The reagent 2′,4′-dihydroxychalcone (>95% purity) was isolated from O. falcata herb, as described by Lu et al (13). The structure of TFC is exhibited in Fig. 1. The compound was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was <1% (v/v). Ham's −12 Nutrient Mixture (F-12) was purchased from Life Technologies (Grand Island, NY, USA). Invitrogen fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell counting kit (CCK-8) was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection kit and Cycletest plus DNA Reagent kit were obtained from BD Pharmingen (San Diego, CA, USA). Other chemicals were of analytical grade from commercial suppliers (Sinopharm Chemical Reagent Co., Ltd., Nanjing, China).

The human prostate cancer PC-3 cell line was purchased from the Cell Bank of China Academy of Sciences (Shanghai, China) and maintained in F-12 culture medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin, at 37°C and supplied with 95% room air and 5% CO₂.

PC-3 cells in the exponential phase of growth were placed in a 96-well plate (5,000 cells/well). Following a 4-h incubation period, the cells were treated with compounds or with the vehicle (vehicle control, 1% DMSO) for 24 h. Then 10 µl of CCK-8 was added and the plates were incubated for another 4 h. The absorbance at 450 nm was recorded by a Benchmark™ Plus microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

The PC-3 cells were incubated with or without TFC for 24 h in a 5% CO₂ incubator at 37°C. Then, the cells were observed for morphological changes using an Eclipse TE2000-U inverted microscope (Nikon Instruments, Melville, NY, USA) and images were captured.

Flow cytometry was used to analyze the cell cycle distribution and apoptosis subsequent to treatment with TFC. Briefly, for the apoptosis analysis assay, the PC-3 cells were placed in a 6-well plate (1×10⁶ cells/well) and treated with various doses of TFC for 24 h. The cells were then collected, washed with phosphate-buffered saline (PBS) and re-suspended in 1X binding buffer. FITC Annexin V and propidium iodide (PI) reagent were added and staining occurred for 15 min prior to flow cytometry analysis. For the cell cycle analysis assay performed subsequent to TFC treatment, the cells were collected and fixed with 70% ethanol overnight. The cells were then handled according to the manufacturer's instructions with Cycletest plus DNA Reagent kit, prior to analysis by flow cytometry. The cells in the G0/G1, S and G2/M phases were gated out as appropriate.

The PC-3 cells were treated with TFC for 24 h and the treated cells were collected, washed three times with PBS and lysed in cell lysis buffer. The cell lysates were separated on a 15% polyacrylamide gel and transferred to a polyvinyl difluoride membrane (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with Block Ace (AppliChem GmbH, Darmstadt, Germany) for 4 h at room temperature, the membrane was incubated overnight with primary antibodies, and then for 2 h with secondary antibodies. All antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The following rabbit anti-human primary antibodies were used for western blot analysis: Anti-p27^Kip1 (monoclonal; clone, D69C12; cat no. 3686); anti-PTEN (monoclonal; clone, D4.3; cat no. 9188); anti-caspase-7 (polyclonal; cat no. 9492); anti-caspase-3 (monoclonal; clone, 8G10; cat no. 9665); and anti-β-actin (monoclonal; clone, 13E5; cat no. 4970). Anti-rabbit IgG conjugated to horseradish peroxidase (cat no. 7074) was used as the secondary antibody. The primary antibodies were used at a dilution of 1:1,000. The secondary antibodies were used at a dilution of 1:3,000 and visualized with an enhanced chemiluminescence system (Amersham Imager 600; GE Healthcare Life Sciences, Little Chalfont, UK).

Statistical analysis was performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). The results were expressed as the mean ± standard deviation. P<0.05 or P<0.01 was considered to indicate a statistically significant difference.

The CCK-8 assay was used as an indirect measure to determine the viability of PC-3 cells exposed to TFC. TFC significantly suppressed the cell viability of PC-3 cells in a dose-dependent manner (Fig. 2). The cell viability was reduced to 90.81, 42.51, 26.52 and 1.51% when cells were treated with 25, 50, 100 and 200 µM TFC, respectively. The half maximal inhibitory concentration (IC₅₀) was 53.82 µM. Morphological changes were observed using microscopy (Fig. 3). The PC-3 cells treated with TFC for 24 h exhibited marked morphological changes. TFC caused individual cells to shrink and separate from neighboring cells. The cells appeared refringent and finally detached from the monolayer by TFC administration.

To determine whether the reduction of cell viability in PC-3 cells was associated with the induction of apoptosis, flow cytometry analysis was used to assess the number of apoptotic cells following TFC treatment. Apoptotic cells were significantly increased in a dose-dependent manner when compared with the control group (Fig. 4). The percentage of apoptotic cells reached 44.58±7.19 and 85.05±2.14% when treated with 50 and 100 µM TFC, respectively. These results demonstrated that TFC inhibited the proliferation of PC-3 cells by inducing apoptosis.

Cell cycle distribution was assessed by flow cytometry. The results demonstrated that TFC treatment caused G0/G1 phase cell cycle arrest in PC-3 cells (Fig. 5). The percentage of cells in the G0/G1 phase significantly increased between 46.06% in the cells not treated with TFC and 89.87% in cells treated with 100 µM TFC. These results indicated that TFC induced apoptosis in PC-3 cells by arresting the cell cycle in the G0/G1 phase.

Caspases are crucial mediators of programmed cell death, termed apoptosis (14). Out of these mediators, caspase-3 and caspase-7 are responsible for the proteolytic cleavage of numerous key proteins, including adenosine diphosphate (ADP)-ribose polymerase and poly(ADP-ribose) polymerase, that induce cell apoptosis (14). In the present study, TFC treatment significantly increased the activation of caspase-3 and −7 (Fig. 6A), demonstrating the importance of caspases in the induction of apoptosis.

The expression levels of the cyclin-dependent kinase inhibitor p27^Kip1 have been revealed to be a reliable prognostic marker in prostate cancer, as low or absent expression of p27^Kip1 correlates with poor prognosis (15). In addition, PTEN, a tumor-suppressor gene and one of the most promising biomarkers for prostate cancer, also acts as a valid and accurate tool for the prediction of prognosis and support of clinical decisions (16,17). To determine the effect of TFC on these biomarkers in prostate cancer, western blot analysis was performed to assess the effect of TFC on the expression of p27^Kip1 and PTEN in PC-3 cells. Notably, the expression of PTEN and p27^Kip1 was significantly upregulated subsequent to treatment with TFC (Fig. 6B).