Apigenin (≥99% pure) and MTT were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against E-cadherin, Snail, vimentin and GAPDH and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibodies were all rabbit-type and monoclonal. The bicinchoninic acid protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA).

The DU145 human prostate cancer cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS, USA), 100 U/ml penicillin and 100 mg/l streptomycin (Sigma, St. Louis, MO, USA). The cultures were then maintained in a humidified atmosphere of 5% CO₂ at 37°C.

The effect of apigenin on the viability of DU145 cells was evaluated using an MTT assay. The DU145 cells (~10×10⁴) were seeded onto 96-well plates. Following incubation overnight, the cells were treated with apigenin at different concentrations (0–80 μM) in dimethyl sulfoxide (DMSO) for 24 and 48 h in a humidified atmosphere of 5% CO₂ at 37°C. Following incubation for the indicated duration, MTT (20 μl of 5 mg/ml) was added to each well and incubated at 37°C for 4 h. The MTT solution in the medium was then removed. To achieve solubilization of the formazan crystal formed in the viable cells, 150 μl DMSO was added to each well prior to measuring the absorbance at 490 nm using an MRX II absorbance reader (Dynex Technologies, Chantilly, VA, USA). The results were expressed as a percentage of growth.

Cells were grown to ~100% confluence in 6-well plates following treatment with apigenin. The cell monolayers were wounded through scraping with a micropipette tip and the wound closure was observed after 24 h. Images were captured under a phase-contrast microscope DP80 (Olympus, Tokyo, Japan) immediately or 24 h after wounding. The experiments were repeated three times.

The invasion and motility assays were performed, as previously described (18) with minor modifications. The cells were plated onto a 6-well plate (8×10⁴ cells/well). Following incubation overnight, the cells were treated with apigenin at different concentrations (0–20 μM) in DMSO for 24 h and harvested. The treated and control cells were suspended in medium at a concentration of 4×10⁵ cells/ml and 0.2 ml of the medium with suspended cells was added to the top chamber of uncoated and Matrigel-coated PET (BD Bioscience, Franklin Lakes, NJ, USA) membranes for motility and invasion assays, respectively (24-well insert; 8 μm pore size; Millipore, Bedford, MA, USA). The medium (0.6 ml), supplemented with 20% FBS, was then added to each well of the plate to act as a chemoattractant in the lower chamber. The cells were incubated for 24 h and any cells that did not migrate through the pores to the lower surface of the membrane were removed using a cotton swab by scraping the upper membrane surface. The cells that migrated to the lower surface of the membrane were fixed in 100% methanol for 5 min and stained with 0.1% crystal violet for 2 min (Sigma). These experiments were performed a minimum of three times.

The cells were plated onto six-well culture dishes at concentrations 80,000 cells/well that were determined to yield 60–70% confluence within 24 h. The cells were then treated with apigenin at a range of concentrations (0–80 μM) in DMSO. After 24 h, the cells were washed twice with phosphate-buffered saline (PBS) and centrifuged at 800 × g for 5 min. The pellet was fixed with 70% ethanol for 1 h at 4°C. Following washing with PBS, the cells were resuspended with propidium iodide solution (0.05 mg/ml) containing RNase and incubated in the dark at room temperature for 30 min. The DNA content was then analyzed using an FC500 flow cytometer (BD Bioscience).

The cells were harvested 24 h after treatment with apigenin. These cells were washed and lysed with lysis buffer (10 mmol/l Tris-hydrochloride, 0.25 mol/l sucrose, 5 mmol/l EDTA, 50 mmol/l NaCl, 30 mmol/l sodium pyrophosphate, 50 mmol/l NaF, 1 mmol/l Na₃VO₄, 1 mmol/l PMSF and 2% cocktail; pH 7.5). The protein concentration in the resulting lysate was determined using a bicinchoninic acid protein assay (Merck KGaA, Darmstadt, Germany). Appropriate quantities of protein (20–30 μg) were separated by electrophoresis on 10–12% Tris-glycine polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were inhibited and incubated overnight with the appropriate primary antibody at dilutions according to the manufacturer’s instructions. The membranes were subsequently washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody at 1:1,000 dilution in Tris-buffered saline-Tween 20 (10 mM Tris-Cl; pH 7.4, 150 mM NaCl and 0.1% Tween-20). The bound secondary antibody was then detected using an enhanced chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL, USA).

Statistical significance was compared between the various treatment groups and the controls using analysis of variance. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using Prism software (GraphPad Prism 6; GraphPad, La Jolla, CA, USA).

The MTT assay revealed that treatment with apigenin with the vehicle DMSO (1 μl/ml) at varying concentrations (10–80 μM) for different time periods (24–48 h), resulted in dose and time-dependent inhibition of DU145 cell growth compared with the untreated control. As is shown in Fig. 1, no significant difference was identified between the untreated control and the vehicle control indicating that DMSO did not affect the proliferation of DU145 cells. Treatment with 10 μM apigenin caused minimal change in the viability of the cells, therefore the migration and invasion assays were performed using concentrations of 0–20 μM apigenin. With increasing concentration and time, a marked reduction in cell viability was observed, particularly at concentrations of 40 and 80 μM. The inhibitory concentration 50% values for apigenin treatment were estimated to be 80.8 and 49.3 μM for 24 and 48 h, respectively. These data indicated that apigenin exerted a significant inhibitory effect on the DU145 cells.

As the low concentration of apigenin did not induce significant DU145 cell death, the DU145 cells were treated with 0–20 μM apigenin to detect whether the migration and invasion potential of the DU145 cells was decreased at the low concentration. Wound healing assays and transwell assays were performed to observe the function. As shown in Fig. 2, the wound healing assays revealed that apigenin-treated cells exhibited retarded wound closure and fewer stained cells in a dose-dependent manner. As shown in Fig. 3, a significant decrease in motility and invasion was observed compared with the untreated control. These results demonstrated that treatment with apigenin caused significant suppression of the migratory and invasive capability of the DU145 cells.

The effect of apigenin on cell cycle perturbations was also assessed. Compared with the untreated controls, treatment with apigenin led to a marked arrest of the DU145 cells in the G2/M phase of the cell cycle. Cell cycle analysis demonstrated that the population of control cells in the G2/M phase was 14.46% and the percentage of cells in the G2/M phase increased significantly following treatment with apigenin at different concentrations for 24 h (Fig. 4A). This increase in the population of cells in the G2/M phase was accompanied by a concomitant decrease in the number of cells in the G1 phase of the cell cycle (Fig. 4B).

EMT is important in metastasis. The loss of E-cadherin is key in EMT (19), therefore the dose-dependent effects of apigenin on the constitutive protein levels of E-cadherin, Snail and vimentin in DU145 cells was examined. The western blot analysis revealed a significant increase in the expression of E-cadherin, while in sharp contrast, the protein expression of Snail and vimentin was significantly decreased by apigenin treatment in a dose-dependent manner (Fig. 5). These results demonstrated that apigenin reversed the process of EMT.