U2OS (p53 wild) and MG63 (p53 null) cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's media with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator containing 5% CO₂ in air. Both cell lines were used at passages 4–10 for all experiments.

Mouse anti-β-actin antibody (cat. no. A5441), MTZ (cat. no. M6545) and the following chemicals and solvents, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base and Tween20, were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Mouse anti-poly(ADP-ribose) polymerase(PARP)1 (cat. no. sc-8007), rabbit anti-FOXO3 (cat. no. sc-11351), mouse anti-Lamin B1 (cat. no. sc-56145) and mouse anti-GAPDH antibodies (cat. no. sc-32233) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti-pAkt (cat. no. 9271), rabbit anti-Akt (cat. no. 4691), rabbit anti-cleaved PARP1 (cat. no. 9541), rabbit anti-cleaved Caspase-3 (cat. no. 9664), rabbit anti-Bax (cat. no. 5023), rabbit anti-Bim (cat. no. 2933), rabbit anti-Bcl2 (cat. no. 2872) and rabbit anti-p27 antibodies (cat. no. 3686) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit (cat. no. 111-035-003) and goat anti-mouse (cat. no. 115-035-003) horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from GenDEPOT (Barker, TX, USA).

Cells (1×10³) were seeded in each well of a 96-well plate and incubated for 18 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following incubation, cells were treated with DMSO (0.1%) as a control vehicle and 0, 0.1, 0.2, 0.5, 0.8, and 1 µM indicated concentration of MTZ for 72 h at 37°C. Following this, 20 µl WST-1 solution (DoGenBio, Seoul, Korea) was added to each well for 4 h. The visible absorbance at 460 nm for each well was then quantified using a microplate reader. The assay was repeated 3 times.

Cells (0.5×10³) were seeded in 6 cm dishes and incubated for 18 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following incubation, cells were treated with DMSO (0.1%) as a control vehicle or the indicated concentration of MTZ for 7 days. The colonies were washed twice with PBS, fixed with 3.7% paraformaldehyde and stained with 1% crystal violet solution in distilled water at room temperature for 15 min. Images were captured using the Chemi-doc detection system (Bio-Rad Laboratoried, Hercules, CA, USA). The assay was repeated 3 times.

Cells were washed 3 times with PBS and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS; pH 8.0) with protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cell lysates were centrifuged (10,000 × g at 4°C for 10 min). The protein concentration was measured using a Bradford protein determination assay (Bio-Rad Laboratories) and 20 µg protein was loaded per the lane. Proteins were separated on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked in 3% non-fat dry milk for 1 h at room temperature and probed with primary antibodies for FOXO3, pAkt, Akt, p27, LaminB1, GAPDH, cleaved caspase3, Bim (EL), Bax, Bcl2, and β-actin. All antibodies were diluted at 1:1,000 and incubated for 1 h at room temperature. Membranes were then probed with HRP-tagged goat anti-mouse or anti-rabbit IgG antibodies, diluted at 1:15,000 and 1:5,000 respectively, for 1 h at room temperature. Chemiluminescence was detected using ECL.

U2OS cells [2×10⁴/well in 100 µl DMEM (Gibco; Thermo Fisher Scientific, Inc.)] were seeded into 96-well plates and incubated at 37°C for 18 h in a CO₂ incubator. Cells in each well were treated separately with DMSO (negative control), LY294002 (Sigma-Aldrich; Merck KGaA) or Wortmannin (positive control; Sigma-Aldrich; Merck KGaA), and small-molecule compounds (20 µM/ml final conc.; http://www.enzolifesciences.com/BML-2843/screen-well-fda-approved-drug-library-v2/) from the FDA-Approved Drug Library (Enzo Life Sciences, Inc., Farmingdale, NY, USA) (totaling 640 drugs) at 37°C and 5% CO₂ for 24 h. Then, cells were fixed and quenched by adding 100 µl 4% formaldehyde (in PBS) at room temperature for 15 min and 100 µl 0.6% H₂O₂ (in PBS) at room temperature for 15 min. To determine the level of the phosphorylated-FOXO3-S318/321 in these cells, ELISA was performed by treating cells with 100 µl blocking buffer [5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST)] at room temperature for 1 h. Subsequent to washing the cells with TBST 3 times, cells were treated with 100 µl primary antibody against phospho-FOXO3-S318/321 (9465; 1:250 dilution in TBST containing 2% BSA; Cell Signaling Technology, Inc.) at 4°C overnight. Subsequent to washing, cells with TBST three times were treated with 100 µl anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution in TBST containing 2% BSA; Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 2 h. Following washing cells with TBST 3 times, cells were treated with 100 µl 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, followed by adding 50 µl ELISA stop solution (2N H₂SO₄; Sigma-Aldrich; Merck KGaA). The optical density of each well was measured by reading the microplate using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 450 nm.

Cells were washed 3 times with PBS and lysed in cytoplasmic fractional buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH 8.0, 50 mM NaCl, 500 mM sucrose, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 0.2% Triton X-100, 1 mM dithiothreitol, 2 µM phenylmethylsulfonyl fluoride and 0.15 U/ml aprotinin] at 4°C for 30 min. Cell lysates were centrifuged (10,000 × g at 4°C for 30 min) and the supernatant was collected for the cytoplasmic fraction. The pellet was washed twice with the washing buffer (10 mM HEPES pH 8.0, 50 mM NaCl, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine and 0.15 mM spermine) and lysed with nuclear fractional buffer (10 mM HEPES pH 8, 350 mM NaCl, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidine and 0.15 mM spermine) at 4°C for 30 min. Lysates were centrifuged (10,000 × g at 4°C for 30 min) and the supernatant was collected for the nuclear fraction. All of buffers used in this experiment were prepared in the Park laboratory (Sejong, Korea), based on the previous report (12).

Cells (5×10⁵) were seeded in 6 cm dishes and incubated for 18 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following incubation, cells were treated with DMSO (0.1%) as a control vehicle and 1 µM MTZ for 2 h sat 37°C. Cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature, permeabilized with Triton X-100 (0.2%), blocked with bovine serum albumin and incubated with the primary antibody against FOXO3 (diluted, 1,500) at room temperature for 1 h, followed by Alexa 594-conjugated anti-mouse secondary antibody (cat. no. A-11037; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) a room temperature for 1 h. Following counterstaining with DAPI at room temperature for 1 h, fluorescence images were captured with a Zeiss LSM510 confocal microscope at magnification, ×100.

Cells (5×10⁵) were seeded in 6 cm dishes and incubated for 18 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following incubation, cells were treated with DMSO (0.1%) as a control vehicle and 0.5 and 1 µM MTZ for 2 h. Cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature and permeabilized with Triton X-100 (0.2%). Apoptosis was determined by enzymatic labeling of DNA strand breaks with a TUNEL assay kit (the DeadEnd Fluorometric TUNEL System; Promega Corporation, Madison, WI, USA), according to the manufacturer's procedures. Fluoroshield Mounting Medium with DAPI (Abcam, Cambridge, UK) was used, and 3 fields of view were imaged using the Olympus CKX53 light microscope (Olympus Corporation, Tokyo, Japan; magnification, ×100).

Cells (5×10⁵) were seeded in 6-cm dishes and incubated for 18 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following incubation, cells were treated with DMSO (0.1%) as control vehicle and 0.5 and 1 µM MTZ for 2 h. Cells were washed 3 times with PBS, trypsinized with Gibco™ Trypsin-EDTA (0.25%) with Phenol red (Gibco; Thermo Fisher Scientific, Inc.) and resuspended in binding buffer from the Annexin V Apoptosis Detection kit I (BD Pharmingen; BD Biosciences). Cells were analyzed using FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) and the data were analyzed by FlowJo (version 10; De Novo Software, Ashland, Oregon, USA). A total of 10,000 events were collected in each run. The percentage of cells that underwent apoptosis was determined using the fluorescein isothiocyanate with propidium iodide from the Annexin V Apoptosis Detection kit I, according to the manufacturer's procedures.

Cells (5×10⁵) were seeded in 6-cm dishes and incubated for 18 h at 37ls that humidified incubator containing 5% CO₂ in air. Following incubation, cells were transfected with control (GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) or FOXO3 siRNA (GE Healthcare Dharmacon, Inc.) using the ratio of 1 nM siRNA: 3 µl Dharmafect (GE Healthcare Dharmacon, Inc.) in 300 µl serum free media for 6 h at 37°C in a humidified incubator containing 5% CO₂ in air. Following this, cell culture media was replaced with the fresh media containing 10% FBS and cells were incubated for 18 h.

Data are expressed as the mean ± standard error of three independent experiments. Differences between groups were analyzed with one-way analysis of variance with Tukey's post-hoc test when the variances were equal with SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). All statistical tests were two-sided. P<0.05 was considered to indicate a statistically significant difference.

To identify small molecules that can activate FOXO3 in U2OS cells, a osteosarcoma cell line, a cell-based enzymatic ELISA was performed to screen small molecules that may significantly reduce the phosphorylation of Ser318/321 in FOXO3 (pS318/321 FOXO3), which is usually located in the cytoplasm of cells (10). Various FOXO3 phosphorylation sites have been identified by a number of kinases, and these serve a key role in regulating the cellular location and transcriptional activity of FOXO3. Among these, Ser318 and Ser322 have been investigated by Rena et al (22). They indicated that the phosphorylation of Ser318 by Akt induces the subsequent phosphorylation of Ser322 by casein kinase 1, which is critical for translocation of FOXO3 from the nucleus into the cytosol. In previous studies (11,12,23), attempts were made to develop a novel cell-based ELISA assay system using a phospho-FOXO3 antibody to screen small molecules causing nuclear localization of FOXO3. Following testing various commercially available phospho-FOXO3 antibodies including pThr32 and pSer253, which are possible Akt-phosphorylation sites identified by Brunet et al (10), pSer318/321 FOXO3 antibody was selected as the best antibody for the cell-based ELISA system, due to a larger difference in the readout value being detected between the positive controls (Wortmannin and LY294002) and the negative control. Following screening small molecule drugs from a commercially available food and drug administration (FDA)-approved drug library (SCREEN-WELL^® FDA approved drug library V2: BML-2843-0100; Enzo Life Sciences, Inc.), MTZ was focused on (Fig. 1), which indicated a decreased level (>50%) of pS318/321 FOXO3, compared with the DMSO vehicle control in U2OS cells (data not shown).

To demonstrate the mechanism by which MTZ treatment decreases pS318/321 FOXO3 in U2OS cells, nuclear fractional western blot analyses with lysates from U2OS cells previously treated with various doses of MTZ were performed. As depicted in Fig. 2A, MTZ treatment reduced the expression of Akt-pS473 (pS473 Akt) in the cytoplasm, in a dose-dependent manner. In addition, the expression of FOXO3 in the cytoplasm was decreased by MTZ treatment, whilst the expression of FOXO3 in the nucleus was significantly increased by MTZ treatment, in a dose-dependent manner. The expression of the cytoplasmic and nuclear p27 protein was significantly increased by MTZ treatment, in a dose-dependent manner. Additionally, it was confirmed that FOXO3 translocates into the nucleus following MTZ treatment, by carrying out confocal microscopy analysis (Fig. 2B). These results indicated that MTZ may downregulate pS473 Akt, leading to the translocation and activation of the FOXO3 protein from the cytoplasm to the nucleus in osteosarcoma cells.

To investigate the anti-cancer activity of MTZ against osteosarcoma cells, U2OS and MG63 osteosarcoma cells were treated with MTZ and growth rate of the cells was measured using the WST-1 and colony formation assays. Cell viability was determined using WST-1 assay, which involved incubating the U2OS and MG63 cells with MTZ (0, 0.1, 0.2, 0.5, 0.8 and 1.0 for 72 h. As depicted in Fig. 3A, MTZ had an inhibitory effect on osteosarcoma cell growth, in a dose-dependent manner. Notably, treatment with 0.5, 0.8 and 1 µM MTZ resulted in a statistically significant (P<0.05) inhibition of cell viability; therefore, based on the data on cell viability/cytotoxicity, it may be concluded that ≥0.5 µM MTZ is the optimal dose for the subsequent experiments. In addition, clonogenic assay results demonstrated that MTZ treatment markedly suppressed the colony-forming ability of osteosarcoma cells (Fig. 3B). Taken together, these results indicated that MTZ displays a potent inhibitory effect on the cell survival/proliferation of osteosarcoma cells. To examine the effect of MTZ on apoptosis in osteosarcoma cells, western blot analyses, TUNEL assays and Annexin V staining analysis were performed. MTZ treatment increased the cleavage of Caspase-3 and PARP1. MTZ treatment also increased the expression of Bax and Bim extra-large (EL) in osteosarcoma cells; however, MTZ treatment decreased the expression of Bcl-2 (Fig. 3C). These results indicated that the mechanism underlying MTZ and its apoptotic activity may be through a Caspase-3-mediated mechanism in osteosarcoma cells, upregulation of the pro-apoptotic Bax and Bim and downregulation of the anti-apoptotic Bcl2. In addition, compared with the DMSO control, MTZ treatment of osteosarcoma cells resulted in an increase in the amount of Annexin V stained cells as well as TUNEL-positive cells, both of which are typical markers of apoptosis (Fig. 4A and B). Thus, the present results indicated that MTZ treatment may induce cellular apoptosis in osteosarcoma cells.

To examine the role of FOXO3 in MTZ-induced apoptosis in osteosarcoma cells, FOXO3 expression was knocked down in U2OS cells by transfecting siRNA against FOXO3 or control siRNA. Additionally, western blot analysis of PARP1, Caspase-3, Bax, Bim EL, Bcl2 and FOXO3 was performed. As depicted in Fig. 5, FOXO3 knockdown markedly attenuated Caspase-3 and PARP1 cleavage and reduced the expression levels of Bax and the Bim EL isoform, compared with the control siRNA. However, MTZ treatment decreased the reduced the expression levels of Bcl-2. Thus, the present results indicated that FOXO3 may serve a key role in MTZ-mediated apoptosis in U2OS cells.