The human PCa cell line LNCaP and human normal prostate cell line RWPE-1 were obtained from Shanghai Institute of Biochemistry and Cell Biology. Both cell lines purchased are certified by the American Type Culture Collection. PCa cells were routinely cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and RWPE-1 cells were cultured according to the manufacture's direction in K-SFM medium (all Gibco; Thermo Fisher Scientific, Inc.), penicillin 100 U/ml and streptomycin 100 U/ml (both Beijing Solarbio Science & Technology Co., Ltd.) at 37°C under an atmosphere of 5% CO₂ in humidified air.

Resveratrol (Res), docetaxel (PC) and bicalutamide were purchased from Sigma-Aldrich; Merck KGaA and were dissolved in 100% DMSO to create stock solutions of 50 mM and stored at −20°C. These were subsequently diluted in RPMI-1640 medium to the desired concentration for the experiments and the final concentration of DMSO that was given to the cells was 0.05%. Antibodies against AKT (cat. no. 4685), phosphorylated (p-)AKT (cat. no. 4058) were obtained from Cell Signaling Technology, Inc. Antibodies against AR (cat. no. 133273), ARV7 (cat. no. 15656) and GAPDH (cat. no. 9485) were purchased from Abcam. GAPDH was used as a loading control. The goat anti-rabbit and anti-mouse IgG-HRP secondary antibodies were purchased from Wuhan Boster Biological Technology, Ltd. The AKT pathway activator (phen) was purchased from Selleck Chemicals, primers (ARV7 and GAPDH) were purchased from Angen Biotech Co., Ltd. SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) and the PrimeScript™ RT Reagent kit with gDNA Eraser (Perfect Real Time) were purchased from Bao Biological Engineering Co., Ltd. (http://www.takara.com.cn). Lipofectamine^® 2000 was purchased from Thermo Fisher Scientific, Inc., and the miRcute miRNA First-Strand cDNA Synthesis kit was purchased from Tiangen Biochemical Technology Co., Ltd.

LNCaP cells in the androgen-dependent logarithmic growth phase were digested with trypsin and inoculated in a medium containing 10% non-androgen (activated charcoal-treated) FBS and cultured at 37°C with 5% CO₂. After 10 generations of culture, bicalutamide (1.0 µM) was added to the culture medium for 20 generations, and then the concentration of bicalutamide was increased to 5.0 µM to continue subculture. By 6 months of subculture, hormone-resistant prostate cancer cells had been constructed successfully.

The ARV7 overexpression vector was based on the PCDNA3.1 plasmid (Guangzhou RiboBio Co., Ltd.). The primers were designed and the RNA fragment of the ARV7 gene was obtained using the cell genome cDNA as a template. The fragment was inserted into multiple cloning sites downstream of the plasmid PCDNA3.1 to obtain the wild-type ARV7-overexpression vector. The PCDNA3.1-ARV7 plasmid and DH5α competent cells mixed culture PCDNA3.1-ARV7. The constructed PCDNA3.1-ARV7 vector and PCDNA3.1 vector were introduced into LNCaP-B cells, respectively, using Lipofectamine 2000 reagent according to the manufacturer's instructions, and ARV7 protein expression was verified using western blotting.

Cell viability was determined using an MTT assay. Briefly, the PCa cell lines LNCaP and LNCaP-B (5×10³ cells/well) were seeded in 96-well plates. Cells were cultured overnight (37°C, 5% CO₂), and the cells were incubated with various concentrations of RES (25, 50, 100 and 200 µM) dissolved in DMSO and PC (5 µM) and various concentrations (200 µl/wells) of RES, RES+ARV7 and RES+phen for 12, 24, 48 and 72 h (at 37°C in 5% CO₂). DMSO (0.05%) was used as the negative control (NC) group. MTT (0.5 mg/ml, 20 µl/well) was added to each well 4 h in advance. After 4 h incubation (at 37°C in 5% CO₂), DMSO (150 µl/well) was added to each well, and the cells were measured at 492 nm using a Multiskan Ascent microplate photometer. Viability was expressed as the percent viable cells compared with vehicle-treated control cells that were arbitrarily assigned as 100% viability.

A flow cytometric assay was conducted to analyze the apoptosis of PCa cells. Cells plated in 6-cm plates were treated with various concentrations of Res (50 and 100 µM), PC (5 µM), RES+ARV7 and RES+phen. After 24-h treatment, cells were washed with phosphate buffer saline and harvested. The apoptosis assay was performed according to the manufacturer's protocol of the Annexin V/7-ADD apoptosis kit (BD Pharmingen™; BD Biosciences). The sedimented cells were resuspended with 500 ul binding buffer based on the instructions. Subsequently, 5 µl Annexin V-7AAD and 5 µl Annexin V-APC were added to the cell suspension and cells were incubated at room temperature for 15 min in the dark. The stained cells were analyzed using BD FACSCanto II flow cytometry (BD Pharmingen™; BD Biosciences) and the data analyses were performed using SPSS 19.0 software (IBM, Corp.).

Total RNA was extracted from LNCaP and LNCaP-B cells treated with various drugs in each group, using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and purity of RNA were measured using UV spectrophotometry (Thermo Fisher Scientific, Inc.). One microgram of the total RNA was used as a template and was reverse transcribed into cDNA using PrimeScript™ RT Reagent kit with gDNA Eraser (Perfect Real Time) to obtain a template of the reaction system. The condition for reverse transcription was at 37°C for 15 min, followed by 85°C for 5 sec. The expression levels of AR, ARV7 and GAPDH in the cells were analyzed using two-step fluorescence quantitative PCR. A 10-µl reaction system was generated. The qPCR reaction was performed using the LightCycler 480 II DNA Amplifier according to the manufacturer's instructions. The reaction conditions were as follows: Pre-denaturation at 95°C for 2 min followed by 95°C for 15 sec, and 60°C for 30 sec was performed for a total of 40 cycles. After completion of the reaction, the relative expression levels of AR and ARV7 in the cells were expressed using 2^−ΔΔCq normalization (26). The designed specific primers for each gene are listed in Table I.

PCa cells were treated with various concentrations of Res or PC for 24 h. Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and total proteins were extracted at 4°C. The total protein concentrations were determined using the BCA assay (Beyotime Institute of Biotechnology). The proteins (20 µg/lane) were loaded on a 12% gel, and resolved using SDS-PAGE with a constant voltage of 80 V. Then the proteins were transferred to PVDF membranes (EMD Millipore) using the Bio-Rad PowerPac Basic Power Supply system. Membranes were blocked in 5% skimmed milk in Tris-Buffered Saline Tween-20, followed by incubation with primary antibodies overnight at 4°C. The dilutions of ARV7, Bax, Bcl-2, AR, AKT, p-AKT and GAPDH were all 1:1,000. Blots were then probed with the IgG-HRP secondary antibody (1:1,000) at room temperature for 1 h, and protein bands were detected using chemiluminescence reagent (EMD Millipore), under the Tanon5200 chemiluminescent imaging system (Tanon Science and Technology Co., Ltd.). ImageJ 1.8.0 software (National Institutes of Health) was used to quantify western blotting images.

All the data analyses were performed using SPSS 19.0 software. All experiments were repeated at least three times and P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate. Data are expressed as the mean ± SD. A Student's unpaired t-test was used for determining the differences between two groups and for more than two group comparisons one-way ANOVA test with a Tukey's post hoc test was used.

Various concentrations of Res (25, 50, 100 and 200 µM) and PC (5 µM) were used to treat LNCaP and LNCaP-B cells for 24, 48 and 72 h. The proliferative capacities of both cell types were significantly inhibited compared with the control, and the effects were time- and dose-dependent (Fig. 1A and B). The results suggested that Res inhibited the proliferation of PCa cells. Nevertheless, following administration of relatively various concentrations of Res (25, 50, 100 and 200 µM) to RWPE-1 cells for 12, 24, 48 and 72 h, the viability of PCa cells was significantly lower compared with that of normal cells after Res treatment after 48 and 72 h (Fig. 1C). To further investigate the molecular mechanism of ARV7 inhibition in PCa cells mediated by Res, an overexpression vector (PCDNA3.1-ARV7) of ARV7 was constructed and transfected it into LNCaP-B cells. Western blot analysis verified that, compared with cells transfected with empty vector (PCDNA3.1), ARV7 protein levels in cells transfected with ARV7 overexpression vector (ARV7) increased significantly (Fig. 1D). It was demonstrated that the Res+ARV7 group and the Res+phen group restored the inhibitory effect of Res on the proliferation of LNCaP-B cells compared with Res treatment alone (Fig. 1E). This suggests that effect of Res on the proliferation of LNCaP-B cells may be associated with the ARV7 and AKT pathways.

Flow cytometry was used to measure the effects of various concentrations of Res on apoptosis in LNCaP and LNCaP-B. Compared with the NC group, after treatment of LNCaP and LNCaP-B cells with various concentrations of Res (50 and 100 µM) for 24 h, the apoptosis level of LNCaP cells increased by approximately 33.3 and 75.9%, respectively, and the apoptosis level of LNCaP-B cells increased by approximately 9.6 and 33%, respectively. (Q1, Q2, Q3, Q4 represented dead cells, late apoptotic cells, viable cells, and early apoptotic cells, respectively.) Therefore, the total number of early and late apoptotic cells in the two cell lines was significantly higher compared with those in the control group. The effect of Res on apoptosis of LNCaP and LNCaP-B cells was concentration-dependent (P<0.05 and P<0.01; Fig. 2A and B, respectively).

The effects of overexpression of ARV7 on Res-induced apoptosis in LNCaP-B cells were also studied. Upregulation of ARV7 suppressed Res-induced apoptosis by approximately 26%. It was reported that Res+ARV7 and Res+phen treatment reversed Res-induced apoptosis in LNCaP-B cells (Fig. 2C). In order to further study the relevant molecular mechanisms, the levels of apoptosis-associated proteins were measured using western blotting. The results showed that all concentrations of Res increased the expression levels of pro-apoptotic proteins Bax and the expression levels of antiapoptotic protein Bcl2 were lower compared with those in control group (Fig. 2D and E). In addition, compared with the effect of Res on PCa cells, Res+ARV7 and Res+phen treatment increased the expression of antiapoptotic protein Bcl2 and decreased the expression of pro-apoptotic protein Bax (Fig. 2F). These results suggested that Res not only inhibits the proliferation of PCa cells, but also induces apoptosis. These results also suggested that the pro-apoptotic effect of Res on LNCaP-B cells is associated with the ARV7 and AKT pathway signaling pathways.

Chemiluminescence detection showed that various concentrations of Res (25, 50 and 100 µM) and PC (5 µM) applied to LNCaP and LNCaP-B cells for 24 h, caused PSA content in both cell lines to be significantly lower compared with that of the control group (*P<0.05 and **P<0.01). Furthermore, it was also revealed that Res decreased PSA levels in LNCaP and LNCaP-B cells in a concentration-dependent manner (Fig. 3). This suggested that Res inhibited the secretion of PSA by LNCaP and LNCaP-B cells.

The effect of various concentrations of Res on mRNA levels in PCa cells was measured using RT-qPCR. It was demonstrated that, after treatment of LNCaP and LNCaP-B cells with various concentrations of Res (25, 50 and 100 µM) and PC (5 µM) for 24 h, expression levels of AR mRNA in the two PCa cells were significantly lower compared with those of the control group (Fig. 4A and B). It was also reported found that, with increasing Res concentrations, the effect of Res on the downregulation of AR mRNA levels in PCa cells was dose-dependent. These results indicated that Res inhibited the expression levels of AR in LNCap and LNCap-B cells in a dose-dependent manner.

The effect of various concentrations of Res on levels of AR protein in PCa cells was measured using western blotting. Expression levels of AR protein in the two PCa cells were significantly lower compared with those in the control group after treatment of LNCaP and LNCaP-B cells with various concentrations of Res (25, 50 and 100 µM) for 24 h. It was also shown that Res was associated with decreases of AR expression in PCa cells in a concentration-dependent manner (Fig. 5A and B). Western blot analysis showed that Res was associated with lower levels of AR protein in LNCaP and LNCaP-B cells.

Studies have shown that PCa castration-resistance is associated with expression levels of ARV7 (27,28). Therefore, expression levels of ARV7 in LNCaP and LNCaP-B cells were examined using western blotting. ARV7 was expressed at low levels in LNCaP cells but positive in LNCaP-B (Fig. 5C). This suggested that the hormone resistance of LNCaP-B may be associated with high expression of ARV7. The effect of Res on ARV7 expression was further examined and it was revealed that Res inhibited ARV7 expression (Fig. 5D). This suggested that the inhibitory effect of Res on hormone-resistant LNCaP-B cells may be associated with the inhibition of ARV7 expression.

Western blotting was used to measure the effect of Res on the AKT signaling pathway in PCa cells. After treatment of LNCaP and LNCaP-B cells with various concentrations of Res (25, 50 and 100 µM) for 24 h, levels of p-AKT in the two PCa cells were significantly lower compared with those in the control group (Fig. 6A and B). Res inhibited the AKT phosphorylation in a concentration-dependent manner. These results suggested that Res may have inhibited AKT phosphorylation in LNCaP and LNCaP-B cells.

The molecular mechanism of Res-mediated ARV7 inhibition in PCa was further investigated. Compared with the Res group, the Res+ARV7 and Res+AKT pathway activator groups showed significantly higher levels of p-AKT (Fig. 6C). These results suggested that overexpression of ARV7 significantly decreased Res-induced phosphorylation of AKT.