Matrine (C₁₅H₂₄N₂O) was purchased from Aladdin Ltd. (Shanghai, China) and dissolved in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA). Fetal bovine serum (FBS), streptomycin, and penicillin were all purchased from Gibco (Grand Island, NY, USA). Anti-MEK1, anti-ERK 1/2, anti-phosphorylated MEK 1/2 (Thr386), anti-phosphorylated ERK 1/2 (Thr202/Tyr204), anti-BCL2, anti-BAX, and anti-β-actin were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). The ERK pathway inhibitor U0126 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MEK expression plasmid pcDNA3.1(+)-MEK1 was purchased from GeneChem Co., Ltd. (Shanghai, China). The RMS cell line RD was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).

RD cells, were cultured in DMEM supplemented with 10% FBS, 100 µg/ml streptomycin and 100 units/ml penicillin in a humidified atmosphere of 5% CO₂ at 37°C. For transient transfection, cells were plated in a 6-well plate at a density of 2×10⁵ cells per well and cultured for 24 h. Lipofectamine^® 2000 liposome transfection kit (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to transfect pcDNA3.1(+)-MEK1 or the empty pcDNA3.1(+) into the cells according to the manufacturer's instructions.

RD cells were plated in 96-well microtiter plates at a density of 5×10³ cells per well and treated with matrine in various doses (0, 0.5, 1.0, 1.5, 2.0, 3.0, and 5.0 g/l) for 24 h. Cell viabilities were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). The absorbance (A) was detected at 490 nm using an ELISA reader. Cell viability rate was calculated as followes: (%)=A₄₉₀, matrine/A₄₉₀, control × 100%. RD cells were treated with or without U0126 for 1 h before treatment with matrine for 24 h. Then, cell viabilities were assessed as described above. For transfection experiments, the cells were treated with or without matrine after transfection for 24 h, and the cell viabilities were assessed as described above.

RD cells in exponential growth phase were plated in 12-well plates at a density of 2×10⁵ cells per well. The cells were treated with matrine (0, 0.5, 1.0, and 1.5 g/l) for 24 h or 1.5 g/l matrine for 24 or 48 h. Apoptosis was measured using Annexin V-FITC/PI double staining (MultiSciences Biotech, Shanghai, China) according to the manufacturer's instructions. The apoptotic cells were detected with flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition and analysis were performed using CellQuest software (BD Biosciences). In addition, RD cells were treated with or without U0126 for 1 h before treatment with matrine for 24 h, and apoptosis was measured as described above. For transfection experiments, the cells were treated with or without matrine after transfection for 24 h, and apoptosis was measured as described above.

RD cells were seeded into a 6-well plate at a density of 2×10⁵ cells per well and cultured overnight to attain 90% confluence. Cell wounds were scratched by a plastic tip, washed twice with medium, treated with matrine (0, 0.25, 0.5, and 0.75 mg/ml) and cultured in serum-free medium for 24 h. Images were captured at 0 and 24 h under an inverted microscope.

The RD cells invasion assay was performed using Transwell chambers (8-µm pore size) coated with Matrigel (Corning Inc., Acton, MA, USA). Cells (1×10⁵) in serum-free medium containing various concentrations of matrine (0, 0.25, 0.5, and 0.75 mg/ml) were seeded into the upper Transwell chambers, while 600 µl medium containing 10% FBS was added to the lower chambers. After 24 h, cells on the upper face of the filter and the Matrigel were removed by a cotton swab, and the cells on the bottom were fixed, stained, and counted.

RD cells were treated with matrine (0, 0.25, 0.5, and 0.75 mg/ml) or U0126 for 48 h, and lysed in lysis buffer [50 mmol/l Tris-HCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA), 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1 mmol/l phenylmethyl sulfonyl fluoride (PMSF)]. The protein extracts were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking in defatted milk [5% in Tris-buffered saline/Tween-20 buffer (TBST)] at 37°C for 2 h, the membrane was incubated overnight at 4°C with primary antibodies against MEK1, ERK1/2, p-MEK1, p-ERK1/2, BCL2, BAX, and β-actin in TBST containing 1% bovine serum albumin and with secondary antibodies for 1 h at room temperature. Signals were detected with ECL detection reagents (GE Healthcare, Piscataway, NJ, USA). The optical densities of the bands on photographic films were obtained using ImageJ software (NIH, Bethesda, MD, USA).

Each experiment was repeated three times, outcome variables were presented as mean ± standard deviation. The difference between multiple groups were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The results of the MTT assay showed that matrine inhibited the proliferation of RD cells (Fig. 1), and increasing the concentration of matrine leads to significant increase of inhibition rate. The results indicated that matrine inhibited effectively the RD cell viability. However, at concentrations below 1 mg/ml, matrine did not significantly reduce the total viable cells. Thus, the concentration range of matrine for the subsequent experiments was chosen depending on these results.

The results of the Annexin V-FITC/PI double staining showed that matrine induced apoptosis of RD cells. After treatment with matrine (0.5,1 and 1.5 mg/ml) for 24 h, apoptosis rates were 22.8±0.78, 26.5±1.21, 40.1±2.17%, respectively. The apoptosis rate significantly increased with increasing drug concentrations (Fig. 2A and B). After treatment with matrine (1.0 mg/ml) for 24 or 48 h, apoptosis rates were 26.5±1.21, 44.7±3.03%, respectively. The apoptosis rate significantly increased with increasing treatment times (Fig. 2C and D).

The motility of RD cells was evaluated by wound healing assay. After treatment with matrine (0, 0.25, 0.5, and 0.75 mg/ml) for 24 h, the migration distance of RD cells were 381.2±10.3, 259.1±8.9, 177.9±14.2, 42.5±3.7 µm, respectively. The migration distance decreased in a dose-dependent manner (Fig. 3A and B). The results suggested that matrine inhibited the motility of RD cells.

The invasiveness of RD cells was evaluated by using Transwell chambers coated with Matrigel. After treatment with matrine (0, 0.25, 0.5, and 0.75 mg/ml) for 24 h, the invasion of RD cells was reduced in a dose-dependent manner (Fig. 3C and D). The results suggested that matrine inhibited the invasive ability of RD cells.

The ERK signaling pathway plays an important role in cell growth, survival and apoptosis (17). Studies showed that the inhibition of the ERK pathway positively reduced RMS cell growth, survival, and epithelial-mesenchymal transition (EMT) (7–9). Thus, we investigated the effect of matrine on ERK phosphorylation in RD cells. The results of the western blot analysis showed that the phosphorylation of MEK1 and ERK1/2 was reduced in a dose-dependent manner, whereas the total MEKand ERK levels did not change (Fig. 4A and B).

In order to further investigate the effect of matrine on the ERK pathway, we treated cells with pcDNA3.1(+)-MEK1 to promote ERK phosphorylation. The results showed that increased phosphorylation of ERK1/2 reversed the effect of matrine on cellproliferation and apoptosis (Fig. 5A-C).

Furthermore, in order to verify whether the effects of matrine were ERK-dependent. U0126 was used to prevent ERK phosphorylation. The results showed that the combined use of matrine and U0126 significantly increased apoptosis (Fig. 6A and B), reduced BCL-2 levels, and increased BAX protein expression (Fig. 6C and D).

Our results proved that matrine significantly inhibited the proliferation and induced apoptosis of RMS cells via the ERK1/2 signaling pathway.