Genistein (≥98% purity), quercetin (≥97% purity) and luteolin (≥98% purity) were purchased from Aladdin Industrial Corp. (Shanghai, China). An Annexin V-FITC/PI apoptosis detection kit (40302ES20) and Hoechst 33342 nucleic acid stain were purchased from Shanghai Qcbio Science and Technologies Co., Ltd. (Shanghai, China). The following reagents were from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China): Propidium iodide (PI), ethidium bromide (EB), dimethyl sulfoxide (DMSO), RNase and materials for western blot analysis, including SDS gel, nitrocellulose membranes, blot filter paper, bovine serum albumin and Ponceau S dye. Human anti-PSCA (ab56338) and anti-β-actin (ab3280) antibodies were obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse antibody (TA130003) was purchased from Origene Technologies, Inc. (Beijing, China).

The prostate cancer cell line DU145, which expresses PSCA (10), was obtained from the National Infrastructure of Cell Line Resource (Beijing, China). DU145 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 mg/ml streptomycin (all Beijing Solarbio Science and Technology Co., Ltd.) at 37°C in a humidified atmosphere of 5% CO₂ in air.

DU145 cells (1×10⁴ cells/well) were plated into 96-well plates and incubated at 37°C for 24 h in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum to allow the cells to attach prior to treatment with flavonoids (genistein, luteolin and quercetin). Each flavonoid compound was dissolved in 0.1% DMSO and made up with RPMI-1640 medium to a final flavonoid concentration of 20, 40, 80 and 100 µM. DU145 cells were individually treated with 20, 40, 80 and 100 µM of each flavonoid compound at 37°C for 24 h in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Cells treated with 0.1% DMSO served as a negative control. Cell viability was measured by counting the cells with a Neubauer chamber (BRAND Trading Co., Ltd., Shanghai), according to the manufacturer's protocol. Cell viability was expressed relative to control cells. Half maximal inhibitory concentration (IC₅₀) values were subsequently determined as the concentration of compound that produced a 50% reduction in cell number, and were determined graphically from the concentration response curve.

Morphological changes characteristic of cell death were observed using a normal inverted light microscope. Cells were individually treated with different concentrations of the three flavonoids for 24 h. Morphological changes of cells were observed at 24 h post-treatment.

DU145 cells (70% confluence in 6-well plates) were starved in 1% FBS-supplemented RPMI-1640 media for 24 h to arrest cells in the G0 phase of the cell cycle, after which they were individually treated with each flavonoid compound (80 µM) for 24 h in 10% FBS-supplemented RPMI-1640 medium. The cells in the control group were treated with 0.1% DMSO under the same conditions. Following flavonoid treatment, cells were trypsinized, washed twice with cold phosphate-buffered saline (PBS) and centrifuged at 110 × g for 5 min at room temperature. The pellet was resuspended in 75% ethanol for 24 h at −20°C. Cells were subsequently centrifuged at 110 × g for 5 min at room temperature and the supernatant containing dead and apoptotic cells was discarded, whereas the pellet was washed twice with cold PBS, suspended in 500 µl PBS and incubated with 5 µl RNase (final concentration, 20 µg/ml) at 37°C for 30 min. Cells were incubated in the dark with PI (final concentration, 50 µg/ml) for 1 h at room temperature and analyzed by flow cytometry using a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA, USA) and WinMDI (version 2.8) software (Scripps Institute, La Jolla, CA, USA).

DU145 cells were seeded into 6-well plates (5×10⁵ cells/well) and individually incubated with each of the three flavonoid compounds (80 µM) at 37°C for 24 h. Cells in the control group were treated with 0.1% DMSO under the same conditions. The cells were incubated with Hoechst 33342 (30 µM in PBS) in the dark for 30 min. Cells were then washed twice with PBS and cell fluorescence was observed using a FluoView FV1000 confocal microscope (Olympus Corp., Tokyo, Japan).

Levels of cell apoptosis were measured using an Annexin V-FITC apoptosis detection kit, according to the manufacturer's protocol. Briefly, cells were individually treated with each of the three flavonoids (80 µM) or DMSO control (0.1%) for 24 h at 37°C. Cells were trypsinized and washed with PBS, then resuspended in Annexin V binding buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH (pH 7.4), 140 mM NaCl and 2.5 mM CaCl₂. Cells were subsequently stained with FITC-conjugated Annexin V and PI at room temperature for 15 min in the dark prior to the addition of binding buffer. The number of apoptotic cells was measured by flow cytometry, as described above. Cells were sorted into intact (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺) and necrotic (Annexin V⁻/PI⁺) cell populations.

Total RNA was isolated from DU145 cells individually treated with each of the three flavonoids (80 µM) or DMSO control (0.1%) for 24 h using the TRIzol reagent-phenol chloroform procedure (Gibco; Thermo Fisher Scientific, Inc.). DNase I (2 U, D7076; Beyotime Institute of Biotechnology, Haimen, China) was added to the RNA and incubated in a 37°C water bath for 20 min. DNAse Inactivation reagent (2 µl, AM1906; Ambion; Thermo Fisher Scientific, Inc.) was added to the RNA and incubated for 2 min at room temperature. The tube was flicked once more during the incubation to redisperse the DNase Inactivation reagent and centrifuged at 10,000 × g for 1 min at 4°C. A total of 2 µg total RNA from each sample, quantified with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc.) was subjected to RT using a SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. For each sample, qPCR was carried out in triplicate in a total reaction mixture of 20 µl [2 µl cDNA, 10 µl SYBR Premix Ex Taq, 0.4 µl ROX Reference Dye II, 0.5 µl of each forward and reverse primer (both 10 µM) and 6.6 µl H₂O] on an ABI PRISM 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers (Sangon Biotech Co., Ltd., Shanghai, China) used were as follows: PSCA forward, 5′-ATCAGGAGGGCCCAGTAAAG-3′ and reverse, 5′-TCCCAGGAACTCACGTCAAC-3′; and β-actin forward, 5′-AACACCCCAGCCATGTACG-3′ and reverse, 5′-ATGTCACGCACGATTTCCC-3′.

qPCR thermal cycling was initiated at 95°C for 10 sec, followed by 40 thermal cycles, each at 95°C for 5 sec and 60°C for 34 sec. Levels of PCSA mRNA expression were measured using the 2^−ΔΔCq method (11) and were normalized to the expression of β-actin in each sample. Melting curves for each qPCR analysis were generated to verify the purity of the amplification product.

DU145 cells (70% confluent) were individually treated with each of the three flavonoid compounds (80 µM) or DMSO control (0.1%) at 37°C for 24 h. Following treatment, the media was aspirated and cells were washed with cold PBS and subsequently incubated with ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na₃VO₄, 0.5% NP-40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride; pH 7.4) over ice for 30 min. Cells were then scraped and the lysate was collected in a microfuge tube. The lysate was cleared by centrifugation at 14,000 × g for 15 min at 4°C and the supernatant (total cell lysate) was immediately used or stored at −80°C. Protein concentration of the supernatant was determined using a Bradford Protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. Proteins (50 µg) from each sample were separated by 10–12% SDS-PAGE and transferred to a nitrocellulose membrane. The blot was blocked in blocking buffer (5% non-fat dry milk and 0.05% Tween-20 in 20 mM TBS; pH 7.6) for 1 h at room temperature. Blots were then incubated with anti-PSCA (1:1,000) overnight at 4°C and anti-β-actin antibodies (1:1,000) for 2 h at room temperature, followed by incubation with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (1:10,000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Beyotime Institute of Biotechnology). β-actin was used as an internal control.

All data are expressed as the mean ± standard error of the mean of at least three independent experiments. Differences between the mean values of multiple groups were analyzed using one-way analysis of variance. Statistical analysis was performed with SPSS 19.0 software (IBM SPSS, Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of flavonoids on the inhibition of cell viability, the cytotoxicity of various concentrations (20–100 µM) of genistein, luteolin and quercetin were evaluated by counting cells in a Neubauer chamber at 24 h post-treatment. As depicted in Fig. 1A, flavonoids inhibited cell viability in a dose-dependent manner (P<0.05). Genistein exhibited the greatest inhibitory effect on cell cycle inhibition with an estimated IC₅₀ value of 33 µM, which was significantly lower compared with that of luteolin (42 µM; P<0.05) and quercetin (78 µM; P<0.01; Fig. 1B). Observations by light microscope also indicated that, following flavonoid treatment, numbers of DU145 cells decreased in a dose-dependent manner, and cell morphology was altered relative to that of control cells. Notably, fewer DU145 cells were observed in cell cultures treated with increasing concentrations of each flavonoid, while swelling and damage of the plasma membrane was observed in the remaining cells (Fig. 1C).

To determine whether the inhibitory effects of flavonoids on cell growth were mediated by alterations in cell cycle progression, synchronized cells were incubated with each flavonoid (80 µM) for 24 h, and the effect of the flavonoids on cell cycle phase distribution was determined. Representative histograms are depicted in Fig. 2. Relative to control cells, cell groups treated with genistein, luteolin and quercetin exhibited a significantly increased population of S phase cells, and a corresponding significant decrease in G1 phase cells (both P<0.01 for genistein; both P<0.05 for luteolin and quercitin). This was consistent with the aforementioned growth inhibitory effects of genistein, luteolin and quercetin.

Flavonoids may have inhibited the viability of DU145 cells through the induction of apoptosis, and thus cell morphologies were assessed to determine the extent of apoptosis. Hoechst 33342 staining demonstrated that treatment with genistein, luteolin and quercetin induced chromatin condensation and fragmentation of the nuclei into oligonucleosomes (Fig. 3A). Apoptotic cells were subsequently quantified using a flow cytometry assay. Individual treatment with each of the three flavonoid compounds (80 µM) for 24 h decreased the proportion of intact cells (Fig. 3B) and increased the proportion of apoptotic cells. Notably, treatment with genistein, luteolin and quercetin decreased the proportions of intact cells from 94.3%, as observed for control cells, to 51.6, 63.8 and 65.9%, respectively. By contrast, genistein, luteolin and quercetin increased the proportions of early apoptotic cells from 2.1% (control) to 26.4, 17.1 and 15.9%, respectively. These data indicate that flavonoids may induce apoptosis in DU145 cells.

To determine whether the inhibitory effects of genistein, luteolin and quercetin were associated with a downregulation in PSCA, RT-qPCR was used to measure the levels of PSCA mRNA expression in DU145 cells following treatment with each of the three flavonoids (80 µM) for 24 h. As depicted in Fig. 4A, levels of PSCA mRNA in DU145 cells treated with genistein, luteolin and quercetin were decreased by 0.46-fold (P<0.01), 0.49-fold (P<0.05) and 0.64-fold (P<0.05), respectively, when compared with controls. Thus, genistein, luteolin, and quercetin may inhibit the expression of PSCA at the mRNA level.

In addition, western blot analysis was conducted to verify whether alterations in the expression of PSCA mRNA led to alterations at the protein level. As depicted in Fig. 4B, genistein, luteolin and quercetin markedly decreased the expression of PSCA at the protein level after 24-h treatment.