Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), polycarbonate membrane filters, polylysine-coated slides, paraformaldehyde, 3,3′-diaminobenzidine, hematoxylin, lysis buffer and PVDF membranes were purchased from Shanghai Abcam Biological Products Co., Ltd (Shanghai, China). Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Shanghai Worship Biological Technology Co., Ltd (Shanghai, China). Matrine was purchased from Shanghai Chuan Xiang Biological Technology Co., Ltd (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Anti-p38 (AB01165), anti-AKT (AB01313) and anti-β-actin (4967L) antibodies were purchased from Cell Signaling Technology, Inc. Anti-p-p38 (sc-166182) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-E-cadherin (ab11512) and anti-N-cadherin (ab11339) antibodies were purchased from Shanghai Abcam Biological Products Co., Ltd (Shanghai, China). Anti-p-AKT (AP0056) was purchased from Shanghai Seebio Biotech, Inc. (Shanghai, China).

The human glioma cell lines U251MG and U87MG (obtained from a cell bank at the Fourth Military Medical University, Xi’an, China) were cultured in DMEM supplemented with 10% FBS. All cells were incubated at 37°C with 5% CO₂.

Cell survival was assessed using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (9). Briefly, cells were plated in 96-well culture plates (3×10⁴ cells per well). The cells were treated with various concentrations of matrine. After 24 h incubation, the cells were washed twice with PBS and incubated with 5 mg/ml MTT (Sigma, St. Louis, MO, USA) for 4 h. Following the incubation period, the cells were washed with PBS and then solubilized with DMSO. The optical density was read on an enzyme-linked immunosorbent assay plate reader (frequency, 3; Victor X2 Multilabel Plate reader, Perkin-Elmer, Waltham, MA, USA).

The in vitro invasion and migration activity was measured according to the methods described previously (10). U87MG cells were pretreated with 0, 0.2, 0.4 and 0.8 mg/ml matrine for 24 h, then surviving cells were harvested and seeded to Transwell chambers (Corning, Inc., Corning, NY, USA) at 2×10⁵ cells/well in serum-free medium and then incubated for 24 h at 37°C. At the endpoint, the cells on the upper side of the inserts were completely removed by swabbing, while the cells on the bottom side of the filter were fixed, stained and measured. For the invasion assay, 50 μl Matrigel (25 μg/ml; BD Biosciences, Bedford, MA, USA) was applied to 8-mm pore size polycarbonate membrane filters.

ICC was performed as described previously (11); cells were seeded on polylysine-coated glass slides, cultured for 2 days, fixed in 4% paraformaldehyde, then incubated with primary antibody. Subsequently, sections were incubated with biotinylated secondary antibody and visualized with 3, 3′-diaminobenzidine; the nuclei were counter-stained with hematoxylin. Negative controls were prepared using the same procedure, but PBS was substituted for primary antibody.

Cells were suspended in lysis buffer (40 mmol/l Tris-HCl, 1 mmol/l EDTA, 150 mmol/l KCl, 100 mmol/l NaVO₃, 1% Triton X-100, 1 mmol/l PMSF, pH 7.5), following treatment with various concentrations of matrine. The proteins were separated by 10% or 8% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were subsequently blocked in defatted milk at room temperature for 1 h and then incubated with antibodies against E-cadherin, N-cadherin, p38, p-p38, AKT, p-AKT or β-actin at 4°C overnight. The membranes were then incubated with a horseradish peroxidase goat anti-mouse or anti-rabbit IgG antibody for 1 h at room temperature. The bands were detected with an enhanced chemiluminescence kit (Amersham, ECL Plus, Freiburg, Germany) and exposed by autoradiography. The densitometric analysis was performed using Image J software (GE Healthcare, Buckinghamshire, UK), and the results were expressed as arbitrary units.

All experiments were repeated three times. The statistical significance of differences throughout this study was analyzed by the one-way ANOVA test to compare differences between treatments, and followed up using Dunnett’s multiple comparison post hoc test. All statistical tests and corresponding P-values were two-sided. P<0.05 was considered to indicate a statistically significant difference. Correlation analysis was performed using the Z-test.

The effects of matrine at various concentrations (0 to 2 mg/ml) on the proliferation of U87MG cells are shown in Fig. 1. At 1 mg/ml, matrine clearly inhibited the proliferation of U87MG cells, while at concentrations below 1 mg/ml, the inhibition effect was not significant. Hence we selected a concentration range of matrine lower than this for all subsequent experiments.

Fig. 2 demonstrates the effect of matrine on the migration and invasion of U87MG cells that were treated with 0, 0.2, 0.4 and 0.8 mg/ml matrine for 24 h in the cell migration and cell invasion assays. The results revealed that matrine reduced the invasion and migration of U87MG cells substantially in a concentration-dependent manner. Matrine also inhibited the invasion of U251MG (Fig. 3).

Cells were treated with 0, 0.2, 0.4 and 0.8 mg/ml matrine for 24 h and then subjected to western blot analysis to test the expression of E-cadherin and N-cadherin in U87MG cells. Fig. 4A and B reveal that matrine significantly reduced the protein levels of N-cadherin and increased the expression of E-cadherin in a concentration-dependent manner compared with the control group. Fig. 4C demonstrates the effect of matrine on the expression of E-cadherin and N-cadherin. From the image, it can be observed that matrine increased the expression of E-cadherin and reduced the expression of N-cadherin. Matrine also increased the expression of E-cadherin and reduced the expression of N-cadherin in U251MG cells (Fig. 5).

In human glioma cells, activation of p38 is required for the invasion process (12). Moreover, the mechanism is correlated with EMT; thus, we investigated the effect of matrine on p38 MAPK and AKT in U87MG cells. Western blot analysis revealed that matrine reduced the phosphorylation of p38 in a concentration-dependent manner (Fig. 6A and B). At the same time, matrine inhibited the phosphorylation of AKT in a concentration-dependent manner (Fig. 6C and D).