Curcumin (95%purity) [(E,E)-1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] was purchased from Sigma-Aldrich (St. Louis, MO, USA), and was stored as a 100 mM stock solution in dimethyl sulfoxide (DMSO) at −20°C until use. The present study was approved by the Ethical Committee of Nanjing Medical University (Nanjing, China) (no. 2014-109).

The human U87 glioblastoma cell line was provided by the China Center for Typical Culture Collection (Shanghai, China). The U87 cells were cultured in low-glucose (1 g/l) Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1 mM sodium pyruvate, 100 units/ml penicillin and 100 mg/ml streptomycin (all purchased from Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C under a humidified atmosphere containing 5% CO₂. The cells were incubated for 24 h, after which the medium was removed and replaced with fresh DMEM containing 20 or 40 µM curcumin or DMSO (untreated control) for 24 or 48 h. In some experiments, proliferating cells were transfected with FoxO1 small interfering (si)RNA (cat. no. sc-35382; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or cyclin-dependent kinase 1 (CDK1) siRNA (Suzhou Ribo Life Science Co., Ltd., Suzhou, China), 6 h post-treatment with Lipofectamine^® 2000 in Opti-MEM serum-free medium (both purchased from Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Then, the Opti-MEM medium was changed into DMEM and cells were cultured for 24 h.

Cells were treated with 20 or 40 µM curcumin for 24 or 48 h. The effects of curcumin on cell viability were determined using the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay. Briefly, following incubation with the 20 or 40 µM curcumin for 24 or 48 h, the cells were incubated with 5 g/l CCK-8 solution for 2 h. Subsequently, the cells were placed in a 96-well microplate reader (BioTek, Winooski, VT, USA) for analysis and the optical density (OD) was detected at 450 nm. Cell viability was evaluated using the following formula: Cell viability (%) = [1 − (OD of the samples/OD of the control)] × 100%.

Cells were treated with curcumin (20 or 40 µM) for 24 h, and the control cells were untreated. The cells (1×10⁶) were subsequently washed in phosphate-buffered saline (PBS) and fixed in ice-cold ethanol. Cell proliferation was determined by 5-ethynyl-2-deoxyuridine (EdU) incorporation using the Cell-Light EdU Imaging Detecting kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China), according to the manufacturer's protocol.

Cells were cultured in DMEM for 24 h, and were then treated with 20 or 40 µM curcumin or DMSO for 24 h. To measure cell cycle distribution, the cells were harvested and fixed in 70% ethanol overnight at 4°C. Cells were washed twice in PBS, and re-suspended in 500 µl PBS containing 20 µg/ml propidium iodide (PI; Sigma-Aldrich) for 30 min in the dark. Cell cycle distribution was analyzed by flow cytometry using CellQuest™ software (version 3.0; BD Biosciences, Oxford, UK) and a FACScan flow cytometer (BD Biosciences).

For flow cytometric analysis, Annexin-V-fluorescein isothiocyanate (FITC) conjugate and binding buffer (Santa Cruz Biotechnology, Inc.) were used as standard reagents. Cells remained untreated, or were treated with 20 or 40 µM curcumin for 24 h. The cells (1×10⁶) were then digested with 0.25% trypsin for 2–3 min, digestion was terminated with FBS heat-inactivated serum (Biological Industries, Kibbutz Beit Haemek, Israel), and the cells were re-suspended in PBS. Following fixing with 70% ethanol and staining with FITC and PI using an Annexin V-FITC/PI Apoptosis Detection kit (Biobyt, Ltd., Cambridge, UK), flow cytometry analyses were performed using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and CellQuest™ software (version 3.0). Subsequently, the cells were analyzed. Fluorescent emission of FITC was measured at 515–545 nm and that of DNA-PI complexes was measured at 564–606 nm. Cell debris was excluded from analysis using an appropriate forward light scatter threshold setting. Compensation was used wherever necessary.

The cytoplasmic and nuclear proteins were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Treated cells were suspended in buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol; 0.15% Nonidet P40; 1% protease inhibitor cocktail), were incubated for 10 min on ice, and were centrifuged at 16,000 × g for 5 min. The supernatant fraction was collected as the cytoplasmic extract. Subsequently, the pellet was washed, re-suspended in buffer B (20 mM HEPES, pH 7.9; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol; 0.5% Nonidet P40; 1% protease inhibitor cocktail) and was agitated for 15 min at 4°C. The supernatant fraction from the centrifugation (16,000 × g at 4°C for 10 min) was collected as the nuclear extract. The nuclear and cytoplasmic expression levels of FoxO1 were determined by western blot analysis.

Using the Compartment Protein Extraction kit (EMD Millipore, Billerica, MA, USA), cytoplasmic and nuclear proteins were isolated from the U87 cells. Cellular fractionation was conducted according to the manufacturer's protocol. Cells were lysed on ice in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) for 20 min in the presence of a protease inhibitor (Roche Diagnostics, Basel, Switzerland). The protein concentration was determined using the Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. Total proteins (20 µg) extracted from the untreated and treated cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electophoretically transferred to a polyvinylidene difluoride membrane (EMD Millipore), according to standard procedures. Membranes were blocked for 1 h with 5% nonfat dry milk in TBS-T buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20), followed by an overnight incubation at 4°C with the following primary antibodies: Rabbit anti-FoxO1 (1:1,000 dilution; cat. no. 2880, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-histone h4 (1:3,000 dilution; cat. no. ab61254, Abcam, Cambridge, UK), anti-phosphorylated (p)-FoxO1 (1:1,000 dilution; cat. no. sc-16307, Santa Cruz Biotechnology, Inc.), anti-CDK1 (1:1,000 dilution; cat. no. 9116, Cell Signaling Technology, Inc.), anti-cyclin G2 (1:1,000 dilution; sc-7266, Santa Cruz Biotechnology, Inc.), anti-cleaved caspase-3 (1:1,000 dilution; cat. no. 9664, Cell Signaling Technology, Inc.), anti-Fas ligand (FasL; 1:1,000 dilution; cat. no. 4273S, Cell Signaling Technology, Inc.) and anti-glyceraldehyde 3-phosphate dehydrogenase (1:1,000 dilution; cat. no. ab9484; Abcam). Blots were subsequently washed with TBS-T and were then incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5,000 dilution; cat. no. sc-2004, Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence reagents (EMD Millipore), and were exposed to X-ray films. Protein expression was semi-quantified using Scion Image Beta 4.02 (Scion Corporation, Torrance, CA, USA).

Treated cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100. After blocking with blocking buffer containing 5% nonfat dry milk, the cells were incubated with rabbit anti-FoxO1 (1:200) overnight at 4°C, followed by incubation with a goat anti-rabbit immunoglobulin G tagged with Alexa Fluor 488 (cat. no. 4412, Cell Signaling Technology, Inc.) for 1 h at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Morphological alterations in U87 cells were observed and documented under a fluorescence microscope (Zeiss LSM 510 META; Carl Zeiss AG, Oberkochen, Germany). Fields were chosen at random from various sections, in order to ensure objectivity of sampling. Morphometric analyses were performed using the Zeiss LSM510 v.3.2 analysis software (Carl Zeiss AG).

All statistical analyses were performed using SPSS software (version 19.0; IBM SPSS, Amronk, NY, USA). Quantitative data is expressed as the mean ± standard deviation and analyzed by one-way analysis of variance followed by the Tukey's post-hoc test. Comparison between the groups was made by analyzing data using a post-hoc method. Representative results from independent experiments are presented in the present study. Student's t-test or analysis of variance was used to statistically analyze the results between the control and treatment groups. P<0.05 is considered to indicate a statistically significant difference.

The present study observed the effects of curcumin on the inhibition of U87 cell viability. Curcumin markedly inhibited the viability of U87 cells in a dose- and time- dependent manner. The inhibitory effects of curcumin were most obvious when used at a concentration of 40 µM (Fig. 1A). To detect whether curcumin was able to affect the proliferation of U87 cells, U87 cell growth was analyzed using the EdU incorporation assay following treatment with various concentrations of curcumin for 24 h. Similarly, cell proliferation was markedly inhibited by curcumin when used at 20 or 40 µM. In addition, the effect of 40 µM curcumin was more obvious than that of 20 µM curcumin (Fig. 1B).

The present study determined the effects of curcumin on cell cycle progression and apoptosis in U87 cells. To determine whether curcumin had an effect on cell cycle distribution and apoptosis, U87 cells were treated with 0, 20 or 40 µM curcumin for 24 h. According to the results of a flow cytometric analysis, an increased number of cells were detected in G₂/M phase, and a corresponding reduction in the number of cells was detected in S phase (Fig. 2A). In addition, the number of apoptotic cells was increased in a dose-dependent manner. Curcumin (40 µM) was able to markedly induce apoptosis of U87 cells (Fig. 2B). These results indicate that curcumin may effectively induce G₂/M arrest and apoptosis in U87 cells.

The expression levels of proteins associated with the cell cycle and apoptosis were also detected in the cells following treatment with 20 or 40 µM curcumin for 24 h. Compared with the control group, cyclin G2 was markedly increased in the U87 cells; however, the expression levels of CDK1 were markedly decreased (Fig. 2C). Furthermore, two apoptotic proteins: Cleaved caspase-3 and FasL, were markedly upregulated (Fig. 2D). These data indicate that curcumin may induce G₂/M cell cycle arrest and apoptosis in U87 cells by decreasing CDK1 expression and increasing the expression of cyclin G2, cleaved caspase-3 and FasL.

To investigate the mechanism underlying regulation of the previously mentioned proteins, the present study detected the expression levels of FoxO1 in the presence of 40 µM curcumin. The expression levels of FoxO1 in the nucleus and cytoplasm were increased in response to curcumin in a dose-dependent manner. Notably, the expression levels of p-FoxO1 were markedly decreased (Fig. 3A). In addition, the nuclear translocation of FoxO1 was detected following treatment with curcumin by immunofluorescence, which demonstrated that the nuclear expression of FoxO1 was markedly increased (Fig. 3B). These findings were in accordance with the results presented in Fig. 3A. These results indicate that FoxO1 may have an important role in curcumin-induced regulation of the cell cycle and apoptosis.

To investigate the role of FoxO1, the present study determined the effects of FoxO1 on the expression of CDK1, cyclin G2, cleaved caspase-3 and FasL in the presence of 40 µM curcumin. Following knockdown of FoxO1 with FoxO1 siRNA, the expression levels of CDK1 were not affected. Following the knockout of CDK1 by CDK1-siRNA, the expression of p-FoxO1 decreased; although the total expression of FoxO1 was not affected, the expression of FoxO1 in the nucleus decreased. However, the expression levels of cyclin G2 were inhibited (Fig. 4A), thus indicating that cyclin G2 may be regulated by FoxO1. In addition, the expression levels of cleaved caspase-3 and FasL were markedly inhibited (Fig. 4B), thus indicating that cleaved caspase-3 and FasL may be downstream of FoxO1. To determine whether FoxO1 was regulated by CDK1, CDK1 expression was knocked down using CDK1 siRNA in untreated cells. In cells transfected with CDK1 siRNA, the expression levels of total FoxO1 were not affected; however, the expression levels of p-FoxO1 were decreased. Furthermore, the expression levels of cyclin G2, cleaved caspase-3 and FasL were increased (Fig. 4C), and the nuclear expression of FoxO1 was increased (Fig. 4D). These results indicate that the nuclear translocation of FoxO1 may be dependent on the effects of CDK1 on FoxO1 phosphorylation.

The present study also tested the function of FoxO1 in U87 cells. Following FoxO1 knockdown with FoxO1 siRNA in the presence of 40 µM curcumin, cell cycle distribution and apoptosis were detected in U87 cells. Curcumin-induced G₂/M arrest and apoptosis were partly inhibited by FoxO1 knockdown (Fig. 5A and B). These data suggest that FoxO1 may have an important role in curcumin-induced G₂/M cell cycle arrest and apoptosis in U87 cells.