Antibodies against DUSP6, matrix metallopeptidase 3 (MMP-3) and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The cell lines (RWPE-1, PC-3, LNCap and DU-145) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RWPE-1 cells (a normal prostate cell line) were maintained in keratinocyte serum-free medium containing 10% fetal bovine serum (FBS). Prostate cancer cells (PC-3, LNCap and DU-145) were cultured in RPMI-1640 medium supplemented with 10% FBS. All cells were cultured at 37°C within a humidified atmosphere containing 5% CO₂.

To generate DU-145 cells overexpressing DUSP6, the cells were transfected with a pcDNA 3.1-DUSP6 vector that was designed and purchased from Sangon Biotech (Shanghai, China). DU-145 cells transfected with an empty pcDNA 3.1 vector were used as a negative control. To silence the expression of DUSP6 in LNCap cells, the cells were transfected with DUSP6 short hairpin RNA (shRNA) vector, purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells transfected with a scrambled-sequence shRNA vector were used as a negative control. G418 (Gibco-BRL, Grand Island, NY, USA) was used to isolate the stable cells.

Total RNA was isolated using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was then obtained through reverse transcription using a cDNA Synthesis kit (Promega Corp., Madison, WI, USA). qPCR was performed using the primers listed in Table I. The qPCR cycling conditions comprised 10 min at 95°C, and 40 cycles of 15 sec at 95°C and 1 min at 60°C. The expression levels of the examined genes were normalized to β-actin and the 2^−ΔΔCt method was used to determine relative quantification (12).

Cells were lysed in radioimmunoprecipitation assay buffer with protease inhibitors (Roche, Mannheim, Germany). A bicinchoninic acid protein assay (Applygen Technologies, Inc., Beijing, China) was then used to determine the protein concentration of each sample. Equal amounts of protein were subsequently separated by SDS-PAGE and the gel was transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% milk and probed for DUSP6 (1:500), MMP-3 (1:500) and β-actin (1:1,000). The membrane was then further incubated with the secondary antibody (Sigma, St. Louis, MO, USA) for 1 h at room temperature. The immunoreactive bands were exposed to a film and quantified by Quantity One software (Bio-Rad).

To detect the expression level of interleukin 8 (IL-8) secreted by the prostate cancer cells, the cell supernatant was collected and an IL-8 ELISA kit (Boster Biological Technology Co., Ltd., Wuhan, China) was used to measure the protein level of IL-8. Samples were assayed in duplicate. The optical density values were read at 450 nm using a microtiter plate reader within 30 min of performing the reaction, and readings were compared with a standard curve. The concentration of IL-8 protein was normalized to the total protein levels.

A 24-well Transwell plate (Costar, Corning, NY, USA) was used for the invasion and migration assays. For the invasion assay, cells were harvested and resuspended in RPMI-1640 medium supplemented with 0.1% bovine serum albumin. The upper chambers were coated with Matrigel™ (BD Biosciences, Franklin Lakes, NJ, USA) and 100,000 cells were placed in the upper chambers. The lower chambers were filled with 600 μl RPMI-1640 medium supplemented with 20% FBS, and cells were allowed to invade for 24 h. The cells on the lower surface of the membranes were fixed with 4% formaldehyde and stained with crystal violet. The number of invaded cells in seven fields was observed and counted under a microscope at a magnification of ×200.

For the migration assay, cells were resuspended at a density of 5.0×10⁵ cells/ml. Cells in 100 μl RPMI-1640 were placed on the top of the chambers. RPMI-1640 supplemented with 20% FBS was added to the lower chambers. The cells were incubated for 24 h at 37°C. Following staining with crystal violet, the number of migrated cells was counted and determined from seven representative fields.

For the growth assay, cells were seeded in a 24-well culture plate at a density of 1.0×10⁴ cells/well. The cells were harvested and counted using a hemocytometer stained with trypan blue on the day indicated. For the MTT assay, cells were seeded in a 96-well culture plate at an initial density of 0.5×10³ cells/well. On the day indicated, MTT was added to the wells. Four hours later, the medium was removed and dimethyl sulfoxide was added. Following agitation for 15 min in the dark, the absorbance was measured at 490 nm.

Dulbecco’s Modified Eagle’s Medium (DMEM) containing 0.6% agarose was added to a six-well culture plate on the bottom layer. Cells (0.8×10³ cells/well) mixed with DMEM containing 0.3% agarose were plated on top to form an upper layer. Cells were plated in triplicate and colony numbers were counted after 14 days.

Male four-week-old BALB/c nude mice (Vital River Laboratories, Beijing, China) were maintained in pathogen-free conditions. All experimental protocols were approved by the Animal Care and Use Committee of Zhengzhou University (Zhengzhou, China). Each mouse was injected subcutaneously with 3.0 × 10⁵ DU-145 Vector or DU-145 pcDUSP6 cells (n=6 for each group). The length (L) and width (W) of the tumors in the mice were measured using calipers each week and the tumor volumes were estimated with the following formula: 0.52 × L × W². After eight weeks the mice were sacrificed, the mean tumor weight was determined and the lungs and livers were dissected and fixed in 4% paraformaldehyde. The tissues were sectioned into slices and stained with hematoxylin and eosin. Each section was observed by microscopy.

Data are presented as the mean ± standard deviation. The data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). The Student’s t test was used to compare two means, whilst nonparametric analysis of variance was performed to compare multiple means. P<0.05 was considered to indicate a statistically significant difference.

To determine the expression of DUSP6, qPCR and western blotting were performed in a normal prostate cell line (RWPE-1) and three prostate cancer cell lines (LNCap, PC-3 and DU-145). The results showed that DUSP6 was significantly expressed in the RWPE-1 cells, whereas the expression of DUSP6 was low in the analyzed prostate cancer cells (Fig. 1A and B).

To investigate the role of DUSP6 in prostate cancer cells, a cell clone stably overexpressing DUSP6 was isolated from DU-145 cells (pcDUSP6) and a cell clone transfected with an empty vector was used as a negative control (Vector). In the LNCap cells, a DUSP6 shRNA vector was used to stably silence the expression of DUSP6 (shDUSP6) and a scrambled-sequence shRNA vector was used as negative control (shVector). The cells stably expressing DUSP6 were analyzed by western blotting for protein expression levels (Fig. 2A) and were used for in vitro invasion and migration assays (Fig. 2B–D). The results showed that the overexpression of DUSP6 significantly suppressed the invasion and migration of DU-145 cells (Fig. 2B and D). Furthermore, as compared with the LNCap shVector cells, LNCap shDUSP6 cells showed increased invasion and migration abilities (Fig. 2C and E). These data indicated that DUSP6 can suppress the invasion and migration of prostate cancer cells in vitro.

The role of DUSP6 in the growth of prostate cancer cells was next evaluated. Using growth and MTT assays, it was found that the overexpression of DUSP6 suppressed the growth of DU-145 cells, whereas DUSP6-knockdown promoted the growth of LNCap cells (Fig. 3A–D). In addition, a soft agar assay was performed to assess the anchorage-independent growth of prostate cancer cells. The results demonstrated that the overexpression of DUSP6 decreased the number of colonies of DU-145 cells, whereas knockdown of DUSP6 increased the number of colonies of LNCap cells (Fig. 3E and F). These data suggested that DUSP6 is involved in the negative regulation of prostate cancer cell growth in vitro.

To explore the functional role of DUSP6 in growth and metastasis in vivo, nude mice were injected subcutaneously with DU-145 Vector cells or DU-145 pcDUSP6 cells. The tumor volume was measured once per week. The data showed that DU-145 pcDUSP6 tumors exhibited a lower growth rate compared with DU-145 Vector tumors (Fig. 4A). After eight weeks, the mice were sacrificed and the mean tumor weight was assessed. The results showed that the mean tumor weight was 5.93±1.20 g for the DU-145 Vector tumors and 3.50±0.66 g for the DU-145 pcDUSP6 tumors (P<0.05) (Fig. 4B). These data indicated that the overexpression of DUSP6 can suppress the growth of prostate cancer cells in vivo. Furthermore, the liver metastasis rate of mice injected with DU-145 Vector cells was 100% (6/6), whilst that of mice injected with DU-145 pcDUSP6 cells was 16.7% (1/6) (Fig. 4C and D), indicating that DUSP6 can suppress the metastasis of prostate cancer cells in vivo.

MMPs are crucial factors in the invasion and metastasis of prostate cancer cells (13). This study revealed that the overexpression of DUSP6 inhibited MMP-3 expression in DU-145 cells, whereas DUSP6-knockdown enhanced MMP-3 expression in LNCap cells (Fig. 5A). A consistent result was observed by examining the protein level of MMP-3 using western blotting (Fig. 5B). IL-8 has been suggested to be associated with cell invasion and proliferation in prostate cancer (14,15). Using qPCR and ELISA, it was found that the overexpression of DUSP6 suppressed the expression of IL-8 in DU-145 cells, whereas DUSP6-knockdown enhanced the level of IL-8 in LNCap cells (Fig. 5C and D). These results suggested that DUSP6 can downregulate the expression of MMP-3 and IL-8 in prostate cancer cells.