Prostate epithelial cells RWPE1 and androgen-independent prostatic carcinoma cells (PC-3) were used in the present study. The PC-3 cell line was obtained from the Center for Experimental Animals of Sun Yat-Sen University. The cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS), supplemented with 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). The RWPE1 cell line was maintained in complete keratinocyte serum-free medium, supplemented with 50 mg/ml of bovine pituitary extract and 5 ng/ml of epidermal growth factor (Gibco). Both cell lines were cultured in a humidified incubator at 37̊C with an atmosphere of 5% CO₂. Matrine was purchased from Melonepharma (Dalian, Liaoning, China). LY294002 was purchased from Cell Signaling Technology (Carlsbad, CA, USA) and was dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions. Antibodies against Akt, p-Akt, P27, CDK4 and Bim were purchased from Cell Signaling Technology. Antibodies against CDK2, Bax, Bcl-2 and p-FoxO3a were purchased from Abcam (Cambridge, MA, USA). Antibodies against FoxO3a were purchased from GeneTex (Irvine, CA, USA).

The cell proliferation rate was determined using an MTS assay (Promega Biosciences, LLC, San Luis Obispo, CA, USA) according to the manufacturer's protocol. Briefly, 5×10³ cells/well were seeded into 96-well plates (Corning, New York, NY, USA) containing 100 µl of culture medium plus different concentrations of matrine and were grown at 37̊C for 24, 48 and 72 h. Subsequently, the MTS reagent was added to each well and incubated in the dark for 2 h and optical densities (ODs) at 490 nm (OD490) were determined using a microplate reader (Multiskan MK3; Thermo Scientific, Shanghai, China).

The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (eBioscience, Inc., San Diego, CA, USA) was used to detect cell apoptosis according to the instructions of the manufacturer. Cells were seeded into 6-well plates at 1.5×10⁵ cells/well in a medium supplemented with 10% FBS for 24 h, followed by the addition of matrine (1.5 g/l), LY294002 (10 µmol/l) or a combination of the two. After 48 h, treated cells were collected and washed twice with chilled phosphate-buffered saline (PBS). The cells were then resuspended in 400 µl of binding buffer and divided into two tubes, adding 5 µl of Annexin V-FITC to one tube according to the manufacturer's instructions. After incubation for 15 min at room temperature in the dark, 10 µl of PI was added. Finally, the stained cells were examined by BD FACSCalibur flow cytometer equipped with CellQuest software (both from BD Biosciences, Franklin Lakes, NJ, USA).

Following treatment with matrine or LY294002 under the same conditions as the apoptosis assay, the cells were resuspended at a concentration of 1.5×10⁵ cells/ml. Subsequently, the cells were fixed in 70% ethanol and stored overnight at 4̊C. The fixed cells were suspended in PI containing RNase A, and then incubated at 37̊C for 1 h. DNA content was analyzed using a fluorescence-activated cell sorter and CellQuest software.

Following treatment with matrine (1.5 or 2.5 g/l) and/or LY294002, cells were washed twice with ice-cold PBS, lysed with RIPA lysis buffer and complete protease inhibitor for 30 min on ice, and then cleared by centrifugation at 12,000 rpm at 4̊C for another 30 min. The total protein concentration in the extracts was assessed utilizing a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA or non-fat dry milk in Tris-buffered saline and Tween-20 (TBST), for 1 h and then probed with antibodies against Akt (9272), p-Akt (2965), P27 (2552), CDK4 (12790), Bim (2933) (1:1,000; Cell Signaling Technology); CDK2 (ab32147), Bax (ab32503), Bcl-2 (ab59348) and p-FoxO3a (ab154786) (1:1,000; Abcam) and FoxO3a (GTX79072) (1:500; GeneTex) at 4̊C with gentle shaking overnight. The membranes were then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:20,000; Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature followed by detection using a chemiluminescence ECL kit (Millipore).

According to the manufacturer's instructions, total RNA was extracted from treated cell samples using TRIzol reagent, before being reverse-transcribed into cDNA using PrimeScript RT Master Mix (both from Takara, Dalian, China). GAPDH was used as an internal control. RT-PCR and data collection were performed on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The results were calculated using the 2^−ΔΔCt method. All results are expressed as the mean ± SEM of three independent experiments. Primers were synthesized by Shenggong Biological Engineering Co., Ltd. (Shanghai, China). The results were normalized with the value detected for GAPDH. Real-time PCR primers were as follows: FoxO3a forward (5′-GCACCTCATCCTTCTTTCAA-3′) and reverse (5′-CATGCGTCACCATCTCTTTT-3′); PI3K forward (5′-AAAGGCGGCTTGAAAGGT-3′) and reverse (5′-GACGATCTCCAATTCCCAAA-3′); and GAPDH forward (5′-TGGTCGTATTGGGCGCCTGGT-3′) and reverse (5′-TCGCTCCTGGAAGATGGTGA-3′).

Statistical analyses were performed using the SPSS software package (version 19.0) and GraphPad Prism Software. Data are expressed as the mean ± standard deviation (SD) and analyzed using one-way ANOVA. All of the p-values were two-sided and p<0.05 was considered to indicate a statistically significant result.

The proliferation of prostate cancer cells was assessed using the MTS assay. Matrine inhibited the proliferation of PC-3 cells, with an IC₅₀ value of 1.78±0.023 g/l at 24 h, 1.40±0.008 g/l at 48 h and 1.17±0.023 g/l at 72 h (Fig. 1A and B). Therefore, the concentration of 1.5 g/l was appropriate for the subsequent experiments. To determine whether matrine affects the proliferation of RWPE1, an MTS assay was performed following matrine treatment at various concentrations. As shown in Fig. 1C and D, similar proliferation inhibition was observed in the RWPE1 cell line. Matrine inhibited the proliferation of RWPE1 cells, with an IC₅₀ value of 2.26±0.039 g/l at 24 h, 1.63±0.061 g/l at 48 h and of 1.28±0.053 g/l at 72 h. The sensitivity of matrine in the PC-3 cells was higher than that in the RWPE1 cells (Fig. 1E).

To further determine whether the antiproliferation effect of matrine on prostate cancer cells was due to cell cycle arrest, cultured prostate cancer cells were treated with matrine, LY294002 or a combination of the two for flow cytometric analysis. As shown in Fig. 2A-D, matrine treatment resulted in an appreciable arrest of PC-3 (Fig. 2A and C) and RWPE1 cells (Fig. 2B and D) in the G₀/G₁ phase of the cell cycle at 74.72 and 71.35%, respectively, after 48 h of treatment compared to the untreated controls (55.87 and 58.58%, respectively). The decrease in the percentage of cells in the S and G₂/M phases of the cell cycle was accompanied by a concomitant increase in the G₀/G₁ cell population. Matrine combined with LY294002 treatment of PC-3 (Fig. 2A and C) and RWPE1 cells (Fig. 2B and D) induced 83.25 and 79.29% arrest in the G₀/G₁ phase of the cell cycle, respectively, compared to the LY294002 control (71.03 and 70.14%, respectively). Compared to the control, the increasing percentage of cell arrest in the G₀/G₁ phase in the PC-3 cells was higher than that in the RWPE1 cells (Fig. 2E).

We assessed the effect of matrine on the expression of P27, CDK2 and CDK4, to determine whether matrine-induced cell cycle arrest of prostate cancer cells was dependent on the regulation of P27, CDK2 and CDK4. The expression levels of CDK2 and CDK4 were decreased in the PC-3 and RWPE1 cell lines after treatment with matrine (Fig. 3A-G). Notably, activation of P27 in cells was accompanied by a parallel decrease in the expression of CDK2 and CDK4. This was particularly evident when compared to cells treated with LY294002, in which matrine triggered prostate cancer cell cycle arrest at the G₀/G₁ phase by upregulating P27 and downregulating CDK4 and CDK2 relative to the protein expression of the PC-3 cells compared to the RWPE1 cells (Fig. 3H-K). The increased expression level of P27 and decreased expression levels of CDK4 and CDK2 in the PC-3 cells were higher than those in the RWPE1 cells.

To gain further insight into the dynamic progression from apoptosis to eventual cell death induced by matrine, Annexin V-FITC/PI double staining was used to assess the cell population undergoing apoptosis after treatment with matrine, LY294002 or a combination of the two for 48 h. The percentage of apoptotic cells, including early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic cells, was 20.11% in the PC-3 cells and 15.55% in the RWPE1 cells after treatment with matrine (Fig. 4A-D). However, <5% of the untreated cells underwent apoptosis under the same conditions (Fig. 4A-D). PC-3 and RWPE1 cells treated with matrine combined with LY294002 resulted in 25.88% cell apoptosis in the PC-3 cells and 18.88% in the RWPE1 cells, compared to 3.11 and 6.89%, respectively, with LY294002 treatment only (Fig. 4A-D). Compared to the RWPE1 cells, the total apoptosis in the PC-3 cells was higher (Fig. 4E).

To further demonstrate that matrine induced cell apoptosis in prostate cancer cells, the proteins Bim, Bax and Bcl-2 were studied. Bim can neutralize pro-survival members such as Bcl-2 and activate Bax, which can finally induce apoptosis. The prostate cancer cell line PC-3 expressed a high Bcl-2 and low Bax level, but the level of Bcl-2 decreased while the level of Bax and Bim increased under the effect of matrine (Fig. 5A). Thus the ratio of the Bcl-2/Bax (Fig. 5B and D) protein was markedly downregulated, which corresponded to the upregulation of Bim (Fig. 5A, C and E). For further verification, cells were treated with matrine combined with LY294002 to confirm that Bim may contribute, at least in part, to the induction of prostate cancer cell apoptosis by regulating the Bax and Bcl-2 relative protein expression in PC-3 cells compared to RWPE1 cells (Fig. 5F-I). The increased expression of Bim and the ratio of Bax/Bcl-2 in the PC-3 cells were higher than those in the RWPE1 cells.

Activation of the Akt signaling pathway plays a critical role through which cancer cells promote cell survival. We were therefore interested in determining whether matrine treatment has an effect on the Akt pathway. The expression levels of p-Akt and Akt were decreased after treatment with matrine for 48 h (Fig. 6A). To further confirm this finding, LY294002 was used to treat the cells for 1 h prior to matrine treatment. As shown in Fig. 6B and C, and F and G the expression levels of p-Akt and Akt were decreased after treatment with matrine and LY294002 compared to the matrine- or the LY294002-only group. A previous study demonstrated that an increase in p-Akt induced increased p-FoxO3a and its dysregulation (15). To determine whether matrine targets the expression of FoxO3a and its phosphorylation in prostate cancer cells, we determined the expression of FoxO3a and p-FoxO3a at the transcriptional (Fig. 7B and D) and translational levels (Fig. 6D and E, and 6H and I). The decreased expression levels of Akt/p-Akt and increased levels of FoxO3a/p-FoxO3a in the PC-3 cells were more obvious than in the RWPE1 cells (Fig. 6J-M). Furthermore, we also detected the expression of PI3K at the transcriptional level in the two prostate cell lines (Fig. 7A and C). The increase in the mRNA levels of FoxO3a in the PC-3 cells was higher than that in the RWPE1 cells, but PI3K was not (Fig. 7E and F). Unfortunately, the exact mechanism causing the different transcriptional and translational levels remains unclear. However, matrine treatment resulted in an increase in FoxO3a and a decrease in p-FoxO3a and PI3K. Furthermore, LY294002 enhanced the effect of matrine, increasing FoxO3a with a concomitant decrease in p-FoxO3a via the PI3K/Akt signaling pathway.