Matrine (chemical formula, C₁₅H₂₄N₂O; molecular weight, 248.36) was purchased from Sun Yat-sen University (Guangzhou, China). Human prostate cancer cell lines DU145 and PC-3 were purchased from the Center for Experiment Animals of Sun Yat-sen University (Guangzhou, China), and cultured at 37°C in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified CO₂ incubator.

The cell proliferation rate was assessed using the MTS assay (Promega, Biosciences, USA) according to the manufacturer's protocols. Briefly, 10,000 cells were seeded in a well into 96-well plates (Corning, New York, NY, USA) containing 100 µl culture medium plus different concentrations of matrine and were grown at 37°C for 24 h. Then 20 µl MTS reagent was added to each well, cells were continuously incubated at 37°C for 4 h, and optical densities (ODs) at 490 nm (OD490) were determined using a microplate reader (Multiskan MK3; Thermo Scientific, Shanghai, China).

In vitro invasion assays were performed with a BD Bio-Coat Matrigel invasion assay system according to the manufacturer's protocol. Cells were seeded 24 h after treatment with different concentrations of matrine for 48 h. Cells suspended in serum-free DMEM-F12 medium (c11330500bt; Invitrogen, Life Technologies) were seeded into the upper chamber, and fetal bovine serum (10%) was added to the bottom chamber. After an incubation for 48 h at 37°C in the presence of 5% CO₂, the cells on the upper side were removed with a cotton swab, and the cells on the bottom side of the filter were fixed, stained and counted.

Cells suspended in serum-free RPMI-1640 medium were seeded into the upper chamber of a Transwell^® well (BD, USA) for 24 h after treatment with different concentrations of matrine for 48 h. The lower chamber of each well was filled with 600 µl of RPMI-1640 medium with 10% fetal bovine serum and incubated for 48 h at 37°C in the presence of 5% CO₂. Cells were fixed and stained, non-migratory cells in the upper chamber were removed, and migrated cells were counted in 10 random high-power fields.

The cell cycle was evaluated using a KeyGen kit from BD. At first, cells were treated with different concentrations of matrine for 48 h, harvested, fixed in 70% pre-chilled ethanol (−20°C) and were set at 4°C overnight. Cells were then re-suspended in propidium iodide (PI) buffer (50 g/ml PI and 100 µg/ml RNase) and incubated at room temperature for 30 min in the dark. Cells were then washed twice (3 min each wash) with 1X PBS and subjected to flow cytometry (BD Calibur, USA). The excitation wavelength was 488 nm and the emitted red fluorescence was collected through a 630 nm long-pass filter. DNA analysis was performed with ModFit software (BD).

Apoptosis was evaluated using the Annexin V/FITC apoptosis detection kit from BD. At first, cells were treated with different concentrations of matrine for 48 h and harvested by twice centrifugation at 1,000 rpm (5 min each spin). Cells were then washed twice (3 min each wash) in binding buffer, 1×10⁶ cells were resuspended in 1 ml of binding buffer containing 1.25 µl of Annexin V-FITC (BD Pharmingen, San Diego, CA, USA) and 10 µl of PI, and incubated for 15 min at room temperature in the dark. Finally, cell cycle analysis was performed by flow cytometry. Scatter plots were performed against the intensities of the FITC fluorescence and PI fluorescence. The scatter plot was divided into four quadrants: the left lower quadrant [Annexin V-FITC (−) and PI (−)] representing viable cells, the left upper quadrant [(Annexin V-FITC (−) and PI (+)] necrotic cells, right lower quadrant [Annexin V-FITC (+) and PI (−)] early apoptotic cells, and right upper quadrant [Annexin V-FITC (+) and PI (+)] late apoptotic cells.

Protein extracts from cells treated with different concentrations of matrine for 48 h were separated on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked and then probed with antibodies against P65 (8242), p-P65 (3033), IKKα (11930), IKKβ (8943), p-IKKα/β (2697), IKBα (4812) and p-IKBα (2859) (1:1,000; Cell Signaling Technology, Beverly, MA, USA) and GAPDH (1:1,000; Kangcheng Biology, Shanghai, China). The blots were then washed with tris-buffered saline/Tween-20 solution and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:20,000; Cell Signaling Technology) at room temperature. After washing, the blots were exposed, stained with ECL Plus (Millipore) and visualized on X-ray film. The mean normalized ODs of protein bands relative to the ODs of the GAPDH bands from the same condition were calculated.

Data are expressed as the mean ± standard deviation (SD) and analyzed using one-way ANOVA. Data concerning cell counts were analyzed by Fisher's exact test (SPSS 17.0; SPSS, Inc., USA). A probability value of <0.05 was considered to indicate a statistically significant result.

After treatment with different concentrations of matrine for different times, both DU145 and PC-3 cells exhibited a dose- and time-dependent growth inhibition (Fig. 1A and B). The growth inhibition rates of both types of cells reached as high as 88% when cells were treated with 6.0 g/l matrine for 72 h (Fig. 1C and D). The effectiveness of matrine to inhibit cell growth increased with treatment times (Fig. 1E and F). Therefore, matrine effectively suppressed the growth of both androgen-independent prostate cancer cell lines DU145 and PC-3.

After treatment with different concentrations of matrine for 48 h, both DU145 and PC-3 cells exhibited significantly reduced abilities of migration and invasion in a dose-dependent manner (P<0.05, ANOVA analysis) (Fig. 2). The inhibitory effects of matrine on the migration of both DU145 and PC-3 cells were near 100% when the cells were treated with 4 g/l matrine for 48 h (Fig. 2A–D). The inhibitory impacts of matrine on the invasion of both DU145 and PC-3 cells were also close to 100% when the cells were treated with 4 g/l matrine for 48 h (Fig. 2E–H). Thus, matrine effectively impaired the migration and invasion of both DU145 and PC-3 cells.

Both DU145 and PC-3 cells treated with matrine showed significantly lower percentages of cells at the S phase and higher percentages of cells at the sub-G1 phase than the untreated controls (Fig. 3). Matrine displayed no significant impact on the percentage of cells at both the G1 and G2 phases (Fig. 3). Therefore, matrine accelerated the process of DNA synthesis and promoted DNA damage and genome instability. The cell population at the sub-G1 phase consists of apoptotic cells. We further analyzed the impact of matrine on cell apoptosis by flow cytometry. Both DU145 and PC-3 cells exhibited significant increases in the apoptotic cell population upon exposure to matrine (Fig. 4). Therefore, matrine suppresses DNA replication and enhances apoptosis.

To determine the impact of matrine on the expression levels of protein involved in the NF-κB signaling pathway, DU145 and PC-3 cells were treated with different concentrations of matrine for 48 h. The levels of P65, p-P65, IKKα/β, p-IKKα/β, IKBα and p-IKBα in both DU145 and PC-3 cells were significantly reduced upon exposure to matrine (Fig. 5). The results suggest that matrine inhibits the proliferation and enhances the apoptosis of androgen-independent prostate cancer cells by regulating the NF-κB signaling pathway.