The LN229 and U87MG human glioma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and then cultured at 37°C in Dulbecco's modified Eagle's medium (Bio-Rad Laboratories, Inc., Hercules, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin (Sigma-Aldrich) and 100 mg/ml streptomycin (Sigma-Aldrich) in a 5% CO₂ saturated humidity incubator.

The LN229 and U87MG cells were seeded in 96-well culture plates at a density of 5×10⁴ cells/well. Following 24 h, they were treated with 12.5, 25 or 50 µM eupatilin (Sigma-Aldrich) for 24, 48, 72 or 96 h. Control group cells were treated with 0.1% dimethylsulfoxide (DMSO; Sigma-Aldrich) in culture medium. Subsequent to treatment application, all cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) solution for 24 h at 37°C. Then DMSO was added in each well and shaken for 10 min at room temperature. The optical density was measured with an enzyme-linked immunosorbent assay reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 570 nm. Each experiment was performed at least three times.

Cell invasion and migration assays were performed with a Transwell chamber (EMD Millipore, Boston, MA, USA) that was placed in a 24-well plate. The cells were pretreated with 0, 12.5, 25 and 50 µM eupatilin for 24 h and then suspended in 50 µl serum-free medium (Sigma-Aldrich). The cells were also used for invasion assays, cells at a density of 6×10³ cells/well were added to the upper chamber and complete medium (Sigma-Aldrich) was added to the lower chamber. The chambers were separated with a polycarbonate membrane was coated with 20 µl Matrigel (BD Biosciences, San Jose, CA, USA). The cells were then incubated for 36 h at 37°C, those remaining in the upper chamber were removed with cotton swabs and the ones on the bottom surface of the membrane were fixed and stained with methanol and Giemsa (Sigma-Aldrich), respectively, and then counted under an optical microscope (×200; CX31; Olympus Corporation, Tokyo, Japan). The migration assay was performed as described above, except that Matrigel was not applied to the membrane.

Cell apoptosis was detected with Annexin V-fluorescein isothiocyanate (FITC; Abcam, Cambridge, MA, USA) and propidium iodide (PI; Abcam) staining followed by flow cytometric analysis. Briefly, U87MG cells were seeded in 24-well culture plates at a density of 4×10⁴ cells/well. Eupatilin at concentrations of 0, 12.5, 25 and 50 µM was added to the plates after 24 h. The cells were cultured for 48 h at 37°C and then harvested via centrifugation at 1,000 × g for 10 min. The cells were incubated with Annexin V-FITC and PI for 15 min at room temperature. Apoptosis was analyzed using flow cytometry (FC500; BD Biosciences).

The total RNA was extracted from the eupatilin-treated U87MG cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance with the manufacturer's protocol. cDNA synthesis was conducted using 5 µg of the total RNA with M-MuLV reverse transcriptase (Clontech Laboratories, Inc., Palo Alto, CA, USA). The genes of interest were amplified with the following primers: Forward: 5′-TCAGCGGGATCCACTGTGAG-3′ and reverse: 5′-ACACAGGCAGGTGAACGAGTTG-3′ for Notch-1; and forward: 5′-CTCCATCCTGGCCTCGCTGT-3′ and reverse: 5′-GCTGTCACCTTCACCGTTCC-3′ for β-actin. β-actin was used as a control. The PCR was run for 30 cycles at 94°C (denaturation) for 30 sec, at 55°C (annealing) for 30 sec and at 72°C (extension) for 20 sec. The experiment was performed for three times. The data obtained was calculated using the comparative Cq method (2^−ΔΔCq) as previously described (12).

The U87MG cells were treated with 0, 12.5, 25 and 50 µM eupatilin for 24 h and then immersed in a lysis buffer containing 40 mmol/l Tris-HCl, 1 mmol/l EDTA, 150 mmol/l KCl, 100 mmol/l NaVO₃, 1% Triton X-100, and 1 mmol/l phenylmethylsulfonyl fluoride (pH 7.5). The protein was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Sigma-Aldrich) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were treated with 5% non-fat milk in Tris-buffered saline (TBS) at room temperature for 1 h and then incubated overnight at 4°C with primary mouse monoclonal anti-human Notch-1 (1:1,500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-373944) or β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-8432). The membranes were then washed three times with TBS and Tween-20 (TBST) for 10 min at room temperature. Subsequently, the membranes were incubated with a bovine anti-mouse horseradish peroxidase-conjugated secondary antibody (1:3,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2370) for 1 h at room temperature and then washed three times for 10 min with TBST and once with TBS. Immunoreactive bands were detected by enhanced chemiluminescence (GE Healthcare Life Sciences, Freiburg, Germany). The optical densities of the bands were quantified using a Gel-Pro Analyzer, version 4.0 (Media Cybernetics, Inc,, Rockville, MD, USA).

The siRNA sequences were as follows: Sense 5′-ACGAAGAACAGAAGCACAAAGGCGG-3′ and antisense 5′-CCGCCUUUGUGCUUCUGUUCUUCGU-3′ for Notch-1; and sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense 5′-ACGUGACACGUUCGGAGAATT-3′ for scramble control. Prior to transduction (24 h), U87MG cells, at a density of 5×10⁴ cells/well, were seeded into 6-well plates and then cultured in 2 ml basic culture medium containing 5% FBS until the cells were 70% confluent. The cells were then transfected with Notch-1 siRNA or the scramble control siRNA, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the protocol described in Côté et al (13). Subsequently, the transfected cells were treated with 0, 12.5, 25 and 50 µM eupatilin for 48 h.

All experiments were performed at least three times and the data were expressed as the mean ± standard deviation. The differences between the sample means were compared using one-way analysis of variance using SPSS, version 19 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of eupatilin on the viability of glioma cells was investigated using an MTT assay. As demonstrated by Fig. 1, higher doses of eupatilin significantly reduced LN229 and U87MG cell viability (P<0.05; Fig. 1A and B) compared with the control group. Additionally, exposing the cells to eupatilin for a longer time period enhanced its effect on cell viability. These observations indicate that eupatilin may inhibit the viability of LN229 and U87MG cells.

The effect of eupatilin on invasion and migration of glioma cells was also determined. First, we examined the effect of eupatilin on invasion of glioma cells using a transwell chamber with Matrigel. As demonstrated in Fig. 2A and B, the treatment of LN229 and U87MG cells with 0, 12.5, 25 and 50 µM eupatilin for 24 h resulted in inhibition of cell invasion in a dose-dependent manner. Subsequently, the effect of eupatilin on migration of glioma cells was also investigated using a Transwell chamber without Martrigel being applied. Fig. 2C and D demonstrates that the number of eupatilin-treated cells that migrated into the lower chamber was significantly reduced, compared with the control group (P<0.05).

Following treatment with eupatilin for 48 h, apoptosis of U87MG cells stained with Annexin V-FITC and PI was determined by flow cytometric analysis. As presented in Fig. 3, the apoptotic rate of the cells in the eupatilin treatment group was significantly higher than in the control group, indicating that eupatilin induced the apoptosis of glioma cells (P<0.05).

Notch-1 is a transmembrane receptor which is often important for the proliferation and invasion of tumor cells (14). The current study determined how eupatilin affects Notch-1 expression using RT-PCR and western blot analysis. The protein and mRNA expression levels of Notch-1 following treatment with different concentrations of eupatilin were significantly reduced compared with the control group in a dose-dependent manner (P<0.05; Fig. 4). Therefore, eupatilin may suppress Notch-1 expression in glioma cells.

In order to further examine the effect of Notch-1 glioma cells, Notch-1 was downregulated in U87MG cells using siRNA. The transfected cells were treated with eupatilin of different concentrations for 48 h. The protein expression levels of Notch-1 were detected by western blot analysis (Fig. 5A). The combination of Notch-1 siRNA and eupatilin treatment led to inhibition of proliferation and invasion of glioma cells to a greater extent than that observed following treatment with eupatilin only (Fig. 5B and C).