Prostate cancer cell lines PC-3, DU145 and LNCaP, the immortalized normal prostate stroma cell line WPMY-1, the immortalized prostate epithelium cell line RWPE-1 and the 293T cells used for lentiviral packaging were obtained from American Type Culture Collection (Manassas, VA, USA). Prostate cancer cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1:100 penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), whereas WPMY-1 and RWPE-1 cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1:100 penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37°C. The medium supplemented with FBS and penicillin/streptomycin was considered as complete medium (CM).

To overexpress CXCL5 in prostate cells, a pReceiver-Lv203 CXCL5 expression vector (cat. no. EX-A1113-Lv203) and a pReceiver-Lv203 empty vector (cat. no. EX-NEG-Lv203) were constructed by GeneCopoeia Inc. (Rockville, MD, USA), and a Lenti-Pac HIV Expression Packaging kit (cat. no. HPK-LvTR-40; GeneCopoeia, Inc. Rockville, MD, USA) was used to produce CXCL5-carrying lentivirus overexpression vector (CXCL5-OE) and mock lentivirus empty vector (mock), according to the manufacturer's instructions. The 293T lenti-viral packaging cells were transfected using the transfection reagent provided in the Lenti-Pac HIV Expression Packaging kit, following the manufacturer's protocol. Briefly, 293T cells (1.5×10⁶ cells) were plated in a 10-mm dish in 10 ml of DMEM supplemented with 10% heat-inactivated FBS. Following incubation at 37°C for 48 h, DNA-EndoFectin Lenti complex was directly added to the dish and the cells were incubated at 37°C for 12 h, after which the culture medium that contains the DNA-EndoFectin Lenti complex was replaced with fresh DMEM supplemented with 5% heat-inactivated FBS. A 1/500 volume of the TiterBoost reagent was added to the culture medium and continued incubation at 37°C for 48 h. The lentiviral particle-containing culture medium was collected and centrifuged at room temperature at 1,000 × g for 10 min. Subsequently, PC-3, DU145, or WPMY-1 cells (2.5×10⁵ cells/well) were incubated in 6-well plates in 1 ml RPMI-1640 or DMEM CM containing lentiviral particle-containing culture medium at a ratio of 1:1 at 37°C for 48 h. To select the stable gene-transfected cells, these cell lines were further cultured and selected by culturing with puromycin (50 ng/ml, Invitrogen; Thermo Fisher Scientific, Inc.) in RPMI-1640 or DMEM CM. The stable CXCL5-OE-transfected cells and mock-transfected cells were isolated and expanded, and CXCL5 expression was confirmed by ELISA. These isolated cells were used for the subsequent experiments.

Total RNA was isolated from 2×10⁷ cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the Quant Reverse Transcriptase (Tiangen Biotech, Beijing, China), both according to the manufacturers' protocol. cDNA was amplified by PCR to determine the mRNA expression levels of CXCL5 and its receptor CXCR2, as well as the expression levels of BAX, NDRG3, ERK, and β-actin. The primers and PCR thermocycling conditions are provided in Table I and II, respectively. PCR products were visualized by electrophoresis in 1.2% agarose gels stained with ethidium bromide. Relative mRNA expression levels were normalized to β-actin mRNA using Quantity One Software, version 4.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were repeated in triplicate.

Cells (1.5×10⁵ cells/well) were seeded into 6-well plates with 1 ml cell culture medium containing 0.5% FBS and incubated at 37°C for 48 h. Cell culture media were collected and the expression levels of CXCL5 protein were measured using a Human CXCL5/LIX/ENA-78 PicoKine ELISA kit (cat. no. EK0728; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Briefly, 96-well ELISA plates were coated with 100 μl of human recombinant CXCL5 protein from the kit at concentrations of 4,000, 2,000, 1,000, 500, 250, 125, 62.5, 31.2, and 0 pg/ml to create a standard curve. A total of 100 μl of the media from the different cell cultures was added to each well and the plates were incubated at 37°C for 90 min. Subsequently, the plates were washed with phosphate-buffered saline (PBS) and incubated with 100 μl anti-human CXCL5 antibody working liquid (provided in the kit) for 1 h at room temperature. The plates were then washed with PBS again and incubated with ABC working solution at 37°C for 30 min. Following the addition of 100 μl 3,3′,5,5′-tetramethylbenzidine color solution to each well, optical density was immediately detected at 450 nm using a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) and the levels of CXCL5 in the media were quantified according to the standard curve. The experiments were repeated at least three times.

Total protein was extracted from cells (3×10⁶) or 20 mg of xenografted tissues using Pierce Lysis Buffer (Thermo Fisher Scientific, Inc.) on ice for 30 min and centrifuged at 14,000 × g for 15 min at 4°C. Protein concentrations in the supernatant were determined using the Bicinchoninic Acid Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). A total of 30 μg of each sample was separated by 12% SDS-PAGE and transferred onto poly-vinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk in PBS for 2 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-BAX (1:1,000; cat. no. TA890001; OriGene Technologies, Inc., Beijing, China), goat polyclonal anti-NDRG3 (1:200; cat. no. sc-19470; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-extracellular signal-regulated kinase 1/2 (ERK1/2; 1:1,000; cat. no. AF0155; Affinity Biosciences, Inc., Cincinnati, OH, USA), rabbit polyclonal anti-CXCR2 (1:500; cat. no. sc-30008; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-IL-18 (1:500; cat. no. sc-7954; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Bcl-2 (1:500; cat. no. sc-492; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-caspase-3 (1:500; cat. no. sc-7148; Santa Cruz Biotechnology, Inc.), and mouse monoclonal anti-β-actin (1:1,000; cat. no. TA811000; OriGene Technologies, Inc.). The membranes were subsequently washed with PBS containing 0.05% Tween-20 and incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit, rabbit anti-goat or goat anti-mouse secondary antibodies (1:10,000; cat. nos. BA1054, BA1060, and BA1050, respectively; Wuhan Boster Biological Technology, Ltd.) at 37°C for 30 min. Immunoreactive protein bands were detected using the Pierce Enhanced Chemiluminescence solution (Thermo Fisher Scientific, Inc.) and the intensity of each target band was normalized to β-actin and quantified using ImageJ software (version 1.45s; National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least three times.

Prostate tissue samples were collected from patients (age, 56–72 years) with prostate cancer (n=13), with benign prostate hyperplasia (BPH; n=3), with cystic dilatation prostate (n=3), and with normal prostate (n=3). Of the 13 patients with prostate cancer, 1 had Gleason score 4 tumor, 2 had Gleason score 5, 1 had Gleason score 6, 2 had Gleason score 7, 3 had Gleason score 8, 3 had Gleason score 9, 1 had Gleason score 10, and of the 13 cases, 7 had metastatic tumors. All patients had undergone surgical treatment at The Affiliated Hospital of Jiamusi University (Jiamusi, China) between June 2013 and June 2015. This study was approved by the Ethics Committee of Jiamusi University (approval no. JMSU-195) and written informed consent was provided by all patients prior to enrolment.

A tissue microarray (TMA) comprising 50 prostate cancer, 20 BPH, and 10 normal prostate tissue samples was purchased from Alenabio (Xi'an, China). Among the 50 cancer tissues, patients were aged between 20 and 87 years and had Gleason scores between 4 and 10 (4 with Gleason score 4; 7 with Gleason score 5; 9 with Gleason score 6; 8 with Gleason score 7; 2 with Gleason score 8; 9 with Gleason score 9; 11 with Gleason score 10) and of the 50 cases, 19 with metastatic tumors).

Tissue samples were fixed in 10% buffered formalin at room temperature for 24 h, embedded in paraffin and sectioned into 5 μm-thick sections. These and the TMA (5 μm) sections were deparaffinized in xylene at room temperature and rehydrated in a descending ethanol series. The sections were subjected to antigen retrieval in a high-pressure cooker with 0.01 M citric acid buffer for 3 min, and endogenous peroxidase activity was quenched by incubating the sections in 3% H₂O₂ in PBS for 30 min at room temperature. The sections were blocked with 20% normal goat serum (cat. no. AR0009; Wuhan Boster Biological Technology, Ltd.) at 37°C for 1 h, followed by incubation with a mouse monoclonal anti-CXCL5 antibody (1:50; cat. no. sc-73931; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following this incubation, the sections were washed with PBS and incubated with a HRP-conjugated secondary goat anti-mouse immunoglobulin G (1:200; cat. no. BA1050; Wuhan Boster Biological Technology, Ltd.) for 30 min at the room temperature. Subsequently, the sections were washed with PBS and incubated with the Avidin-Biotin Enzyme Complex (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at room temperature, followed by incubation with 3,3′-diaminobenzidine solution at the room temperature for 5 min for colorimetric determination of protein expression, and then counterstained with hematoxylin at room temperature for 15 sec. For quantification, Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used to determine the average optical density and relative area of total CXCL5 expression in each tissue section within three random fields at ×400 magnification under an Olympus BX-60 Epifluorescence microscope (Olympus Corporation, Tokyo, Japan).

To determine the effects of exogenous, autocrine and paracrine CXCL5 expression on the proliferation of prostate cancer cells, the following three experimental treatments were conducted: i) Exogenous assay, in which PC-3 and DU145 cells were seeded (2×10³ cells/well) into 96-well plates and incubated in CM overnight at 37°C in a 5% CO₂ incubator; following removal of the culture media, 100 μl of CM containing different concentrations of recombinant CXCL5 (0, 10, 20, 40 or 60 ng/ml; PeproTech, Inc.) was added to each well; ii) autocrine assay, in which the untransfected parental, mock-transfected, and CXCL5-OE-transfected PC-3 and DU145 cells were seeded (2×10³ cells/well) into 96-well plates and incubated in 100 μl of CM; and iii) paracrine assay, in which parental, mock-transfected and CXCL5-OE-transfected WPMY-1 cells were seeded (5×10⁵ cells/well) into 100 mm cell culture dishes and incubated in CM at 37°C for 48 h. The WPMY-1 CM was collected and subsequently added to PC-3 and DU145 cell cultures (containing 2×10³ cells/well in 96-well plates) at different dilutions (20, 50, 80 or 100%). The three groups of cells were incubated at 37°C in a 5% CO₂ incubator for 72 h without any subsequent medium change. Cell proliferation was then determined by using the Cell Counting Kit-8 (CCK-8; Boster Biotechnology), following the manufacturer's protocol. Briefly, 10 μl CCK-8 reagent was added into each well and cells were incubated at 37°C for 1 h. The optical density was measured at 450 nm using a spectrophotometer (BioTek Instruments, Inc.). Each assay was repeated at least thrice.

PC-3 and DU145 cells were seeded (100 cells/well) into 24-well plates and incubated with CM containing 60 ng/ml (for PC-3 cells) or 20 ng/ml (for DU145 cells) CXCL5 for 12 days (according to pre-test experiments; data not shown) at 37°C with 5% CO₂ until the colonies were visible. The aforementioned CXCL5-containing medium was replaced every 3 days. The colonies were stained with 0.01% crystal violet solution at room temperature for 15 min and counted under an Olympus BX-60 Epifluorescence microscope BX-60 (Olympus Corporation).

The Transwell migration assay was performed using 24-well chambers with polycarbonate membrane filters (pore size, 8 μm; Corning, Inc., Corning, NY, USA), which was modified from our previously described protocol (16). The effects of exogenous, autocrine, and paracrine CXCL5 exposure on the migratory capcity of prostate cancer cells was determined as follows: i) Exogenous assay, in which PC-3 cells (2×10⁴ cells/well) or DU145 cells (5×10³ cells/well) were seeded into the upper Transwell chamber with serum-free RPMI-1640 medium; 600 μl RPMI-1640 medium with 10% FBS and various concentrations of CXCL5 (0, 10, 20, 40, or 60 ng/ml) was added into the lower chamber; ii) Autocrine assay, in which parental, mock-transfected, and CXCL5-OE-transfected PC-3 cells (2×10⁴ cells/well) or DU145 cells (5×10³ cells/well) were seeded into the upper chamber with serum-free RPMI-1640 medium; 600 μl RPMI-1640 medium with 10% FBS was added into the lower chamber; and iii) Paracrine assay, in which PC-3 cells (2×10⁴ cells/well) or DU145 cells (5×10³ cells/well) were seeded into the upper Transwell chamber containing serum-free RPMI-1640 medium; 600 μl of the conditional medium prepared by dissolving different ratios of culture media from parental, mock-transfected, and CXCL5-OE-transfected WPMY-1 cells (20, 50, 80 or 100%) in DMEM containing 10% FBS was added into the lower chamber. In addition, a co-culture system was used in the paracrine assay, in which PC-3 cells (2×10⁴ cells/well) or DU145 cells (5×10³ cells/well) were seeded into the upper chamber containing RPMI-1640 medium, with parental, mock-transfected, or CXCL5-OE-transfected WPMY-1 cells (3×10⁴ cells/well) were cultured in the lower chamber containing 600 μl of DMEM containing 10% FBS. For all experimental groups, cells were incubated at 37°C in a 5% CO₂ incubator for 48 h; subsequently, the upper surfaces of the membranes were scraped with a cotton swab to remove the non-migrating cells, and the filters were washed with PBS, fixed and stained with 95% ethanol containing 0.5% crystal violet solution at room temperature for 30 min. The filters were then washed with PBS and the migrating cells were counted in five random fields using a bright field microscope at ×50 magnification.

Male nude mice (nu/nu; age, 5 weeks; weight 13–16 g; n=7) were purchased from Charles River Laboratories International, Inc. (Beijing, China). The mice were housed in cages under laminar-flow HEPA-filtered hoods at 25°C with a 12-h light/dark cycle and were provide with sterile rodent chow and sterile water ad libitum. Mock and CXCL5-overexpressing PC-3 cells were cultured and harvested at the exponential growth phase, washed with PBS, and resuspended in serum-free RPMI-1640 medium. The trypan blue exclusion assay was used to ensure that cell viability was >99% prior to injection. PC-3 cells (1×10⁶ cells in 100 μl culture medium) were subcutaneously injected into male athymic mice; each mouse was injected with CXCL5-overexpressing cells into one side of the flank, and the mock control cells were injected into the other side for simultaneous comparison. The animals were sacrificed with CO₂ at week 8 post-injetion and the tumor cell xenografts were harvested for analysis and comparison. The experiments were repeated three times.

All data are presented as the mean ± standard deviation. Comparisons between two groups were analyzed using Student's t-test, and comparison among multiple groups was analyzed by analysis of variance followed by Student-Newman-Keuls post hoc test or by Kruskal-Wallis test with a Nemenyi test pairwise comparison post hoc test. All statistical analyses were performed using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered statistically significant.

The expression levels of CXCL5 mRNA were examined by RT-PCR in the prostate cancer cell lines PC-3, DU145 and LNCaP, in the normal prostate stroma cell line WPMY-1, and in the normal prostate epithelium cell line RWPE-1. The results demonstrated that CXCL5 mRNA expression was high in PC-3, DU145, WPMY-1, and RWPE-1 cells and expression was low in LNCaP cells (Fig. 1A). ELISA was used to determine whether CXCL5 protein was produced and secreted by these cell lines, and the results revealed that each of these cell lines secreted CXCL5 protein (Fig. 1B), which suggested that CXCL5 may function through the autocrine and paracrine systems in prostate cells. In addition, a significantly increased level of CXCL5 secretion was observed in CXCL5-overexpressing PC-3, DU145, and WPMY-1 cells compared with their respective parental and mock controls (Fig. 1B), which may lay the foundation for subsequent autocrine and paracrine assays. Our previous study demonstrated a high expression level of CXCR2 protein in DU145, LNCaP, and RWPE-1 cells and low CXCR2 expression in PC-3 and WPMY-1 cells (16), which suggested that CXCL5 and CXCR2 expression may lack specificity in prostate cells.

Furthermore, CXCL5 protein expression levels were also examined in tissue specimens from patients with prostate cancer, BPH or cystic dilatation prostate, as well as in normal prostate tissue. CXCL5 expression was significantly higher in prostate cancer tissues (Fig. 2A–E; Table III), compared with compared with expression in the non-cancerous tissue specimens, including cystic dilatation prostate tissues (Fig. 2F), normal prostate tissues (Fig. 2G–H), and BPH tissues (Fig. 2I). The average optical density and relative area data indicated that the levels of CXCL5 protein expression in both non-metastatic cancer tissues (non-METs) and METs were significantly higher compared with non-prostate cancer tissues (non-PCa), which indicated that the increased expression of CXCL5 mayb be associated with prostate cancer development and progression (Table III). However, CXCL5 expression was not associated with Gleason scores or age of the patients (data not shown).

Owing to the tendency of LNCaP cells to form colonies in culture instead of a cell monolayer, these cells were not used in subsequent experiments. Tumor cell proliferation, colony formation and migratory ability were assessed in PC-3 and DU145 cell lines following treatment with various concentrations of exogenous recombinant CXCL5 protein (0, 10, 20, 40 and 60 ng/ml). A significant increase in proliferation was observed following treatment with 60 ng/ml (PC-3 cells) or with 10 and 20 ng/ml (DU145 cells) of exogenous CXCL5 (Fig. 3A), although DU145 cells exposed to higher concentrations of exogenous CXCL5 (for example, 60 ng/ml) had a significant decrease in proliferation compared with untreated control cells. As the 60 and 10–20 ng/ml doses of exogenous CXCL5 were indicated as the optimal concentrations for regulation of PC-3 and DU145 cell proliferation, respectively, 60 ng/ml was used for PC-3 and 20 ng/ml for DU145 cells in the subsequent colony formation assays; the treated PC-3 and DU145 cells exhibited a notable increase in colony formation (Fig. 3B and Table IV).

Transwell migration assays were conducted on PC-3 and DU145 cells following treatment with various concentrations of exogenous CXCL5 (0, 10, 20, 40 and 60 ng/ml). The results demonstrated that CXCL5 at a dose of 20 or 40 ng/ml for PC-3 cells or 10, 20 and 40 ng/ml for DU145 cells significantly promoted tumor cell migration capacity compared with respective cells treated with 0 ng/ml of CXCL5 (Fig. 3C and D).

Results from CCK-8 assays demonstrated that the proliferation of CXCL5-overexpressing PC-3 and DU145 cells was increased compared with that of parental or mock control cells (Fig. 4A). CXCL5 overexpression also significantly increased the migratory capacity of PC-3 and DU145 cells compared with that of parental and mock cells (Fig. 4B and C).

Furthermore, a stable CXCL5-overexpressing WPMY-1 prostate stromal cell line was established and various concentrations (20, 50, 80 and 100%) of the conditional medium from parental, mock or CXCL5-overexpressing WPMY-1 cells was subsequently prepared and added to the PC-3 and DU145 cell cultures. PC-3 cells cultured with 100% and DU145 cells cultured with either 80 or 100% CXCL5-overexpressing WPMY-1 conditioned medium exhibited a significant increase in tumor cell proliferation compared with the proliferation of cells cultured with similar concentrations of conditional medium from parental or mock WPMY-1 cells (Fig. 5A); no significant differences in tumor cell proliferation were identified for cells culutred with 20, 50 or 80% of the WPMY-1 CM. Similar phenomena were also observed in prostate cancer cell migration assays; for example, PC-3 and DU145 cells cultured with 100% CXCL5-overexpressing WPMY-1 conditional medium exhibited a significant increase in migration capacity compared with cells cultured with similar concentrations of conditioned medium from parental or mock WPMY-1 cells (Fig. 5B and C). In addition, PC-3 and DU145 cells that were co-cultured with CXCL5-overexpressing WPMY-1 cells exhibited an increase in number of migrating cells compared with the number of migrating tumor cells co-cultured with parental or mock WPMY-1 cells (Fig. 5D and E).

To explore the underlying gene expression of the CXCL5-mediated prostate cancer cell malignat pheotypes, the mRNA and protein expression levels of known tumor-related genes were examined. The results demonstrated that the expression levels of NDRG3, ERK1/2, and CXCR2 mRNA and protein were significantly increased, whereas the expression levels of BAX mRNA and protein were significantly reduced in CXCL5-overexpressing PC-3 cells compared with those of parental and mock control cells (Fig. 6A–D).

To assess the in vivo effects of CXCL5 overexpression on prostate cancer cells, a nude mouse xenograft assay was performed by subcutaneous injection of CXCL5-overexpressing and mock-treated PC-3 cells into nude mice. Eight weeks following tumor cell injection, tumor xenografts were resected from the mice; weight of tumor cell xenografts was significantly higher in mice implanted with CXCL5-overexpressing PC-3 cells compared with those implanted with mock PC-3 cells (Fig. 7A and B). Western blot analysis of the tumor xenografts revealed that the levels of IL-18 and Bcl-2 proteins were increased, whereas caspase-3 levels were decreased in CXCL5-overexpressing PC-3 xenografts compared with mock PC-3 xenografts (Fig. 7C and D).