The LnCaP and PC-3 human prostate cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum, L-glutamine (5 mmol/l), non-essential amino acids (5 mmol/l), penicillin (100 U/ml) and streptomycin (100 U/ml) (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ atmosphere. Berberine was purchased from Sigma-Aldrich (St. Louis, MO, USA). epidermal growth factor (EGF) was obtained from Cell Signaling Technology Inc., (Beverly, MA, USA).

Cell proliferation was determined using an MTT viability assay, the most commonly used assay for determining cell growth and death. The MTT survival assay has been described in detail in a previous study (17). Briefly, exponentially growing cells were recultured (5,000 cells/well) overnight in 96-well tissue culture plates. Up to 20 μl of MTT (Sigma-Aldrich) was directly added to the media in each well, with a final concentration of 2 mg/ml. Following a 4 h incubation, the medium containing MTT was discarded and 120 μl dimethylsulfoxide was added for 10 min. The absorbance was measured using an enzyme-linked immunosorbent assay reader (Bio-tek, Inc., Winooskie, VT, USA) at 570 nm, with the absorbance at 630 nm as the background correction. The cell viability was expressed as the percentage of untreated controls. All the experiments were performed at least three times.

The cells were removed from the culture dishes with trypsin and collected into centrifuge tubes together with the culture medium. All the contents were centrifuged for 5 min at 1,800 × g. The supernatant was decanted, washed once with 1X phosphate-buffered saline (PBS) and centrifuged for another 5 min. The cells were finally fixed with 5 ml of pre-cooled 70% ethanol for at least 4 h. The fixed cells were centrifuged and washed with 1X PBS. Following centrifugation, the cell pellets were resuspended in 500 μl propidium iodine (10 μg/ml) containing 300 μg/ml RNase (Sigma-Aldrich). The cells were then incubated on ice for 30 min and then filtered with a 53-μm nylon mesh. The cell cycle distribution was calculated from 10,000 cells using ModFit LT software (Becton-Dickinson, San Jose, CA, USA) using FACS Calibur (Becton-Dickinson).

Apoptosis analysis was undertaken by a flow cytometer (Beckman, Irvine, CA, USA) and an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, Sparks, MD, USA) was used to measure the percentage of apoptosis induced by berberine. The cells were removed with trypsin and collected into centrifuge tubes together with the culture medium. A total of 5 μl Annexin V-FITC solution and 10 μl PI (1 μg/ml) were added to the cells for 30 min away from the light. Flow cytometry and Annexin V-FITC apoptosis analysis were performed as previously described (18). The apoptotic rates were calculated from 10,000 cells using ModFit LT software with FACS Calibur.

The cell lysates were prepared, and western blot analysis was performed as previously described (19). Equal aliquots of the total cell protein (50 μg per lane) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and then blotted using primary antibodies against β-actin (C-4), phospho (p)-EGFR (Tyr 1092 m), EGFR (C-20) and prostate-specific antigen (PSA; C-19) (all from Santa Cruz Biotech, Santa Cruz, CA, USA; 1:1,000 dilution) followed by incubation with secondary horseradish peroxidase-labeled goat anti-mouse (GAM-007) and goat anti-rabbit (SC-2004) IgG antibodies. The protein bands were visualized using an enhanced chemiluminescence system (Union Bioscience Corporation, Hangzhou, China) with prestained markers as molecular size standards. At least three independent experiments were performed for each cell type observed.

Data are presented as the mean ± standard deviation. The experimental results of the treated and control groups were compared using two-tailed Student’s t-test. All the statistical tests were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

The antiproliferative effects of berberine on LnCaP and PC-3 human prostate carcinoma cells were investigated. The cells were treated with 20, 100 and 200 μM berberine for 24, 48 and 72 h. Berberine treatment in LnCaP cells resulted in a significant reduction in cell proliferation at 20 μM (P<0.05) and 100 μM (P<0.05) after 72 and 24 h treatment, respectively (Fig. 1A). Similar effects were observed in PC-3 cells (Fig. 1B). However, PC-3 cells responded more rapidly compared with the LnCaP cells, with a reduction in viability observed at 20 μM (P<0.05) after 24 h treatment. These data indicate that berberine has a cytotoxic effect on prostate carcinoma cells, in p53 wild-type (LnCaP) and p53-mutated (PC-3) cells.

Based on this preliminary data in which a growth inhibitory effect of berberine was observed in LnCaP and PC-3 cells, a time point of 48 h after treatment was selected in order to determine the possible inhibitory effect of berberine on cell cycle progression and apoptosis. The LnCap cells demonstrated that an increased number of cells had undergone G1 cell cycle arrest at 100 μM (P<0.005) 48 h following treatment, compared with DMSO treatment. Simultaneously, the reduction of the G2/M phase was observed in a dose-dependent manner, indicating that berberine induces G1 phase cell cycle arrest in LnCap cells. Similar results were obtained in PC-3 cells (Fig. 2A). An Annexin V assay was used to determine the effect of berberine on apoptosis in prostate cancer cells. Treatment of LnCap cells with berberine resulted in a significant increase in the number of apoptotic cells at a concentration of 100 μM (P<0.05), compared with the control cells (Fig. 2B). A similar effect was observed in the PC-3 cells.

PSA is a well-characterized target gene of the androgen receptor (20). In the present study, the effect of berberine on PSA expression in prostate cancer cells was determined. Using western blot analysis, it was identified that a low dose of berberine (20 μM) resulted in marginal decrease in PSA protein levels and the inhibition of PSA at a dose of 100 μM or higher in LnCaP cells (Fig. 3A). Berberine also decreased PSA expression in PC-3 cells (Fig. 3B).

PC-3 cells were selected to determine the effects of berberine on the regulation of EGFR expression and activity. Addition of berberine resulted in a decrease in the total EGFR and phosphorylated EGFR levels (Fig. 4A). Furthermore, EGF-stimulated EGFR activation was also inhibited by berberine (Fig. 4B).