Butein (cat. no. B3803) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan), dissolved in dimethyl sulfoxide (DMSO) and used for the treatment of cells. The broad spectrum caspase inhibitor, Z-VAD-FMK (cat. no. G7232), was purchased from Promega Corp. (Madison, WI, USA). Antibodies against survivin (cat. no. 2808), Akt (cat. no. 9272), IκB kinase (IKK)α (cat. no. 2682), IKKβ (cat. no. 2684), phospho-Akt (Thr308) (cat. no. 13038), phospho-Akt (Ser473) (cat. no. 4060), phospho-IκBα (Ser32 and 36) (cat. no. 9246), phospho-IKKα/β (Ser176/180 and Ser177/181) (cat. no. 2694), RelA (cat. no. 8242), phospho-RelA (Ser536) (cat. no. 3033), cleaved caspase-3 (cat. no. 9664), -8 (cat. no. 9496), -9 (cat. no. 9501) and poly(ADP-ribose) polymerase (PARP) (cat. no. 9541) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against Bcl-2 (cat. no. MS-597), CDK2 (cat. no. MS-617), CDK4 (cat. no. MS-299), CDK6 (cat. no. MS-398), cyclin E (cat. no. MS-870), retinoblastoma protein (pRb) (cat. no. MS-107) and actin (cat. no. MS-1295) were obtained from Neomarkers, Inc. (Fremont, CA, USA). Antibodies against XIAP (cat. no. M044-3), cyclin D1 (cat. no. K0062-3), heat shock protein (HSP)70 (cat. no. SR-812C), phospho-pRb (Ser780) (cat. no. M054-3S) were purchased from Medical & Biological Laboratories, Co. (Aichi, Japan). Antibodies against cyclin D2 (cat. no. sc-593), c-IAP2 (cat. no. sc-7944), IκBα (cat. no. sc-371), JunB (cat. no. sc-46) and JunD (cat. no. sc-74), and NF-κB subunits p50 (cat. no. sc-114X), p52 (cat. no. sc-298X), RelA (cat. no. sc-109X), c-Rel (cat. no. sc-70X) and RelB (cat. no. sc-226X), and AP-1 subunits c-Fos (cat. no. sc-52X), FosB (cat. no. sc-48X), Fra-1 (cat. no. sc-605X), Fra-2 (cat. no. sc-604X), c-Jun (cat. no. sc-45X), JunB (cat. no. sc-46X) and JunD (cat. no. sc-74X) for supershift assay were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against HSP90 (cat. no. 610418) was from BD BioSciences (San Jose, CA, USA).

The HTLV-1-infected MT-4 and HUT-102, and ATLL-derived TL-OmI T-cell lines, were cultured in RPMI-1640 medium (cat. no. 30264-56, Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (cat. no. 09367-34, Nacalai Tesque, Inc.). Cells were maintained in a 37°C, 5% CO₂ humidified incubator. The study also included peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor. PBMC proliferation was induced with 20 µg/ml of phytohemagglutinin (PHA) (cat. no. L8754, Sigma-Aldrich Co., St. Louis, MO, USA).

The cell proliferative and toxic effects of butein were determined by the water-soluble tetrazolium (WST)-8 uptake method. Cells were plated (1×10⁴ cells per well) in 96-well microtiter plates in triplicate and treated with various concentrations of butein. After incubation for 24–72 h, 10 µl of the WST-8 reagent (cat. no. 07553-44, Nacalai Tesque, Inc.) was added to each well. After 4 h, WST-8 reduction was measured at 450 nm using a 680 XR microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Values were normalized to untreated control samples. The 50% growth inhibitory concentration (IC₅₀) value was calculated by dotting the data points to a logistic curve using the CalcuSym software (version 2.0; Biosoft, Cambridge, UK).

Cells were treated with vehicle or butein for 24–72 h and then permeabilized by incubation on ice for 20 min with 100 µg/ml of digitonin, and treated with phycoerythrin-conjugated APO2.7 antibody (cat. no. IM2088, Beckman Coulter, Inc., Marseille, France) for 15 min at room temperature. After staining with APO2.7 antibody, apoptosis was determined by Epics XL flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). In addition, for analysis of morphological changes in nuclei, cells were stained by 10 µg/ml of Hoechst 33342 (cat. no. 346-07951, Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and observed under a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany).

Caspase activity was measured using Colorimetric Caspase Assay kits (cat. nos. 4800, 4805 and 4810, Medical & Biological Laboratories, Co.). Briefly, cell extracts were recovered using the cell lysis buffer supplied with the kit and assessed for caspase-3, -8 and -9 activities using colorimetric probes. The assay kits are based on detection of chromophore ρ-nitroanilide after cleavage from caspase-specific labeled substrates. Colorimetric readings were performed in an automated microplate reader.

Cells were stained with the CycleTEST Plus DNA Reagent kit (cat. no. 340242, Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA). The cell cycle distribution was analyzed for 10,000 collected cells by an Epics XL flow cytometry that uses the MultiCycle software (version 3.0; Phoenix Flow Systems, San Diego, CA, USA). The population of nuclei at each phase of the cell cycle was determined and apoptotic cells with hypodiploid DNA content were detected in the sub-G₀/G₁ region.

Whole cell extracts were prepared by subjecting butein-treated cells to lysis in lysis buffer [62.5 mM Tris-HCl (pH 6.8) (cat. no. 35434-21, Nacalai Tesque, Inc.), 2% sodium dodecyl sulfate (cat. no. 31607-65, Nacalai Tesque, Inc.), 10% glycerol (cat. no. 17045-65, Nacalai Tesque, Inc.), 6% 2-mercaptoethanol (cat. no. 21438-82, Nacalai Tesque, Inc.) and 0.01% bromophenol blue (cat. no. 021-02911, Wako Pure Chemical Industries, Osaka, Japan)]. Lysates were spun to remove insoluble material. The supernatants were collected and kept at -80°C. Protein concentrations were determined using a commercial kit (DC Protein Assay, cat. no. 5000116JA, Bio-Rad Laboratories, Inc.). Equal amounts of protein extracts (20 µg) were resolved on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). After electrophoresis, the proteins were electrotransferred onto polyvinylidene difluoride membranes (cat. no. IPVH00010EMD, Millipore, Darmstadt, Germany), blotted with the relevant antibodies. Finally, blots were hybridized with horseradish peroxidase-conjugated secondary anti-mouse (cat. no. 7076, Cell Signaling Technology, Inc.) or anti-rabbit IgG antibody (cat. no. 7074, Cell Signaling Technology, Inc.) and developed using an enhanced chemiluminescence reagent (cat. no. RPN2232, Amersham Biosciences Corp., Piscataway, NJ, USA).

TRIzol reagent (cat. no. 15596026, Invitrogen Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from cells. RNA (1 µg) was reverse transcribed into cDNA using a PrimeScript RT-PCR kit (cat. no. RR014A, Takara Bio Inc., Otsu, Japan). The sequence-specific primers used for RT-PCR were as follows: for IκBα, forward 5′-GCCTGGACTCCATGAAAGAC-3′, reverse 5′-CAAGTG GAGTGGAGTCTGCTGCAGGTTGTT-3′; and for GAPDH, forward 5′-GCCAAGGTCATCCATGACAACTTTGG-3′, reverse 5′-GCCTGCTTCACCACCTTCTTGATGTC-3′. PCR amplifications were performed in a programmable thermal cycler. Results were analyzed at 30 cycles to obtain results at exponential levels of amplification.

To determine NF-κB and AP-1 activation, we prepared nuclear extracts from butein-treated cells and performed EMSA, as previously described (14). Nuclear extracts were incubated with ³²P-labeled probes. The top strand sequences of the oligonucleotide probes or competitors were as follows: for a typical NF-κB element of the interleukin-2 receptor α chain (IL-2Rα) gene, 5′-GATCCGGCAGGGGAATCTCCCTCTC-3′ and for the consensus AP-1 element of the IL-8 gene, 5′-GATCGTGATGACTCAGGTT-3′. The above underlined sequences are the NF-κB and AP-1 binding sites, respectively. In competition experiments, nuclear extracts were preincubated for 15 min with 100-fold excess of unlabeled oligonucleotides. For supershift assays, nuclear extracts were incubated with antibodies against NF-κB or AP-1 subunits for 45 min at room temperature before the complex was analyzed by EMSA. The dried gels were visualized.

Five-week-old female C.B-17/Icr-severe combined immune deficient (SCID) mice were obtained from Kyudo, Co. (Tosu, Japan). To induce malignancy, 1×10⁷ HUT-102 cells suspended in sterile RPMI-1640 medium (200 µl) were inoculated subcutaneously into the postauricular region of SCID mice. The mice were divided at random into two treatment groups (n=6, each). Butein was solubilized in soybean oil (cat. no. 190-03776, Wako Pure Chemical Industries) and administered at a dose of 0.7 mg/kg intraperitoneally three times a week, and the treatment was continued for 27 days, beginning on the day after cell inoculation. The control group received the vehicle only. The tumor diameter was measured weekly with a shifting caliper and tumor volume was calculated. The mice were also weighed weekly, beginning on day 0. All mice were sacrificed on day 28. The tumors were excised and their weight measured. At the end of the study, blood samples were collected and the sera were separated by centrifuging blood and then stored at −80°C until assayed for secreted soluble IL-2Rα [soluble cluster of differentiation 25 (sCD25)] and sCD30. This experiment was performed according to the Guidelines for Animal Experimentation of the University of the Ryukyus (Nishihara, Japan), and was approved by the Animal Care and Use Committee of the University of the Ryukyus (reference no. 5837).

The tumor specimens were collected from the control mice group and 0.7 mg/kg butein group, fixed in formalin (Wako Pure Chemical Industries) solution, dehydrated through graded ethanol series (Japan Alcohol Selling Co., Tokyo, Japan) and embedded in paraffin (cat. no. 09620, Sakura Finetek Japan Co., Tokyo, Japan). The paraffin-embedded specimens of ATLL tumors were stained with hematoxylin and eosin (H&E, cat. nos. 234-12 and 1159350025, Merck, Darmstadt, Germany) and examined histologically. Analysis of DNA fragmentation by TUNEL testing was performed using a commercial kit (cat. no. 11684817910, Roche Applied Science, Penzberg, Germany). Cells were examined under a light microscope (Axioskop 2 Plus) with an Achroplan 40×/0.65 lens (both from Zeiss, Hallbergmoos, Germany). Images were acquired with an AxioCam 503 color and AxioVision LE64 software (Zeiss).

Serum concentrations of human sCD25 (cat. no. DR2A00, R&D Systems, Inc., Minneapolis, MN, USA) and human sCD30 (cat. no. SK00582-01, Aviscera Bioscience, Inc., Santa Clara, CA, USA) were measured by enzyme-linked immunosorbent assay (ELISA), according to the protocol supplied by the manufacturer.

Values are shown as mean ± standard deviation (SD). Data of two groups were compared by the Student's t-test. Differences were considered significant at P<0.05.

To determine the in vitro effects of butein in ATLL cells, two HTLV-1-infected T-cell lines (MT-4 and HUT-102) and an ATLL-derived T-cell line (TL-OmI) were cultured in the presence of butein (1.56–100 µM). As shown in Fig. 1, 24–72 h culture with butein significantly reduced cell proliferation and viability in a concentration- and time-dependent manner, with IC₅₀ values at 72 h of 7.0 µM for HUT-102, 7.9 µM for MT-4 and 9.8 µM for TL-OmI. In contrast, the IC₅₀ was 27.8 µM for normal PBMCs. Butein suppressed the proliferative response of PBMCs to the T cell mitogen PHA, with IC₅₀ of 18.5 µM. Although the IC₅₀ values for HTLV-1-infected T-cell lines were lower than those for normal PBMCs, obvious selective cytotoxic activities of butein on HTLV-1-infected T-cell lines have not been demonstrated.

First, the effect of butein on cell morphology was examined using microscopy. Cells cultured with butein for 48 h demonstrated morphologies characteristic of apoptosis, such as chromatin condensation and nuclear fragmentation (Fig. 2A). To further investigate the mode of butein-induced cell death, HTLV-1-infected T-cell lines were treated with 6.3–25 µM butein for 24 h. Butein induced concentration-dependent increases in the sub-G₀/G₁ population (Fig. 3A and B). We also assessed the expression of 38-kDa mitochondrial membrane antigen APO2.7 during apoptosis (15). Apoptotic cells (APO2.7-positive) increased after treatment with butein concentration- and time-dependently (Fig. 2B and C). Collectively, these results indicate that butein induces apoptosis of HTLV-1-infected T-cell lines. In addition to apoptosis, the same experiment also showed that butein dose-dependently increased the percentage of cells in the G₀/G₁ phase and also reduced the percentage of cells in the S phase (Fig. 3A and B). Considered together, the results showed the arrest of more cells at the G₀/G₁ phase of the cell cycle.

The apoptotic process is executed by a member of the highly conserved cysteine proteases, caspases. Initiator caspase-8 and -9 or effector caspase-3 are synthesized as inactive proenzymes and become finally activated by proteolytic cleavage (16). To identify the mechanism underlying butein-induced apoptosis of HTLV-1-infected T cells, we investigated cleavage of caspase-3, -8 and -9, and also PARP, a substrate for caspase-3. Exposure of MT-4 and HUT-102 cells to butein (12.5–100 µM for 48 h) caused concentration-dependent increase in the cleavage of caspase-3, -8 and -9, and PARP (Fig. 4A), as well as increase in activities of caspase-3, -8 and -9 (Fig. 4B). To further explore the significance of the latter activation, a broad spectrum caspase inhibitor, Z-VAD-FMK, was used to suppress the effect of butein. Pretreatment of cells with Z-VAD-FMK partially attenuated butein-induced inhibition of cell viability (Fig. 4C), suggesting that butein-mediated cytotoxic effect is mediated, at least in part, through a caspase-dependent apoptosis pathway.

Cell cycle progression is tightly regulated by interaction between CDKs and cyclins (17). To identify the molecular mechanism underlying butein-induced G₀/G₁ arrest, cells were cultured with butein (12.5–100 µM) for 48 h. They were harvested for protein extraction and western blot analysis. As shown in Fig. 5A, levels of CDK4, CDK6, cyclin D2 and cyclin E proteins were significantly decreased in butein-treated MT-4 and HUT-102 cells, compared to DMSO-treated cells. Butein also inhibited the expression of cyclin D1 in HUT-102 but not in MT-4 cells. However, butein had no significant effect on CDK2 protein level. Furthermore, butein induced dephosphorylation of pRb protein (Fig. 5A, top panels) and changed the hyperphosphorylated form to a hypophosphorylated form of pRB (Fig. 5A, second panels). The deregulation of apoptosis is often studied by the expression of anti-apoptotic proteins. There was a dose-dependent decrease in the protein expression of survivin and XIAP, but not Bcl-2, in butein-treated MT-4 and HUT-102 cells. Butein also inhibited the expression of c-IAP2 in HUT-102 but not in MT-4 cells (Fig. 5B). The effects of butein seem to slightly differ dependent on cell lines.

NF-κB forms a family of transcription factors that regulate numerous biological processes, including immune response, inflammation, cell growth, survival and development. The NF-κB proteins are usually sequestered in the cytoplasm by a family of inhibitors, including IκBα. IκBα degradation is mediated through its phosphorylation by IKK, a trimeric complex composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ (18). Incubation of MT-4 and HUT-102 cells with butein resulted in significant dose-dependent inhibition of phosphorylation and upregulation of IκBα protein (Fig. 6A). To determine whether butein-mediated upregulation of IκBα is due to transcriptional activation of IκBα, we examined the expression of IκBα mRNA in cells treated with butein by RT-PCR. As shown in Fig. 6B, butein treatment had no influence on IκBα mRNA, suggesting that butein induces the inhibition of degradation of IκBα protein. Next, we measured total and phospho-protein levels of IKKα and IKKβ to evaluate the possible inhibitory mechanism of butein on IκBα protein phosphorylation. Immunoblot analysis demonstrated butein dose-dependent suppression of phospho-protein levels of IKKα and IKKβ in MT-4 and HUT-102 cells. Total protein levels of both IKKα and IKKβ were reduced in HUT-102 cells, while total protein level of IKKβ alone was reduced in MT-4 cells (Fig. 6A). Thus, the effects of butein seem to slightly differ dependent on cell lines.

NF-κB is a complex of proteins, in which various combinations of NF-κB proteins constitute active NF-κB heterodimers that bind to specific DNA sequences. Thus, to show that the band visualized by EMSA in HTLV-1-infected T-cell lines was indeed NF-κB, nuclear extracts from MT-4 and HUT-102 cells were incubated with antibodies to the NF-κB subunits and analyzed by EMSA. The results showed shifts of the bands to higher molecular masses (Fig. 6C), suggesting that the activated complex consisted of p50, RelA and RelB. The addition of excess unlabeled NF-κB caused complete disappearance of the band, whereas the addition of AP-1 oligonucleotide had no effect on the DNA binding. We also investigated whether butein could inhibit constitutive NF-κB in HTLV-1-infected T-cell lines. MT-4 and HUT-102 cells were cultured in the presence of different concentrations of butein for 24 h and then analyzed for NF-κB activation. Butein dose-dependently inhibited constitutive NF-κB activation in HTLV-1-infected T-cell lines (Fig. 6D). These results indicate that butein can suppress constitutively active NF-κB in HTLV-1-infected T cells.

AP-1 is a group of dimeric transcription factor complexes composed of members of the Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun (c-Jun, JunB and JunD) families, which play central roles in the proliferation and transformation of T cells (6). EMSA showed supershifted band for AP-1, as detected with specific antibodies to JunB and JunD in MT-4 and HUT-102 cells (Fig. 7A). As shown in Fig. 7B, butein produced a significant and dose-dependent suppression of AP-1 activation in HTLV-1-infected T-cell lines. As shown in Fig. 7A, JunB and JunD are functional components of AP-1 transcription factor in HTLV-1-infected T-cell lines. The experiment demonstrated that butein inhibited AP-1 signaling pathway through dose-dependent suppression of JunB and JunD protein levels (Fig. 7C).

Akt is a component of an essential pathway for cell survival and growth during development and carcinogenesis. It regulates cell survival by directly targeting XIAP and by indirectly modulating survivin levels (19,20). Akt also regulates cell cycle and proliferation by indirectly modulating the levels of cyclin D1, cyclin D2 and cyclin E (21). Since butein decreased the levels of XIAP, survivin, cyclin D1, cyclin D2 and cyclin E in the treated cells, we determined its effect on Akt protein. Butein at 50–100 µM for MT-4 and 12.5–100 µM for HUT-102 cells, caused a dose-dependent inhibition of Akt protein expression. Treatment of MT-4 cells with butein at 50–100 µM and HUT-102 cells with butein at 12.5–100 µM caused a dose-dependent inhibition in Akt phosphorylation at Thr308. Butein at 12.5–100 µM for MT-4 and 25–100 µM for HUT-102 cells, also inhibited the phosphorylation of Akt at Ser473 (Fig. 8). RelA is phosphorylated at Ser536 by Akt and such phosphorylation enhances RelA trans-activation potential (22). Butein also caused inhibition of RelA phosphorylation (Fig. 8). The molecular chaperone HSP90 is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. HSP90 client proteins include signaling proteins (IKKs and Akt) and cell cycle regulators (CDKs) (23). Therefore, we investigated the effect of butein on the expression of HSP90. As shown in Fig. 8, butein reduced HSP90 protein levels, but increased HSP70 levels.

Butein significantly inhibited tumor growth in SCID mice inoculated with HTLV-1-infected HUT-102 cells (Fig. 9A, left panels). In the control group, the average tumor volume of 1,545 mm³ was reached in 28 days after HUT-102 cell inoculation (Fig. 9B, left panel). At the same time point, the average tumor volume in the butein-treated group was 727 mm³. Tumor tissues were removed, photographed (Fig. 9A, right panel), and weighed. The mean tumor weight of butein-treated mice was significantly lower than that of vehicle-treated mice (Fig. 9B, middle panel). In addition, the body weight of mice remained stable with no significant differences between the two groups (Fig. 9B, right panel). At post-inoculation day 28, quantitative ELISA used to determine the circulating levels of surrogate tumor markers sCD25 (24) and sCD30 (25) secreted by HUT-102 tumor xenografts showed 42% and 59% decrease in their levels, respectively, in butein-treated mice, compared with the control group (Fig. 9C). Considered together, our results showed that treatment with butein resulted in significant decreases in serum sCD25 and sCD30 of SCID mice.

Finally, H&E and TUNEL staining assays were performed to assess whether the inhibitory effect on tumor growth induced by butein was due to an increase in apoptotic cell death. H&E staining showed a significant increase in apoptotic tumor cells in butein-treated mice, compared with the control group, which were characterized by cytoplasmic condensation, chromatin hyperchromatism and condensation, and nuclear fragmentation. TUNEL assays confirmed these findings and demonstrated increased number of TUNEL-positive tumor cells in butein-treated mice (Fig. 9D).