Du145 (ATCC^® HTB-81™) and PC3 (ATCC^® CR L-1435™) human prostate cancer cell lines (American Type Culture Collection, Manassas, VA, USA), which were preserved in liquid nitrogen until use, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Gibco). The cells were plated in 24-well plates for 8 h prior to transfection or drug treatment. The pcDNA3.1 control (mock) plasmid and pcDNA3.1-Pdcd5 were constructed and then transfected into prostate cancer cells for 24 h using Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). In brief, Lipofectamine 2000 and the Pdcd5 plasmid were each added to separate opti-MEM solutions (Gibco; 100 μl) and incubated for 5 min. The two solutions were then combined and incubated at room temperature for a further 15 min prior to addition to the plated cells for transfection for 6 h at 37°C, and 5% CO₂. The medium was then replaced with fresh DMEM plus 10% FBS and penicillin-streptomycin.

Cell viability and proliferation were determined using an MTT assay (Sigma, St. Louis, MO, USA), as previously described (25,26). In brief, following transfection of Pdcd5 or the mock plasmid, 5×10⁵ prostate cancer cells were seeded into 96-well plates and incubated with various concentrations of cisplatin (0, 10, 50 and 250 μM; Sigma) for 24, 48 and 72 h. Following incubation, 10 μl of 5 mg/ml MTT reagent was added to the medium and cells were incubated for a further 4 h. The medium was then drained and cells were treated with 150 μl dimethyl sulfoxide for 15 min. The plates were then analyzed using a microplate reader (Thermo Scientific, Waltham, MA, USA) at a test wavelength of 490 nm.

Following transfection, cisplatin-treated Du145 cells were subjected to Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual labeling followed by fluorescence-activated cell sorting (FACS) analysis (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), performed according to the manufacturer’s instruction, in order to determine apoptosis. Each example recorded >10,000 events.

Whole cell extracts were prepared and separated using SDS-PAGE as previously described (27–29). Protein (30–60 μg) was then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked using 1% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20 for 1 h. The filters were then incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal Bax immunoglobulin (Ig)G (1:1,000), mouse monoclonal Bcl-2 IgG1 (1:1,000), rabbit polyclonal caspase-3 IgG (1:2,000), rabbit polyclonal cleaved caspase-3 IgG (1:2,000) and mouse monoclonal β-actin IgG1 (1:2,000), which were all purchased from Santa Cruz Biotechnology, Inc. β-actin was used as an internal control. PVDF membranes were then washed three times with phosphate-buffered saline with 0.1% Tween20 and then incubated with goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, Inc.) conjugated with peroxidase for 1 h at room temperature. Membranes were then washed and signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) was used for analysis.

All experiments were performed a minimum of three times. Data were analyzed using SPSS 11.5 software (SPSS, Inc., Chicago, IL, USA). Values are presented as the mean ± standard deviation. P<0.01 was considered to indicate a statistically significant difference.

Previous studies have demonstrated that Du145 cells were highly resistant to cisplatin treatment 30,31). In the present study, an MTT assay was used to determine the viability of Du145 cells in order to evaluate the anti-tumor activity of cisplatin. As shown in Fig. 1, different concentrations of cisplatin were used to treat Du145 cells for 24, 48 and 72 h. The results demonstrated that there was no significant decrease in cell viability at any concentration of cisplatin (Fig. 1B); however, at 48 and 72 h post-transfection of Pdcd5, the survival rate of Du145 cells was significantly inhibited compared with that of the untreated cells.

In order to further examine the effects of cisplatin on cellular proliferation in the presence of Pdcd5, Du145 cells were transfected for 48 h with pcDNA3.1-Pdcd5 in order to overexpress Pdcd5 and then incubated with various concentrations of cisplatin for 24, 48 and 72 h. An MTT assay was the used to determine cell viability of the prostate cancer cells. As shown in Fig. 2A, Pdcd5-transfected cells treated with 50 μM cisplatin revealed marked cell death compared with those of the untreated and mock vector-transfected cells. In addition, the rates of cell survival demonstrated time- and dose-dependent decreases compared with those of the untreated mock vector- and Pdcd5-transfected cells (Fig. 2B). This therefore indicated that Du145 cells transfected with Pdcd5 had an increased sensitivity to cisplatin.

As shown in Fig. 3, an MTT assay was also used to determine the IC₅₀ values in the Du145 and PC3 prostate cancer cell lines. Following transfection of Pdcd5, Du145 and PC3 cells were exposed to various concentrations of cisplatin for 48 h. The IC₅₀ values were 114.1 and 50.6 μM in Du145 and PC3 cells, respectively, indicating the synergistic effects of transfection of Pdcd5 and cisplatin treatment.

FACS analysis using Annexin V-FITC and PI staining was performed in order to determine whether the cisplatin-induced decrease in cell survival was due to apoptosis. As shown in Fig. 4, the apoptotic rate of Du145 cells transfected with Pdcd5 was significantly increased compared with that of the control group following cisplatin treatment for 24 h. In addition, the apoptotic rate of untreated Pdcd5-overexpressing cells was significantly increased compared with that of the untreated control cells. These results were consistent with those determined by MTT assays.

Western blot analysis was performed in order the detect the expression levels of several apoptosis-associated proteins. As shown in Fig. 5, following treatment with cisplatin for 24 h, the expression levels of cleaved caspase-3 in Pdcd5 transfected cells was markedly increased compared with those of cells transfected with the mock plasmid. In addition, there was no significant difference in Bcl-2 expression in Pdcd5-transfected Du145 cells following cisplatin treatment; however, the protein expression levels of Bax were significantly increased. Therefore, following cisplatin treatment, the Bcl-2/Bax ratio was significantly decreased in Pdcd5-transfected Du145 cells. In conclusion, Pdcd5-transfected Du145 cells demonstrated a significantly increased level of chemosensitivity to cisplatin and a time- and dose-dependent increase in the rate of apoptosis following cisplatin treatment.