U87MG and T98G human glioma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Normal human astrocytes (NHA) were purchased from ScienCell Research Laboratories, Inc., (Carlsbad, CA, USA). Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; both Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA), and were treated with various concentrations (1, 5, 10, 20 and 40 µM) of arctigenin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 24 or 48 h at 37°C before being subjected to further analyses. Dimethyl sulfoxide (DMSO; 0.5%) was used as the vehicle control. In specific cases, the cells were pre-treated for 1 h with 10 µM Z-DEVD-FMK (Calbiochem; EMD Millipore, Billerica, MA, USA), a general inhibitor of caspase-3, prior to their exposure to arctigenin.

Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich; Merck Millipore). The cells were seeded into 96-well plates (2×10⁴ cells/well) and incubated with 1, 5, 10, 20 or 40 µM arctigenin for 48 h. Subsequently, MTT solution was added to each well and the cells were incubated in the dark at 37°C for 4 h. The formazan crystals were solubilized in 100 µl DMSO and the plates were evaluated using a multiwell spectrophotometer (VersaMax Microplate Reader; Molecular Devices, LLC, Sunnyvale, CA, USA) at a wavelength of 570 nm. All experiments were performed in triplicate and the relative cell viability (%) was normalized to the vehicle-treated control cells.

Cells were seeded into 96-well plates (5×10³ cells/well) and allowed to adhere overnight. Arctigenin was added to the culture medium and incubated with the cells for 48 h. BrdU reagent (5 µM; Sigma-Aldrich; Merck Millipore) was added to each well and the cells were incubated for an additional 2–4 h. The cells were then incubated with a mouse anti-BrdU monoclonal primary antibody (1:300; cat. no. B8434) for 2 h at 37°C and a fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG secondary antibody (1:2,000; cat. no. F9137; both Sigma-Aldrich; Merck Millipore) for 30 min at room temperature. The cells were subsequently stained with DAPI (Sigma-Aldrich; Merck Millipore) prior to imaging using fluorescence microscopy. The number of BrdU-positive cells was determined amongst 500 randomly selected tumor cells.

Cells (1,000 cells/well) seeded into 6-well plates and exposed to arctigenin (10 or 20 µM) for 48 h at 37°C, and were subsequently incubated in fresh medium for an additional 10 days at 37°C. The cells were then fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma-Aldrich; Merck Millipore). The colonies containing >50 cells were identified and counted using a phase-contrast microscope.

A total of 2×10⁴ cells were suspended in serum-free DMEM containing various concentrations of arctigenin (10 or 40 µM) and added to the Matrigel-coated upper chambers of Transwell inserts. Medium supplemented with 10% FBS was added to the lower chambers. Following incubation for 48 h at 37°C, the cells on the upper surface of the filters were removed using cotton swabs. The cells that had invaded to the lower surface of the filters were fixed in 70% ethanol, stained with Giemsa (Sigma-Aldrich; Merck Millipore) and counted.

Following treatment with arctigenin for 48 h, the cells were harvested by trypsinization, washed twice with PBS and fixed in ice-cold 70% ethanol. The cells were then washed again, subjected to RNase A at a final concentration of 0.05 mg/ml (Sigma-Aldrich; Merck Millipore), and incubated with 10 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck Millipore) at 4°C for 15 min in the dark. Cell cycle analysis was conducted using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

In order to analyze cell apoptosis in arctigenin-treated cells, FITC-labeled annexin V/PI staining was performed using the FITC-labeled Annexin V Apoptosis Detection kit, according to the manufacturer's instructions (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). Briefly, 1×10⁶ cells/well were washed with PBS and resuspended in 500 µl binding buffer containing 2.5 µl annexin V-FITC and 5 µl PI. The cells samples were subjected to flow cytometry using a FACSCalibur flow cytometer and analyzed using CellQuest™ software (version 5.1; BD Biosciences). A total of 20,000 events from each cell sample were obtained.

The cells were collected and lysed in lysis buffer (50 mmol/l Tris-HCl, pH 7.4; 150 mmol/l NaCl; 1% NP-40 and 0.5% sodium deoxycholate), supplemented with 1 mM phenylmethanesulphonylfluoride, 10 mg/ml aprotinin and 10 mg/ml leupeptin (all Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was evaluated using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China), and 40 µg total protein/lane was separated using 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, followed by incubation with various primary antibodies (Table I) at 4°C overnight. Membranes were subsequently washed three times with Tris buffer (pH 7.4). The membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Table I). Subsequently, the membranes were incubated with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Chalfont, England) according to the manufacturer's instructions, and the blots were visualized by exposure to X-ray films in the dark. Densitometric analyses were conducted using Scion Image software (version 4.1; Scion Corporation, Frederick, MD, USA) using β-actin for normalization.

Data are presented as the mean ± standard deviation. All analyses were performed using one-way analysis of variance followed by Tukey's post hoc test, using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

At all the concentrations investigated, arctigenin treatment was observed to significantly suppress the viability of U87MG and T98G glioma cells, as compared with vehicle-treated control cells (Fig. 1A). Furthermore, this suppression was determined to be concentration-dependent. Treatment with 40 µM arctigenin decreased the viability of U87MG and T98G cells by 47% (P=0.0027) and 40% (P=0.0012), respectively. By contrast, arctigenin (≤40 µM) was not observed to result in significant cytotoxicity to NHAs (Fig. 1A). Unless otherwise indicated, two concentrations for arctigenin (10 and 40 µM) were used in the following experiments.

The antiproliferative activity of arctigenin in human glioma cells was further investigated using BrdU incorporation assays (Fig. 1B). The results demonstrated that treatment with arctigenin (10 and 40 µM) significantly inhibited the proliferation of U87MG (P=0.0305 and P=0.0046, respectively) and T98G (P=0.0279 and P=0.0113, respectively) cells. The effect of arctigenin on the anchorage-independent growth of glioma cells was also assessed using clonogenic assays. As presented in Fig. 1C, exposure of U87MG and T98G cells to arctigenin induced a significant 60–75% reduction in colony formation, as compared with vehicle-treated cells (P<0.05).

The effect of arctigenin on the invasive capacity of glioma cells was also examined. As presented in Fig. 1D, arctigenin was not observed to have a significant effect on the invasive ability of U87MG and T98G cells through Matrigel-coated membranes (P=0.324 vs. vehicle-treated cells).

To elucidate the mechanisms underlying the antiproliferative activity of arctigenin, PI staining was performed to evaluate the cell cycle progression in response to arctigenin exposure (Fig. 2A and B). The results demonstrated that treatment of U87MG cells with arctigenin significantly increased the proportion of cells in the G₀/G₁ phase (71.2±1.9 vs. 57.8±1.4%; P=0.0143) and reduced the number of cells in the S phase (12.4±0.7 vs. 25.3±1.2%; P=0.005) compared with the vehicle-treated cells. Similar results were noted in T98G cells following treatment with arctigenin, indicating that arctigenin is able to induce cell cycle arrest at the G₀/G₁ phase.

Following exposure of the U87MG and T98G cells to arctigenin, changes in the gene products that regulate cell cycle progression were examined. Following treatment with 10 and 40 µM arctigenin, western blot analysis revealed increased protein expression levels of p21 (P=0.0226 and P=0.0102, respectively), retinoblastoma protein (RB; P=0.0147 and P=0.0079, respectively) and p53 (P=0.0368 and P=0.0054, respectively) in U87MG cells (Fig. 3A and B). By contrast, following treatment with 10 and 40 µM arctigenin, the protein expression levels of cyclin D1 (P=0.0412 and P=0.0308, respectively) and CDK4 (P=0.0375 and P=0.0116, respectively) were significantly decreased in U87MG cells. However, no significant variations in the expression levels of p27, CDK2, S-phase kinase-associated protein 2 (SKP2) and CDK6 were observed between the vehicle- and arctigenin-treated glioma cells. Similar results were detected in T98G cells in response to treatment with arctigenin (Fig. 3A and B).

Flow cytometric analysis revealed that treatment with arctigenin (10 and 20 µM) significantly induced apoptotic response in U87MG and T98G cells (P<0.0001; Fig. 4A). Western blot analysis also demonstrated that increased expression levels of the apoptosis mediator cleaved caspase-3 were present in the arctigenin-treated cells (Fig. 4B). Furthermore, the expression levels of the pro-apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X-protein (BAX) were significantly increased following treatment with 10 and 40 µM arctigenin in U87MG (P=0.0042 and P<0.0001, respectively) and T98G (P=0.0015 and P<0.0001, respectively) cells, compared with the vehicle-treated cells. However, no significant difference was observed in the protein expression levels of the anti-apoptotic protein Bcl-2 between the vehicle-treated and the arctigenin-treated cells.

In order to further evaluate the significance of caspase activation in glioma cells following arctigenin treatment, the general caspase-3 inhibitor, Z-DEVD-FMK, was used. As presented in Fig. 4C, arctigenin-induced apoptosis was significantly suppressed by the pre-treatment of glioma cells with Z-DEVD-FMK, indicating that the arctigenin-induced apoptosis of glioma cells may be mediated via caspase-3 activation.