CCL20 and CXCR4 Interaction Figure 2. Regulation of CCL20 expression and function. (A–B) CXCR4 expression levels of PC3-CXCR4.5 cells 48 hours following the transfection with control non-specific siRNA and specific anti-CXCR4 siRNA. The cells were stained for the control and CXCR4 antibodies and evaluated by FACS. Semi-quantitative RT-PCR analysis of CXCR4 and CCL20 mRNA of the siRNA-transfected PC3-CXCR4.5 cells 48 hours following the transfection. b-actin confirmed comparable loading of RT-PCR products in each lane. Control and CXCR4 siRNA-transfected PC3-CXCR4.5 cells at 48 hours post-transfection were incubated with CXCL12 500 ng/ml for an additional 48 hours. CCL20 secretion was assessed by ELISA. The results represent the average of triplicates6SD (** P,0.05). (C) CCR6 expression in prostate cancer cell lines PC3, LaNCAP, 22Rv1 and DU145 was evaluated by FACS and PCR whereas CCL20 expresion was evaluated by PCR and ELISA assays. Purple line represents mouse IgG control antibody, green line represents staining with CCR6 monoclonal antibody. (D) PC3 and PC3-CXCR4 cells were incubated with various concentration of CCL20 for 6 days. Following 3 days of incubation, the medium with or without CCL20 was renewed. On day 6, the cells were harvested and viable cells were counted using PI staining and FACS analysis. In addition, in order to determine proliferation of PC3 cells, the cells were labeled with BrdU (10 mM) during the last 16 hours of incubation and processed for BrdU detection using specific anti-BrdU FITC-conjugated antibody and FACS analysis. Data is presented as mean6SD from triplicates (** P,0.05). Data is representative of two separate experiments. (E–F) PC3 and PC3-CXCR4.5 cells either untreated or treated with various concentrations of CCL20 were placed on collagen I- or fibronectin-coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). In addition, PC3 cells (E) that demonstrated increased adhesion to collagen I and fibronectin in response to stimulation with CCL20, were co-incubated with CCL20 and PTX (100 ng/ml) and were allowed to adhere to collagen I- and fibronectin-coated plates. doi:10.1371/journal.pone.0005125.g002 CCL20 promotes the growth and adhesion of CCR6- expressing tumor cells in vitro We hypothesized that the CCR6-CCL20 axis may be involved in tumor progression via paracrine and/or autocrine mechanisms. To assess biological behavior resulting from CCL20-mediated activation in PC3 cells, we studied the effect of CCL20 on the growth and survival of PC3 cells in culture. PC3 cells were treated with various concentrations of CCL20. The number of viable cells following six days of incubation was detected using propidium iodide (PI) and FACS analysis. Treatment of cells with CCL20 increased the number of viable PC3 cells in the culture at a concentration of 5 ng/ml (1.35-fold increase, p,0.04) and at a concentration of 50 ng/ml (1.75-fold increase, p,0.0004); where- as at the highest concentration of CCL20, 250 ng/ml, we found no change in the number of viable cells compared to control non- stimulated PC3 cells (Fig. 2D). To further verify these results, CCL20-treated cells were loaded with 5-bromo-2-deoxyuridine (BrdU). Cell proliferation was tested by staining for BrdU incorporation using specific anti-BrdU antibody and FACS analysis. Consistent with previous results, CCL20 induced the incorporation of BrdU in the cells at a concentration of 5 and 50 ng/ml (1.76-fold increase, p,0.0002), whereas treating the cells with higher concentration of CCL20 did not change the level of BrdU incorporation (Fig. 2D). In accordance with the previous PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5125