanthin and fucoxanthinol against HTLV-1-infected T-cell lines and primary ATL cells in vitro prompted us to evaluate their in vivo anti-ATL effects in SCID mice bearing an HTLV-1-infected T-cell line, HUT-102. We used fucoxanthinol, a metabolite of fucoxanthin, and found that it also inhibited tumor formation in vivo. During the period from day 0 to 28, the control mice showed signs of severe disease, including piloerection. In contrast, mice treated with fucoxanthinol showed no significant adverse effects and tolerated the dose well. We conclude that fucoxanthinol could be potentially beneficial as a chemotherapeutic or chemopreventive agent in ATL. Further clinical studies are necessary to assess its effects on fresh primary ATL cells.
We are indebted to the patients with ATL and the control subjects who provided blood samples for these studies. We also thank Dr. M. Maeda for providing ED-40515(-) and the Fujisaki Cell Center, Hayashibara Biomedical Laboratories (Okayama, Japan) for providing HUT-102.