Cell lines and cell culture
Cells from HTLV-1-transformed T cell lines MT-2, MT-4, SLB-1 and HUT-102; ATL-derived T cell lines MT-1 and TL-OmI; and uninfected T cell lines Jurkat, CCRF-CEM and MOLT-4 were cultured in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 1% penicillin/streptomycin (Nacalai Tesque, Inc.) and 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel). The MT-2, MT-4 and MOLT-4 cells were provided by Dr. Naoki Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan). The SLB-1 cells were obtained from Dr. Diane Prager (UCLA School of Medicine, Los Angeles, CA, USA). The TL-OmI and CCRF-CEM cells were obtained from Dr. Masahiro Fujii (Niigata University, Niigata, Japan). The HUT-102, MT-1 and Jurkat cells were provided by Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan).
Reagents
Pimozide and z-VAD-FMK were purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and Promega Corp. (Madison, WI, USA), respectively. N-acetyl-L-cysteine (NAC) and necrostatin-1 were obtained from Wako Pure Chemical Industries (Osaka, Japan) and Abcam (Cambridge, UK), respectively.
Cell growth and cytotoxicity assays
A water-soluble tetrazolium (WST)-8 assay kit (Nacalai Tesque, Inc.) was used to assess cell proliferation and toxicity. Cells were cultured in 96-well plates and treated with pimozide at different concentrations for up to 72 h. After the WST-8 reagent was added to each well and the contents were incubated, the absorbance was measured at 450 nm using a Wallac 1420 Multilabel Counter (PerkinElmer, Inc., Waltham, MA, USA). Triplicate wells were used for each culture condition. The optical density of each sample was compared to that of the control.
Cell cycle analysis
The CycleTEST Plus DNA Reagent kit (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA) was used for staining with propidium iodide (PI), which is used to label nuclear DNA. The DNA content of individual nuclei was analyzed using the Epics XL Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) and the MultiCycle software (version 3.0; Phoenix Flow Systems, San Diego, CA, USA). Histograms of PI fluorescence intensity were produced, and the percentage of total cells at each phase of the cell cycle was determined.
Apoptosis analysis
The cells were treated with pimozide for up to 48 h, followed by permeabilization by incubation with digitonin, following which the cells were labeled with a phycoerythrin-conjugated APO2.7 antibody (1:10; Beckman Coulter, Inc., Marseille, France), as described previously [7]. The percentage of apoptotic cells was quantified immediately after staining using an Epics XL Flow Cytometer. In addition, to evaluate the morphological features of the nuclei, the cells were stained with Hoechst 33342 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and observed under a DMI6000 microscope (Leica Microsystems, Wetzlar, Germany).
Caspase activity assay
The activity levels of caspase-3, caspase-8 and caspase-9 were quantified using Colorimetric Caspase Assay kits (Medical & Biological Laboratories, Co., Nagoya, Japan) in accordance with the manufacturer’s instructions. In brief, the cells were lysed in the lysis buffer provided with the kit, and the cell lysates were incubated with the respective caspase-specific labeled substrates. The chromophore ρ-nitroanilide released upon cleavage from the substrates was quantified using a Wallac 1420 Multilabel Counter. Caspase activity was measured as the ratio between the colorimetric output in the treated sample and that in the control, with the value of the latter set to 1.
Reverse transcriptase (RT)-PCR
Total RNA was isolated from the cultured cells using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Reverse transcription was performed using a PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan). PCR was performed using a combination of individual sequence-specific primer sets. The following primers were used: 5′-TGTACAATACGCGCTACAGCTCCA-3′ (sense) and 5′-ATGCACTCGTTCTGGTCTGCGTTA-3′ (antisense) for the dopamine D2 receptor gene; 5′-TCTGTGCCATCAGCATAGACAGGT-3′ (sense) and 5′-TAAAGCCAAACAGAAGAGGGCAGG-3′ (antisense) for the dopamine D3 receptor gene; 5′-TCTTCGTCTACTCCGAGGTCCA-3′ (sense) and 5′-TGATGGCGCACAGGTTGAAGAT-3′ (antisense) for the dopamine D4 receptor gene; 5′-GCCAAGGTCATCCATGACAACTTTGG-3′ (sense) and 5′-GCCTGCTTCACCACCTTCTTGATGTC-3′ (antisense) for GAPDH. The PCR amplification products were separated by electrophoresis in an agarose gel and stained using ethidium bromide.
Quantification of reactive oxygen species (ROS)
The generation of intracellular ROS was detected using CellROX, a fluorescence probe. After exposure to pimozide at different concentrations for 24 h, the cells were incubated with the CellROX Green reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min in the dark and washed with phosphate-buffered saline. The changes in CellROX Green-induced fluorescence were analyzed using an SH800 Flow Cytometer (Sony Biotechnology Inc., Tokyo, Japan).
Western blot analysis
The cultured cells were lysed using a lysis buffer containing 62.5 mM Tris-HCl (pH 6.8) (Nacalai Tesque, Inc.), 2% sodium dodecyl sulfate (SDS; Nacalai Tesque, Inc.), 10% glycerol (Nacalai Tesque, Inc.), 6% 2-mercaptoethanol (Nacalai Tesque, Inc.) and 0.01% bromophenol blue (Wako Pure Chemical Industries). After protein quantitation using the DC Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), the cell lysates (20 mg) from each sample were resolved using SDS-polyacrylamide gel electrophoresis prior to their transfer to polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany) and probing with primary antibodies (1:1,000). The blots were then incubated with horseradish peroxidase-conjugated secondary anti-mouse (1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA) or anti-rabbit (1:1,000; Cell Signaling Technology, Inc.) IgGs. Immunoreactivity was quantified using an enhanced chemiluminescence reagent (Amersham Biosciences Corp., Piscataway, NJ, USA). The antibodies against Bcl-2, Bcl-xL, Bax, cellular inhibitor of apoptosis (c-IAP) 1, survivin, histone H2AX, phospho-H2AX (Ser139), activating transcription factor (ATF) 4, STAT3, phospho-STAT3 (Tyr705), STAT5, phospho-STAT5 (Tyr694), phospho-IkBa (Ser32/36), IκB kinase (IKK) a, IKKb, phospho-IKKa/b (Ser176/180 and Ser177/181), Akt, phospho-Akt (Thr308), phospho-Akt (Ser473), cleaved poly(ADP-ribose) polymerase (PARP), ae well as cleaved caspase-8, caspase-9 and caspase-3 were obtained from Cell Signaling Technology, Inc. Antibodies against cyclin E, cyclin-dependent kinase (CDK) 2, CDK4, CDK6, p21, p53 and actin were obtained from Neomarkers, Inc. (Fremont, CA, USA). Antibodies against the X-linked inhibitor of apoptosis protein (XIAP) and phospho-retinoblastoma protein (pRb) (Ser780) were obtained from Medical & Biological Laboratories, Co. Antibodies against cyclin D2, p27, myeloid cell leukemia-1 (Mcl-1), c-IAP2, GADD45a, IkBa, JunB and JunD were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). An antibody specific for c-Myc was obtained from Wako Pure Chemical Industries.
Xenograft tumor model
HUT-102 cell suspensions (1 ´ 107/0.2 ml of RPMI-1640 medium) were injected subcutaneously into 5-week-old female C.B-17/Icr-severe combined immunodeficient (SCID) mice (Japan SLC, Inc., Hamamatsu, Japan) on day 0. The mice were randomly divided into control and treatment groups (n = 9, each). Either pimozide (25 mg/kg) or a solvent (0.5% methylcellulose, Wako Pure Chemical Industries) was administered via oral gavage five times per week between days 1 and 28. The tumor size was measured weekly using shifting calipers to calculate the tumor volume using the following formula: p/6 ´ h ´ l ´ w [8]. The body weight of each mice was also measured weekly. At the time of sacrifice on day 28, the xenograft tumors and blood samples were collected rapidly. The weight of each tumor was measured, and the sera were stored at −80 °C until they were assayed for human soluble interleukin-2 receptor a (sIL-2Ra). The studies were approved by the Animal Care and Use Committee at the University of the Ryukyus (A2019149).
Measurement of serum sIL-2Ra levels
The serum sIL-2Ra levels were measured in mice treated or untreated with pimozide using enzyme-linked immunosorbent assay (ELISA) for human sIL-2Ra (Diaclone SAS, Besançon, France) in accordance with the manufacturer’s instructions.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using a Student’s t-test or ANOVA along with the Tukey-Kramer test. Differences were considered significant at P < 0.05.