Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning Cellgro (Mediatech, Inc., Manassas, VA, USA), and antibiotics (streptomycin and penicillin) were purchased from Corning Incorporated (Corning, NY, USA). OM was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China), and the purity (over 98%) was confirmed by high-performance liquid chromatography (HPLC). OM was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10 mg/ml, stored at −20°C, and then freshly diluted with culture medium for each experiment. An Annexin V-FITC Apoptosis Detection kit and a Cycletest Plus DNA Reagent kit were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

The U251MG human malignant glioma cell line was obtained from the Institute of Brain Science of Harbin Medical University (Harbin, China). The cells were cultured in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin at 37°C in an incubator with a humidified atmosphere of 5% CO₂ and 95% air.

A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay was performed to explore the cytotoxic effects of OM and/or erlotinib on human glioblastoma cells. U251MG cells were seeded in a 96-well plate at a density of 1,000 cells/well and then treated with OM at various concentrations (0, 0.25, 0.5, 1, 2 and 4 mg/ml) for 24, 48 and 72 h, or treated with 2 µM erlotinib in the absence or presence of 1 mg/ml OM for 48 h. Following the treatment with OM and/or erlotinib, 10 µl of CCK-8 was added to each well, and the cells were incubated at 37°C for 1 h. A microplate reader (Tecan Group Ltd., Männedorf, Switzerland) was used to measure the absorbance of each well at 450 nm to assess the proliferation.

An Annexin V-fluorescein isothiocyanate (FITC) assay was employed to quantify apoptotic cells by flow cytometry. U251MG cells were seeded into 6-well plates at a density of 2×10⁵ cells/well, cultured overnight, and then cultured with OM at a final concentration of 0.5, 1, or 2 mg/ml or with DMSO for 48 h. Then, the cells were harvested by trypsin, washed with ice-cold PBS twice, collected, fixed and stained using an Annexin V-FITC/PI double fluorescence apoptosis detection kit according to the manufacturer's instructions. The samples were then analyzed by flow cytometry (BD FACSCalibur System; BD Biosciences).

DNA fragmentation was performed using a One-Step TUNEL kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's recommendations. Briefly, U251MG cells were exposed to OM (0.5, 1 or 2 mg/ml) for 48 h and then fixed in 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were washed with PBS three times and permeabilized for 2 min on ice and then the cells were resuspended in TUNEL working solution. Following incubation for 1 h in a humidified atmosphere at 37°C in the dark, the cells were counterstained with DAPI staining solution for 5 min at room temperature, and then, TUNEL-positive cells were visualized under a fluorescence microscope. DAPI was used for the nuclear staining.

The cell cycle phase distribution was evaluated by flow cytometric analysis of the DNA content of cells with the BD Cycletest Plus DNA Reagent kit (BD Biosciences) according to the manufacturer's instructions. In brief, cells were plated in 6-well plates and treated with different concentrations of OM (0.5, 1 and 2 mg/ml) for 48 h, harvested, fixed in 70% pre-chilled ethanol (−20°C) and maintained at 4°C overnight. Then, the cells were resuspended in propidium iodide (PI) buffer (50 g/ml PI and 100 µg/ml RNase) and incubated for 30 min shielded from light at room temperature. The cells were then washed twice with PBS. The percentages of cells in each phase of the cell cycle were determined by BD FACSCalibur flow cytometer (BD Biosciences, Fraklin Lakes, NJ, USA) and analyzed by ModFit LT version 4.1 software (Verity Software House, Topsham, ME, USA).

Cell invasion assays were performed using a Corning Matrigel invasion assay system according to the manufacturer's instructions. Following treatment with OM at various concentrations (0.25, 0.5, 1 and 2 mg/ml) for 24 h or 48 h, cells were seeded for 24 h and suspended in serum-free DMEM. Then, the cells were placed in the upper part of each chamber, whereas the lower chambers contained FBS (10%). After incubation in a humidified 5% CO₂ atmosphere at 37°C for 24 or 48 h, the cells in the upper chamber and on the Matrigel were mechanically removed with a cotton swab, and the cells on the bottom side of the filter were fixed in 4% paraformaldehyde for 30 min at room temperature. Subsequently, the cells were washed with PBS three times and stained with 0.1% crystal violet for 15 min at room temperature. Then, the cells were washed three times with PBS and counted under a inverted phase contrast microscope (magnification, ×200).

Following treatment with different concentrations of OM (0.5, 1 and 2 mg/ml), or with 2 µM erlotinib in the absence or presence of 1 mg/ml OM for 48 h, cells were collected, washed with ice-cold PBS and then lysed in lysis buffer [5 mmol/l EDTA, 40 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 1% NP-40, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 5 mg/ml benzamidine and 0.5 mmol/l phenylmethylsulfonyl fluoride]. The protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). An equal amount of protein (60 µg) from each group was separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA), blocked with 5% skim milk in TBS-Tween 20 (0.05%, v/v) for 1 h, and then incubated with primary antibodies at 4°C for 16 h. After washing with TBS-T, the membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. GAPDH or β-actin was used as protein loading controls. After three washes, the target proteins were visualized using an enhanced chemiluminescent (ECL) kit (Thermo Fisher Scientific; Pierce, Rockford, IL, USA). The band intensities were assessed with the Gel-Pro Analyzer Software version 4.0 (Media Cybernetics, Rockville, MD, USA). Primary antibodies against cyclin D1 (1:1,000; cat. no. 2922), CDK4 (1:1,000; cat. no. 12790), CDK6 (1:1,000; cat. no. 13331), p-Akt (Ser473; 1:500; cat. no. 9271), Akt (1:500; cat. no. 9272), STAT3 (1:1,000; cat. no. 12640), p-STAT3 (Ser727; 1:1,000; cat. no. 9134), caspase-3 (1:1,000; cat. no. 9662), Bax (1:1,000; cat. no. 2774), Bcl-2 (1:1,000; cat. no. 2872), GAPDH (1:1,000; cat. no. 2118) and β-actin (1:1,000; cat. no. 4970) were purchased from Cell Signaling Technology (Danvers, MA, USA). EGFR (1:1,000; cat. no. GTX628887), p-EGFR (Tyr1068; 1:1,000; cat. no. GTX132810), mTOR (1:1,000; cat. no. GTX101557) and p-mTOR (Ser2448; 1:1,000; cat. no. GTX132803) antibodies were purchased from GeneTex Inc. (Irvine, CA, USA). Secondary antibodies goat anti-rabbit IgG HRP (1:5,000; cat. no. ab6721) and goat anti-mouse IgG HRP (1:5,000; cat. no. ab205719) were purchased from Abcam (Cambridge, UK).

All statistical analyses were carried out using the SPSS 22.0 statistical software package (IBM Corp., Armonk, NY, USA). Data were expressed as the mean ± standard deviation (SD). Differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) multiple comparison test. Differences between two groups were assessed using the Student's t-test. For each measurement, at least three independent experiments were performed. P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effects of OM on cells were first evaluated by the CCK-8 cell viability assay. U251MG cells were incubated with OM at various concentrations for 24, 48 and 72 h, and the results revealed that cell viability was reduced in a dose- and time-dependent manner in U251MG cells (Fig. 1).

To reveal the underlying mechanisms of OM in the inhibition of the growth of glioblastoma cells, we performed cell cycle analysis. Cells of the glioblastoma cell line U251MG were exposed to different concentrations of OM for 48 h, and flow cytometric analysis was then performed. Our data revealed that OM led to cell accumulation in the G1 phase with a concomitant decrease in the number of cells in the S phase in a dose-dependent manner (Fig. 2A). The results indicated that the mechanism of the OM-induced suppression of glioblastoma cell viability involved arresting the cell cycle at the G0/G1 phase.

Furthermore, since cyclin D1, CDK4 and CDK6 are the key regulators of the G0/G1 phase of the cell cycle, we performed western blot analysis to determine the expression level of the proteins in OM-treated glioblastoma cells. The results revealed that the protein expression levels of cyclin D1, CDK4 and CDK6 were significantly reduced by OM (Fig. 2B), indicating that OM arrests glioma cells at the G0/G1 phase by downregulating CDK4, CDK6 and cyclin D1. Collectively, our data revealed that OM could potently arrest the proliferation of glioblastoma cells.

To evaluate whether OM induced the apoptosis of glioma cells, cells were stained with Annexin V-FITC/PI and then analyzed by flow cytometry. The results revealed that after treatment with various concentrations of OM for 48 h, the percentage of apoptotic cells was significantly increased in a dose-dependent manner compared to that of the control cells (Fig. 3A).

Furthermore, as fragmented nuclei are typical characteristics of apoptosis, we performed DAPI staining and a TUNEL assay to detect alterations in the nuclei. The results revealed that after treatment with various concentrations of OM for 48 h, the number of TUNEL-positive cells was significantly higher in the OM-treated cells (Fig. 3B). Furthermore, to reveal the potential mechanism of OM-induced apoptosis in glioma cells, we assessed the protein expression levels of Bax, Bcl-2, and caspase-3, which are important regulators of apoptosis, by western blot analysis. The results revealed that the expression levels of Bax and caspase-3 were significantly increased, whereas the level of Bcl-2 was decreased after incubation with OM for 48 h, thus leading to an increased ratio of Bax to Bcl-2 (Fig. 3C). Collectively, the results indicated that OM had pro-apoptotic effects on glioblastoma cells.

Since invasion ability is one of the pathophysiological features of malignant gliomas, an assay using Matrigel-coated Transwell membranes was performed to evaluate the effects of OM on invasion. As displayed in Fig. 4, the percentage of invading cells on the lower side of the membrane was decreased in a dose-dependent manner after treatment with OM for 24 h (Fig. 4B) and 48 h (Fig. 4A) compared with that in the control group. The results indicated that the invasion of glioblastoma cells could be significantly suppressed by OM.

EGFR amplification is one of the most common genetic alterations in glioblastoma. Gain-of-function of EGFR can lead to upregulation of the PI3K/Akt/mTOR signaling pathway, which is involved in cell survival and proliferation. Therefore, we assessed whether the EGFR/PI3K/Akt/mTOR pathway is altered in glioblastoma cells treated with different concentrations of OM for 48 h. As displayed in Fig. 5, OM significantly decreased the expression levels of p-EGFR, p-Akt and p-mTOR in a dose-dependent manner, without affecting total EGFR, Akt and mTOR levels.

To further test whether downregulation of the EGFR pathway is responsible for the OM-induced inhibition of glioma cell proliferation, we treated the cells with erlotinib, an inhibitor of EGFR, in the absence or presence of OM for 48 h. The results indicated that in the presence of both erlotinib and OM, the levels of p-EGFR, p-Akt and p-mTOR were significantly lower than those in the presence of erlotinib or OM alone (Fig. 6A). Additionally, compared with the treatment with either erlotinib or OM alone, the combined treatment with erlotinib and OM exhibited a stronger effect that inhibited the proliferation of glioma cells (Fig. 6B).

Upregulated STAT3 activity is associated with many human tumors, and STAT3 suppression can mediate tumor regression (21). Therefore, we assessed the expression levels of p-STAT3 and total STAT3 in glioblastoma cells treated with OM for 48 h. Western blot analysis demonstrated that OM significantly decreased the expression levels of p-STAT3, but did not affect the total STAT3 level (Fig. 7).