Androgen receptor (AR)-negative PCa cell lines PC-3 (bone metastatic) and DU145 (brain metastatic) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, San Diego, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂ in a humidified incubator. Androgen-sensitive but independent AR-positive PCa cell line C4-2 (bone metastatic) was maintained in T-medium supplemented with 5% FBS. 2HF was obtained from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). For 2HF treatment, a stock solution (0.02 M in DMSO) was added into the culture medium with 1% FBS to achieve the appropriate concentration (10, 20 or 40 μmol/l) and then incubated with the cells, whereas DMSO solution without 2HF was used as the control.

To evaluate the sensitivities of different prostate cancer cells to 2HF treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) proliferation assays were performed to determine cell viability. Different PCa cells were plated at a density of 2×10³ cells/well in 96-well plates for 24 h and then treated with different concentrations of 2HF (0, 10, 20 and 40 μM) for the indicated time. After the exposure period, 20 μl MTT was added to each well for another 5 h incubation at 37°C in 5% CO₂. Thereafter, the medium containing MTT was removed, and 150 μl DMSO was added to solubilize the formazan crystals. The absorbance (OD) was then measured at a wavelength of 590 nm by a microplate autoreader (Bio-Tek Instruments, Vinooski, VT, USA). Independent experiments were repeated in triplicate.

The colony formation assay was used to determine the clonogenicity capabilities in vitro as described previously (12). The indicated cells were treated with different concentrations of 2HF (0, 10, 20 and 40 μM) for 36 h. Then an equal number of cells were seeded in 6-well plates to form colonies for at least two weeks and fresh medium was changed every 3–4 days. The plates were then washed with ice-cold PBS, fixed with 4% paraformaldehyde, stained in crystal violet solution for 15 min at room temperature and washed with distilled water to remove excess dye. The number of colonies was counted for each sample.

PC-3, DU145 or C4-2 cells (1–3×10⁶) were plated on a 6-cm dish for 24 h, and treated with different concentrations of 2HF (0, 10, 20 and 40 μM) for another 24 h. After that, cells were harvested, washed with PBS and then subjected to Annexin V and PI staining using an Annexin V-FITC apoptosis detection kit (Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions and immediately analyzed by flow cytometry (FACSCalibur, BD Biosciences, NJ, USA).

The indicated cells were treated with 2HF for 36 h and then total cellular protein lysates were prepared with RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate] containing proteinase inhibitors, 1% Cocktail and 1 mmol/l PMSF (Sigma, St. Louis, MO, USA). A total of 20–40 μg of protein was separated by 10–12% SDS-PAGE and transferred to nitrocellulose membranes. Following blocking of the non-specific binding sites on the membranes with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (pH 7.6, TBST) for 1 h at room temperature, the membranes were then incubated with the desired primary antibody overnight at 4°C. Antibodies for western blotting were rabbit anti-PARP (CST-9532; Cell Signaling Technology, Danvers, MA, USA) at a 1:500 dilution, rabbit anti-phosphorylated-AKT (Ser473, CST-4060, 1:1,000), rabbit anti-AKT (CST-9272, 1:1,000), mouse anti-phosphorylated-STAT3 (Tyr705, CST-4113, 1:1,000), rabbit anti-STAT3 (CST-8232, 1:1,000), rabbit anti-Mcl-1 (CST-5453, 1:500), rabbit anti-Bcl-2 (SC-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution, rabbit anti-Bax (SC-493, 1:200) and rabbit anti-caspase-3 (CST-9665, 1:1,000). After being washed with TBST, membranes were incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h and visualized with an ECL chemiluminescent detection system (Pierce, Rockford, IL, USA). Loading differences were normalized using monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody.

For the reporter gene assay, cells seeded in 24-well plates were transfected with 200 ng STAT3-responsive luciferase reporter plasmid pLucTKS3 or the control plasmid pLucTK (13) and 1 ng of the pRL-SV40 Renilla luciferase construct (as an internal control) for 24 h and then subjected to 2HF, PI3K inhibitor LY 294002 or STAT3 inhibitor Stattic treatment. Cell extracts were prepared 36 h after treatment and the luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega) as described previously (14). Relative luciferase activity is represented as mean ± SEM from each sample after normalizing with the control.

Sixteen male BALB/c-nu mice at the age of 3–4 weeks and weighing 15–20 g were purchased from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and were acclimated for one week before beginning the experiment. All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Single cells (5×10⁶) were subcutaneously inoculated into both flanks of the mice. Once the tumors were established (100–150 mm³), the mice were randomly divided into two groups. The mice (eight in each group) received 2HF (100 mg/kg body weight, daily) in 100 μl corn oil by oral gavage once per day. Control groups were treated with corn oil only. Body weight and tumor volume were measured two times per week. Calipers were used to measure the two dimensions of the tumors. Thirty days after treatment, mice were sacrificed by cervical dislocation, and tumors were removed and weighed.

Xenografts were harvested for staining and IHC was carried out with Dako Autostainer Plus system (Dako, Carpinteria, CA, USA) as previously described (14). Briefly, sections were deparaffinized, rehydrated and subjected to antigen retrieval in citrate buffer (10 mM, pH 6.0) for 5 min and then endogenous peroxidase and alkaline phosphatase activities were blocked with Dual Block for 10 min. The slides were then incubated overnight at 4°C with p-AKT (Ser473, CST-4060), p-STAT3 (Tyr705, CST-4113), Bcl-2 (SC-492) and cleaved caspase-3 (CST-9661) antibodies (1:75 dilution). After washing, this was followed by incubation with EnVision secondary antibody for 30 min at room temperature. Signals were detected by adding substrate hydrogen peroxide using diaminobenzidine (DAB) as a chromogen followed by hematoxylin counterstaining.

All assays were repeated in triplicate in three independent experiments and quantitative data are presented as means ± SEM. The differences between two groups were compared by the 2-tailed Student’s t-test. In all cases, a P-value of <0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 18.0 (SPSS Inc, Chicago, IL, USA).

The progression to androgen independence is a multi-factorial process by which cells acquire the ability to both survive in the absence of androgens and proliferate using non-androgenic stimuli for mitogenesis. Therefore, targeting tumor cell growth may still play an important role in CRPC therapy. As shown in Fig. 1B, we initially showed that 2HF inhibited in vitro cell proliferation in a dose-dependent manner in PC-3 cells, which were derived from bone metastatic lesion and are androgen-independent. Furthermore, we performed a colony formation assay to analyze the potential effects of 2HF on the viability of PC-3 cells. Indeed, 2HF significantly inhibited the clonogenicity of PC-3 cells with a decreased number of colonies, in a dose-dependent manner (Fig. 1C). To rule out cell-specific effects, we repeated our MTT and colony formation assays in another PCa cell line, DU145, which was also androgen-independent and brain metastatic. Consistently, 2HF suppressed in vitro cell proliferation and clonogenicity of DU145 cells in a dose-dependent manner (Fig. 1B and C). Moreover, similar results were also observed in another bone metastatic, androgen-independent but androgen receptor (AR)-positive PCa cell line C4-2 (Fig. 1D), since the PC-3 and DU145 cells were AR-negative. All of these findings suggest that 2HF targets androgen-independent CRPC tumor growth in vitro, which does not depend on the status of AR expression.

Apoptosis is one of the important mechanisms by which anticancer agents inhibit the growth of cancer cells. To understand the mechanism by which 2HF inhibits the viability of prostate cancer cells, PC-3 and DU145 cells were exposed to various concentrations of 2HF for 24 h, and the population of apopototic cells after Annexin V/PI staining was determined by flow cytometry. As the representative data show in Fig. 2A, 2HF treatments at 10, 20 and 40 μM resulted in 8.82, 11.85 and 21.5% apoptotic cells in the PC-3 cells, respectively, while the baseline of the control cells was 7.35%. In addition, 2HF treatment at 10, 20 and 40 μM induced 6.42, 12.75 and 27.39% apoptosis in the DU145 cells, respectively, while the baseline of the control cells was 5.45% (Fig. 2B). Moreover, similar results were observed in C4-2 cells after 2HF treatment (Fig. 2C). Correspondingly, 2HF gradually increased the cleavage of poly-PARP, a chromatin-associated enzyme that plays an important role in DNA repair and the recovery of cells from DNA damage, in a dose-dependent manner in both PC-3 and DU145 cells (Fig. 3A and B). Consistent with the results, total caspase-3 proteins decreased accordingly after 2HF treatment, indicating the cleavage of caspase-3. Collectively, these results indicate that the induction of apoptotic cell death by 2HF probably occurs through a caspase-dependent pathway.

To further investigate the mechanisms of 2HF targeting prostate tumor growth, we performed western blot analyses to detect the expression of proteins related to apoptosis. Indeed, 2HF treatment modulated the expression of members of the BCL-2 gene family (15), which include protective proteins involved in the mitochondrial apoptotic pathway. As shown in Fig. 3A, following the treatment of PC-3 cells with 10, 20 and 40 μM 2HF for 48 h, the expression levels of Mcl-1 and Bcl-2 proteins were significantly downregulated in a dose-dependent manner, while Bax protein was gradually upregulated after treatment. Similar results were also observed in the DU145 cells (Fig. 3B).

PTEN deletions and mutations lead to aberrant activation of the PI3K/AKT pathway in prostate cancer (16), which is involved in the regulation of cancer cell growth, motility, survival and metabolism. Thus inactivation or inhibition of PI3K/AKT signaling pathways provide a new substantial strategy to target CRPC using small molecules or plant extracts (17,18). In the present study, we observed that 2HF treatment markedly inhibited the phosphorylation of AKT on the site of serine 473 in both PC-3 and DU145 cells (Fig. 4A and B). 2HF treatment also inhibited the phosphorylation of STAT3 (Fig. 4A and B), another critical signaling transduction protein, which is constitutively activated in prostate cancer by phosphorylation of its tyrosine 705 residue (19). After phosphorylation, STAT3 homodimerizes and translocates to the nucleus where it binds to specific STAT3 response elements of target gene promoters to regulate transcription. Indeed, using a specific STAT3-responsive luciferase reporter (13), we further demonstrated that 2HF suppressed STAT3 transactivation in both PC-3 and DU145 cells based on a luciferase reporter gene assay, which was also dose-dependent (Fig. 4C and D). Therefore, 2HF inhibited AKT/STAT3 signaling and the expression of the BCL-2 family in the prostate cancer cells.

To further dissect the roles of AKT and STAT3 signaling in the inhibitory effects of 2HF on PCa cells, we applied the PI3K inhibitor LY294002 or STAT3 inhibitor Stattic to pretreat PC-3 cells and analyzed the changes in Bcl-2 protein expression and cell apoptosis. As shown in Fig. 5A, western blotting data clearly indicated that LY294002 treatment alone significantly decreased p-AKT (Ser473), p-STAT3 (Tyr705) and Bcl-2 protein expression in the PC-3 cells and it also enhanced the inhibitory effects of 2HF on AKT phosphorylation, STAT3 phosphorylation and Bcl-2 protein expression (Fig. 5A). Consistently, it also increased the inhibitory effects of 2HF on STAT3-responsive luciferase activity (Fig. 5B). Moreover, inhibition of PI3K/AKT also significantly enhanced the induction of PC-3 cell apoptosis after 2HF treatment (Fig. 5C). In contrast, Stattic treatment had no effects on AKT phosphorylation in PC-3 cells (Fig. 5A), indicating that the STAT3 pathway was activated as downstream signaling of PI3K/AKT. However, inhibition of STAT3 activity also decreased Bcl-2 protein expression and increased cell apoptosis (Fig. 5A and C). Moreover, we observed that additional Stattic treatment further decreased Bcl-2 protein expression and increased cell apoptosis of PC-3 cells induced by 2HF treatment (Fig. 5A and C).

We also generated xenograft models to show the inhibitory effects of 2HF on prostate tumor growth in vivo. As shown in Fig. 6A, oral 2HF treatment significantly decreased the tumor burden of subcutaneous PC-3 xenografts (left and middle panel), and the average tumor volume was much smaller (right panel, P<0.05). Furthermore, IHC staining data also supported the observation in the cell lines in vitro. p-AKT (Ser473), p-STAT3 (Tyr705) and Bcl-2 protein expression levels in the subcutaneous PC-3 xenograft tissues with or without 2HF treatment were detected by IHC staining. Consistent with our findings in vitro, higher expression levels of p-AKT (Ser473), p-STAT3 (Tyr705) and Bcl-2 were detected in the PC-3 xenograft samples, indicating hyperactivation of AKT/STAT3/Bcl-2 signaling in metastatic CRPC, while 2HF treatment suppressed the phosphorylation of AKT and STAT3 and potentially inactivated STAT3 transcriptional activity and decreased the expression of its target gene Bcl-2 (Fig. 6B). We also observed that 2HF treatment increased the cleavage of caspase-3 in the PC-3 xenografts using an IHC-specific cleaved-caspase-3 antibody (Fig. 6B), indicating the incidence of cell apoptosis in vivo after 2HF treatment.