PC-3 human prostate carcinoma cells were initially stored frozen in our laboratory (Department of Orthopedics, Tonji Medical College, Wuhan, China) prior to cultivation in RPMI-1640 medium (Hyclone Laboratories, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA), 1×10⁵ U/l penicillin and streptomycin (Hyclone Laboratories, Inc.) in an incubator at 37°C with 5% CO₂.

Cell motility was assessed using an in vitro wound healing assay. PC-3 cells were plated in a six-well plate and grown until confluent. Monolayers of confluent PC-3 cells were then scarred, and the repair was monitored using an inverted microscope (Nikon TE200-S; Nikon Corporation, Tokyo, Japan), following 12 h pretreatment with 10 μM RSG (Sigma Aldrich, St. Louis, MO, USA) then 24 h exposure to 100 ng/ml CXCL12 (R&D Systems Inc., Minneapolis, MN, USA). Wound width was measured at 0 and 24 h following the start of incubation with or without RSG and in the absence or presence of CXCL12. Experiments were performed in triplicate and data are presented as the mean ± standard deviation.

The invasive potential of PC-3 cells was quantified using a Matrigel™-coated Transwell system, as described previously (10). The chamber (Corning Inc., Corning, NY, USA) contained a polycarbonate membrane filter with a pore size of 8 μm, which was coated with Matrigel and inserted into a 24-well culture plate. PC-3 cells were then seeded in the top chamber of the Matrigel. Following pre-incubation with or without RSG (10 μM) for 12 h, Transwell chambers were then placed into the wells of a 24 well plate, in which either basal medium or basal medium supplemented with 100 ng/ml CXCL12 was added for 24 h. After 48 h incubation, PC-3 cells on the upper surface of the filters were removed using cotton swabs. Cells that had invaded the lower surface of the membrane were fixed using methanol and stained with crystal violet. Each experiment was performed in triplicate and cells were counted in five fields in each well using light microscopy. The invasive ability of PC-3 cells was assessed relative to the invasive ability of the untreated control cells.

Cells were grown on six-well culture plates, washed with 1X phosphate-buffered saline (PBS) and harvested in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin and 1 mM phenylmethanesulfonyl fluoride (Cell Signaling Technology Inc., Beverly, MA, USA). The cell extractions were collected and centrifuged at 10,000 × g for 10 min at 4°C before the supernatants were collected as cell lysates. Equal concentrations of total cell lysate were resolved using 10% SDS-PAGE (Boster, Wuhan, China) and transferred to a polyvinylidene fluoride (PVDF) transfer membrane (Merck Millipore, Billerica, MA, USA). Non-specific binding sites were blocked using 5% non-fat dry milk in 1X Tris-buffered saline containing 0.1% Tween 20 (TBST; Boster), followed by incubation with primary antibodies against the proteins of interest in 3% bovine serum albumin (BSA; Boster)-TBST [phosphorylated Akt (p-Akt), CXCR4] or 5% non-fat dry milk-TBST (Akt, β-actin). Subsequently, the membranes were incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G; Wuhan Boster Biological Technology Ltd, Wuhan, China). Immunoblots were visualized using SuperSignal™ West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies raised against human Akt, p-Akt (Ser473) and β-actin (rabbit monoclonal IgG) were purchased from Cell Signaling Technology. The antibody raised against human CXCR4 (rabbit polyclonal IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

PC-3 cells were plated in six-well plates for all experiments and allowed to grow for 48 h prior to any treatment. GW9662 (Santa Cruz Biotechnology, Inc.) was added 2 h prior to any other treatment. Total RNA was extracted using TRIzol^® reagent according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). A total of 1 μg RNA was reverse-transcribed according to the kit’s instructions (Invitrogen Life Technologies). qPCR was performed using TransStart Green qPCR SuperMix (TransGen Biotech Co., Beijing, China) using an iQ5 Sequence Detection System (Bio-Rad Laboratories Inc., Berkley, CA, USA). Results were normalized to those obtained for GAPDH. Each sample was analyzed in triplicate and the experiment was repeated twice. The theshold cycle (CT) value (the cycle number at which the fluorescence crosses the threshold) was measured and 2^−ΔCT (ΔCT = CT − CT_GAPDH) was defined as the quantity of the amplified fragment. The primer sequences are listed in Table I.

Data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS 11.0 statistics software (SPSS, Inc., Chicago, IL, USA). Statistical significance was analyzed using one-way analysis of variance. Where significance was observed, a Dunnett’s post-hoc test was used to determine the statistical significance of the differences between the treated- and untreated-groups, with a value of P<0.05 considered to indicate a statistically significant difference.

A previous study has shown that CXCR4 expression is significantly higher in human PCa tissue than in hyperplastic prostate tissues (11). This finding suggests that CXCL12 may exhibit an autocrine regulatory role via its receptor, CXCR4, in the regulation of PCa cell migration, invasion and metastasis (12). Therefore, the initial investigations in the present study focused on the effect of RSG on the expression of CXCR4 in PC-3 cells. It has been reported that TZDs activate PPARα and PPARδ receptors at concentrations >10 μM (13); therefore, concentrations of ≤10 μM were utilized in the present investigations. It was observed that when PC-3 cells were incubated with various RSG concentrations for 24 h, or for various time periods with 10 μM RSG, the expression of CXCR4 was suppressed in a dose- and time- dependent manner, respectively (Fig. 1A and B). This downregulation was not due to a decrease in cell viability, as ~90% of PC-3 cells were viable under these conditions (data not shown).

It was hypothesized that the suppression of CXCR4 expression may occur at the transcriptional level. Therefore, following PC-3 cell treatment with RSG for various time-periods, the mRNA was extracted for qPCR analysis. As shown in Fig. 1D, RSG was found to downregulate CXCR4 mRNA expression in a time-dependent manner, with a significant reduction observed from 6 h following exposure. CXCR4 mRNA expression was inhibited at all RSG doses tested, with 22, 27 and 36% inhibition observed following 12 h exposure to 0.1, 1 and 10 μM RSG, respectively (Fig 1C).

Although RSG was found to affect CXCR4 expression in PC-3 cells, the mechanism by which this was achieved was unclear. To determine whether the reduction in expression of CXCR4 by RSG was dependent upon the activation of PPARγ, PC-3 cells were treated with the PPARγ antagonist, GW9662. The downregulation of CXCR4 mRNA and protein expression induced by RSG in PC-3 cells was attenuated upon addition of GW9662 (Fig. 1). These results indicate that RSG is capable of downregulating CXCR4 expression in a manner dependent upon PPARγ activation. In combination, these data suggest that RSG may inhibit CXCR4 expression in PC-3 cells, in a PPARγ-dependent manner.

This study further investigated a potential correlation between RSG-induced downregulation of CXCR4 and PCa cell migration. An in vitro wound healing assay revealed that PC-3 cells migrated more rapidly upon treatment with CXCL12 and that this effect was abolished upon treatment with RSG (Fig. 2).

To elucidate the inhibitory effect of RSG upon CXCL12-induced invasion in PC-3 cells, chamber invasion assays were performed. RSG was observed to suppress the invasion of PC-3 cells across the Matrigel-coated filter in a dose-dependent manner, with 10 μM RSG found to inhibit 52% of cell invasion. RSG was also observed to suppress the CXCL12-induced invasion of the PC-3 cells (Fig. 3). These results indicate that RSG markedly inhibits the CXCL12-induced invasion of PC-3 cells.

It has previously been indicated that the signaling proteins, PI3K and Akt, may be associated with the expression of matrix-metalloproteinases (MMPs) and metastasis induction (14). To further elucidate the signal transduction pathways responsible for CXCR4 expression and PC-3 cell migration and invasion, the activation of Akt, a signaling component of pathways coupled to G-protein-coupled chemoattractant receptors, was examined. Upon rapid stimulation (30 min) of PC-3 cells with 100 ng/ml CXCL12, an increase in Akt phosphorylation at Ser473 was observed, resulting in the activation of the enzyme. Furthermore, it was observed that concomitant addition of 10 μM RSG for 30 min downregulated the CXCL12-induced phosphorylation of Akt in a dose-dependent manner in PC-3 cells (Fig. 4A). This finding suggests that the inhibition of migration and invasion associated with RSG treatment in PC-3 cells may partly occur through the suppression of PI3K pathways.

In addition to the PI3K/Akt pathway, other signaling pathways downstream of the activated CXCR4/CXCL12 axis were investigated. PC-3 cells were treated with various concentrations of RSG for 12 h and the expression of MMP-2, MMP-9 and vascular endothelial growth factor (VEGF) mRNA were then analyzed using qPCR, respectively. As demonstrated in Fig. 4, RSG significantly downregulated MMP-2, MMP-9 and VEGF mRNA levels in a dose-dependent manner.