SSd was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Rabbit anti-human cleaved-caspase-3, Bcl-2-associated X protein (Bax), p21, p53, cytochrome-c, mouse anti-human B-cell lymphoma 2 (Bcl-2) and β-actin primary antibodies were purchased from Cell Signaling Technology, Inc. (Shanghai, China). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse and anti-rabbit) were purchased from Santa Cruz Biotechnology, Inc. (Beijing, China). Trypsin, Hoechst 33258, Rhodamine 123 (Rho-123), penicillin and streptomycin were purchased from Sigma-Aldrich (Beijing, China).

The DU145 human prostate cancer cell line (ATCC HTB-81) was obtained from the American Type Culture Collection (Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a CO₂ incubator (37°C, 5% CO₂, 95% humidity).

Cell viability was determined by an MTT assay as previously described (19). Briefly, 100 μl cell suspension (1×10⁵ cells) was seeded in 96-well plates and incubated for 12 h, and then the cells were exposed to SSd (0, 1, 2.5, 5, 10, 20 or 50 μM) for 24 h. Following treatment, 20 μl MTT (5 mg/ml) was added and the cells were incubated at 37°C for 4 h. The culture medium was removed and 150 μl DMSO was added to each well to dissolve the formazan crystals. The absorbance (570 nm) was measured using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Waltham, MA, USA). The percentage of viable cells was determined using the following formula: Cell viability (%) = (A570 treated / A570 control) × 100; and the IC₅₀-values were calculated using GraphPad Prism, version 5 (GraphPad Software, Inc., La Jolla, CA, USA).

DU145 cells were incubated with different concentrations (3, 9 or 15 μM) of SSd for 24 h. Morphological changes of the cells were visualized under a phase contrast microscope (1×71; Olympus Corporation, Tokyo, Japan), recorded with a charge-coupled device (CCD) camera (DP72; Olympus Corporation) and analyzed using DP2-BSW software, version 2.2 (Olympus Corporation). For mouse splenocyte morphological study, the cells were analyzed by trypan blue (0.4%) staining prior to the microscopic visualization.

Cell apoptosis was detected by flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, DU145 cells cultured in six-well tissue culture plates were treated with different concentrations of SSd for 12 h. Following treatment, the cells were collected and washed twice with ice-chilled phosphate-buffered saline (PBS). The cell pellets were stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions. Late apoptosis was defined as Annexin V-positive/PI-positive and early apoptosis was defined as Annexin V-positive/PI-negative as determined by flow cytometry (Epics XL; Beckman Coulter, Miami, FL, USA).

Following exposure to the test compound for 12 h, the DU145 cells were harvested and washed twice with PBS. The cells were then stained with Hoechst 33258 (50 μg/ml) at 37°C for 10 min in the dark. After the staining, the cells were washed twice with PBS and analyzed using a fluorescence microscope (1×71; Olympus Corporation) installed with a CCD camera (DP72; Olympus Corporation) and analyzed using DP2-BSW software (Olympus Corporation). Apoptotic cells were defined as cells exhibiting nuclear shrinkage and chromatin condensation.

DU145 cells were treated with different concentrations (3, 9 and 15 μM) of SSd for 12 h, trypsinized, washed twice with PBS and fixed in 70% ice-cold ethanol overnight. The fixed cells were rinsed twice with PBS and then stained with 50 μg/ml PI (containing 100 μg/ml RNase A) using a Cell Cycle and Apoptosis Analysis kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Cell cycle phase distributions of nuclear DNA were assayed using flow cytometry (Epics XL; Beckman Coulter) and CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

DU145 cells cultured in six-well tissue culture plates were treated with different concentrations of SSd. The cells were then stained with 10 μmol/l 2′,7′-dichlorofluorescein-diacetate using a ROS Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. The cells were then collected, washed three times with PBS and assayed using flow cytometry (Epics XL; Beckman Coulter) as described previously (20).

DU145 cells were treated with different concentrations of SSd for 12 h. The cells were trypsinized, collected in a centrifuge tube, and then stained with Rho-123 (10 μg/ml) at 37°C for 30 min in the dark. Following staining, the cells were washed three times with PBS and assayed using flow cytometry (Epics XL; Beckman Coulter).

DU145 cells were treated with different concentrations of SSd for 12 h and then cell extracts were prepared using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). The cell lysates (containing 40 μg protein) were subjected to SDS-PAGE and analyzed by western blotting using various antibodies according to standard protocols. The proteins were visualized using an Enhanced Chemiluminescence Plus kit (Millipore Corporation, Billerica, MA, USA).

All data are expressed as the mean ± standard error of the mean from at least three independent experiments. Statistical analysis was performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The inhibitory effects of SSd (chemical structure is shown in Fig. 1A) on the proliferation of DU145 human prostate carcinoma cells were determined using an MTT assay. As shown in Fig. 1B, SSd treatment induced concentration-dependent proliferation inhibition of the DU145 cells. At 24 h, maximal inhibition was achieved with 50 μM SSd, which inhibited 80% of the DU145 cells proliferation, and the IC₅₀-value of the inhibition was ~10 μM. Morphological changes of the DU145 cells treated with SSd were visualized under a phase contrast microscope, which revealed a reduced number of adherent cells accompanying an increased number of floating cells in the culture medium compared with those in the culture medium containing the untreated cells (Fig. 1C). Furthermore, the effect of SSd on primarily cultured mouse splenocytes was investigated and trypan blue analysis indicated that SSd had little toxicity on the cells compared with the control cells (Fig. 1D).

Cell cycle arrest and apoptosis are major causes of cell proliferation inhibition (20–22). To investigate the mechanisms responsible for SSd-induced inhibition of DU145 cell proliferation, the cell cycle distribution affected by SSd was measured. DU145 cells were exposed to different concentrations of SSd for 12 h and then the cell cycle distributions were determined using PI staining and flow cytometric analysis. The data indicated that SSd caused a significant accumulation of DU145 cells in G0/G1 phase compared with that of the control cells. Compared with that of the DMSO control, the proportions of cells in G0/G1 phase were increased from 51.7 to 53.3, 56.8 and 60.3% following treatment with 3, 9 and 15 μM SSd, respectively (Fig. 2A). To elucidate the molecular mechanism underlying the G0/G1 phase arrest induced by SSd, several key proteins involved in the G1 phase transition were investigated in DU145 cells. The cells were treated with 3, 9 and 15 μM SSd for 12 h and the expression levels of p53 and p21 proteins were analyzed by western blotting. The data showed that SSd treatment significantly increased the expression levels of p53 and p21 compared with those of the control cells (Fig. 2B).

Subsequently, the effect of SSd on the induction of apoptosis of DU145 cells was investigated. Cell apoptosis, a type of programmed cell death, is characterized by nuclear condensation, cell shrinkage, membrane blebbing and DNA fragmentation (23), with nuclear condensation being a key characteristic (24). Morphological changes of the cell nuclei were observed using Hoechst 33258 staining. The results revealed that treatment of DU145 cells with SSd resulted in significant levels of nuclear condensation: 3, 9 and 15 μM SSd treatment increased the percentage of cleaved nuclei from 5.13±0.84 (in the DMSO control group) to 10.01±1.71, 24.50±1.82 and 51.44±2.39%, respectively (Fig. 3A).

To further quantify the SSd-induced apoptotic effect, the treated cells were stained with Annexin V-FITC/PI and assayed using flow cytometry. A concentration-dependent increase in the percentages of necrotic (Annexin V-positive, PI-positive) and apoptotic (Annexin V-positive, PI-negative) cells was observed. Treatment of the DU145 cells with 3, 9 and 15 μM SSd for 12 h increased the rate of apoptosis from 7.37±2.39 to 27.26±2.68, 46.43±4.43 and 75.77±3.01%, respectively, with <2% of cells being necrotic (Fig. 3B). Notably, ~75% of the cells were apoptotic in the 15 μM SSd treatment group after 12 h, with a concomitant <10% increase in the G1 phase proportion (Fig. 2A). G1 phase arrest may only minimally account for the antiproliferative effect of SSd observed in DU145 cells.

The mitochondria-mediated intracellular signaling pathway is characterized by increased ROS generation, MMP dissipation and release of cytochrome c from the mitochondria (25). A study indicated that SSd induced cellular ROS accumulation in cervical (HeLa and Siha), ovarian (SKOV3) and non-small cell lung (A549) cancer cell lines (9). Thus, the present study investigated the effect of SSd on the mitochondrial signaling pathway. ROS generation in DU145 cells was detected using a ROS Assay kit. As shown in Fig. 4A, following incubation with 3, 9 or 15 μM SSd for 30 min, the ROS levels in the SSd-treated group remained almost unchanged compared with those in the control group.

As depolarization of the MMP is a characteristic feature of apoptosis (26,27), the MMP in DU145 cells was subsequently determined using Rho-123 staining and flow cytometry assay. The DU145 cells were exposed to different concentrations of SSd (3, 9 and 15 μM) for 12 h prior to Rho-123 staining. The results showed that SSd reduced the MMP in a concentration-dependent manner, from 98.15±1.84 (in the DMSO control group) to 93.17±3.91, 78.01±5.87 and 22.21±3.41%, respectively (Fig. 4B).

Disruption of the MMP may lead to the release of cytochrome c from the intermembrane space to the cytosol, which leads to the activation of procaspase-3 (28–30). To further define the apoptotic pathway, the levels of cytosolic cytochrome c and activated caspase-3 in DU145 cells were determined. The western blotting results indicated that SSd increased the cytochrome c levels in the cytosol and induced cleavage of caspase-3 (Fig. 5).

The Bcl-2 family proteins regulate apoptosis by controlling mitochondrial membrane stability (31). The Bcl-2 family contains anti-apoptotic, e.g. Bcl-2-associated agonist of cell death (Bad), BH3 interacting domain death agonist (Bid), Bax and Bcl-2-like 11 (apoptosis facilitator) (Bim), and pro-apoptotic, e.g. Bcl-2 and B-cell lymphoma-extra large (Bcl-xL), proteins, and the ratios of anti-apoptotic and pro-apoptotic proteins determine the fate of cells (32,33). The present study investigated the effect of SSd on the expression levels of Bcl-2 and Bax. As shown in Fig. 6, SSd induced elevated levels of Bax, while it reduced levels of Bcl-2 in the DU145 cells compared with those in the control cells.