The human GBM cell line LN-18 was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin (100 U/ml) and 1% streptomycin (100 µg/ml). The culture was maintained at 37°C, in a humid atmosphere containing 5% CO_2.

DMEM and FBS were acquired from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Vitexin was purchased from Selleck Chemicals (Houston, TX, USA) and stored at −20°C following preparation of the stock solution at 300 mM vitexin dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Antibodies against cleaved-PARP (cat. no. 5625), Akt (cat. no. 4685), mTOR (cat. no. 2983), phosphor (p)-Akt (cat. no. 4060), p-mTOR (cat. no. 5536), GAPDH (cat. no. 5174) and anti-rabbit immunoglobulin G, horseradish peroxidase-linked antibody (cat. no. 14708) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Viability of GBM cells following treatment with vitexin was assessed using Cell Counting Kit-8 (CCK8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were plated in 96-well plates at a density of 5×10⁵ cells/ml for 24 h and subsequently treated with different concentrations of vitexin (0, 10, 20, 40, 80 and 160 µM). Following incubation for another 24 or 48 h at 37°C, cells were washed with PBS twice and cultured with CCK8 solution for 2 h according to the manufacturer's instructions. The absorbance was measured at a wavelength of 450 nm using a microplate reader iMark (Molecular Devices, LLC, Sunnyvale, CA, USA) and IC₅₀ values were calculated using SPSS software 19.0 statistical software package (IBM Corp., Armonk, NY, USA).

The influence of vitexin on the proportion of cells at differing stages of the cell cycle was examined using flow cytometry. Cells were plated in a 6-well plate at a density of 5×10⁶ cells/ml and treated with different concentrations of vitexin (0, 20, 40 and 80 µM) for 24 h at 37°C. Cells were digested using trypsin at 37°C for 1 min and centrifuged at 600 × g for 5 min at room temperature following washing with PBS, fixed in 75% ethanol at 4°C overnight and subsequently stained with propidium iodide assay kit (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min in the dark. The cell cycle distribution was analyzed using an Accuri C6 flow cytometer (BD Biosciences) and the resulting data were presented following analysis with ModFit LT software v3.1 (FACSCalibur; BD Biosciences).

Morphological characteristics of apoptotic cells were observed using Hoechst 33342 staining. Cells were incubated in 6-well plates with a coverslip at a density of 5×10⁶ cells/ml and treated with 40 µM vitexin for 24 h at 37°C. Following incubation, cells on the coverslip were washed with PBS three times and fixed in 4% paraformaldehyde for 20 min at room temperature and washed again with PBS. Subsequently, 0.1% Triton X-100 was used to permeabilize the cells and Hoechst 33342 solution (5 µg/ml) was added to stain the nucleus of apoptotic cells, for 10 min at room temperature without light. A fluorescence microscope (Olympus Corporation, Tokyo, Japan) was used to observe morphological alterations in the stained cells.

Apoptosis detection was identified using a Annexin V-fluorescein isothiocyanate (FITC)/PI assay kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's instructions. In brief, the GBM cells were incubated in 6-well plates at a density of 5×10⁶ cells/ml and treated with 0, 20, 40 and 80 µM vitexin. Following incubation for 24 h at 37°C and two washes in ice-cold PBS, cells were collected and re-suspended in annexin-binding buffer. Subsequently, cells were stained by adding Annexin V-FITC and PI at room temperature for 15 min without light. Apoptosis analysis of each sample was performed using an flow cytometer with Accuri C6 software (BD Biosciences).

Protein samples were extracted by adding radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) to lyse GBM cells. Protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Proteins, 30 µg/lane, were added and separated on 8–12% SDS-PAGE prior to being transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, membranes were blocked using 5% skimmed milk at room temperature for 1 h. Primary antibodies (1:1,000; cleaved-PARP, Akt, phospho-Akt, mTOR, phospho-mTOR and GAPDH) were added to the membranes and incubated at 4°C overnight. Subsequently, horseradish peroxidase-conjugated secondary antibody (1:5,000) was added for 1 h at room temperature. Detection of proteins was performed using an Enhanced Chemiluminescence kit (EMD Millipore).

Data are presented as the mean ± standard deviation of three independent experiments and analyzed by one-way analysis of variance and Dunnett's post hoc test were used in order to compare differences among groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA).

The influence of vitexin on proliferation of human GBM cells was assessed by CCK-8 assay (Fig. 1). Cells were cultured with different concentrations of vitexin (0, 10, 20, 40, 80 and 160 µM) for 24 or 48 h. Following incubation, cell viability significantly decreased in a dose and time dependent manner. IC₅₀ values for GBM cells were 32.32 and 25.32 µM following 24 and 48 h of incubation, respectively. The aforementioned results indicated that vitexin suppressed cell proliferation in human GBM cells in a dose- and time-dependent manner.

To investigate whether vitexin inhibited GBM cell proliferation by inducing cell cycle arrest, cell cycle progression was assessed by flow cytometry. Following treatment with vitexin at 20–80 µM for 24 h, cells were collected to perform cell cycle analysis (Fig. 2A and B). Compared with the control group, a significant increase in the percentage of the cell population in the G2/M phase, and a decrease in the number of cells in G1 phase, were observed in cells treated with vitexin. The aforementioned data indicated that vitexin exhibited an effect on cell cycle progression by inducing cell cycle arrest at G2/M phase.

Cell apoptosis is a consequence of cell cycle arrest. Therefore, the present study further investigated the influence of vitexin on cell apoptosis of human GBM cells. Hoechst 33258 staining was performed to observe nuclear morphological alterations characteristic of apoptotic cells, following treatment of GBM cells with 40 µM vitexin for 24 h. GBM cells treated with vitexin exhibited features including cell shrinkage, chromatin condensation and nuclear fragmentation, compared with the control group (Fig. 3A). Subsequently, Annexin V/PI double staining was used to assess the proportion of apoptotic cells. The results indicated a dose-dependent increase in the number of early and late apoptotic cells (Fig. 3B and C). Western blotting was performed to determine the expression level of the intracellular apoptosis-associated protein, cleaved-PARP. PARP recruits DNA repair proteins by binding to DNA breaks. Levels of cleaved-PARP markedly increased following vitexin treatment. The aforementioned results demonstrated that vitexin induced cell apoptosis to inhibit cell proliferation.

The aforementioned in vitro results indicated that vitexin treatment resulted in cell cycle arrest and cell apoptosis to inhibit cell viability in human GBM cells. To further investigate the underlying molecular mechanism of the anti-tumor effect of vitexin, the present study investigated the alterations in the Akt/mTOR signaling pathway. Western blotting was performed to measure the expression levels of Akt, mTOR, p-Akt, and p-mTOR proteins. The results demonstrated a dose-dependent decrease in phosphorylation of Akt and mTOR following treatment with vitexin (Fig. 4). These results indicated that vitexin inhibited activation of the Akt/mTOR signaling pathway, which may contribute to vitexin-induced cell cycle arrest and apoptosis.