results, we found that PC3-CXCR4.5 cells, which constitutively express high levels of CCL20, where inhibited in their growth when treated with increasing concentrations of CCL20 (Fig. 2D). Concentration of 250 ng/ml even decreased the number of PC3- CXCR4.5 viable cells (p,0.006). Adhesion of cancer cells to extracellular matrix (ECM) components is a step that is associated with tumor seeding, invasion and spreading. In order to investigate the effect of CCL20 on PC3 cell adhesion to the ECM proteins, we tested the adhesion of PC3 cells to the fibronectin and collagen I in response to increasing concentrations of CCL20. As shown in Fig. 2E, elevated doses of CCL20 slightly increased the adhesion of cells to fibronectin (500 ng/ml of CCL20 promoted 1.45-fold increase, p,0.01), and significantly increased the adhesion of cells to collagen type I in a dose-dependent manner (Fig. 2E). Upon activation by CCL20 at concentration of 50 ng/ml PC3, adhesion to collagen I was 2.2-fold elevated (p,0.001); concentration of 500 ng/ml caused a 2.09-fold increase in adhesion (p,0.003). Treatment with pertussis toxin (PTX) prevented the CCL20- induced increase in PC3 cell adhesion to fibronectin (p,0.0009) and collagen I (p,0.002) (Fig. 2E). In contrast to PC3 cells , adhesion of CCL20-producing PC3-CXCR4.5 cells to collagen I and fibronectin was slightly decreased following stimulation with increased doses of CCL20 (Fig. 2F). The exposure of chemokine receptors to high concentrations of chemokines often results in the rapid attenuation of receptor responsiveness and a reduced biological response. This process termed ‘desensitization’ is the consequence of a combination of different mechanisms. These mechanisms include receptor phos- phorylation followed by uncoupling of the receptor from heterotrimeric G proteins and internalization of cell surface receptors to intracellular endocytic vesicles [24]. It is therefore possible that at high levels of CCL20, the chemokine receptor CCR6 is desensitized due to internalization and/or phosphoryla- tion [25]. Indeed preliminary data suggest that high levels of CCL20 can stimulate the internalization of CCR6. In addition to PC3 cells, we tested the effect of CCL20 activation on biological function of other human prostate cancer cell lines - LNCaP, 22RV1 and DU145 cells. In vitro proliferation and adhesion of the cells to collagen were tested in the absence or presence of different concentrations of CCL20. In agreement with CCR6 expression pattern, LNCaP, 22RV1 and DU145 cells that do not express CCR6, did not respond to CCL20 stimulation and no increase in proliferation or adhesion was observed (data not shown). We therefore concluded that CCL20 can stimulate both PC3 cell proliferation and adhesion to collagen type I in a dose dependent manner. In order to further determine the involvement of CCL20 in cancer development in vitro, we introduced the CCL20 gene into prostate PC3 cells which express the CCR6 receptor. PC3 cells were stably transfected with vector encoding CCL20, and different clones overexpressing CCL20 were obtained. The levels of CCL20 were quantified using PCR and ELISA assay (Fig. 3A). Clone numbers 7 and 8 demonstrated the highest levels of CCL20 secretion (5,000 and 1,030 pg/ml, respectively) whereas clone numbers 10 and 30 showed moderate levels of CCL20 secretion (100 and 320 pg/ml, respectively). Next, we tested the growth of PC3 clones that overexpress CCL20. The number of viable cells following six days of incubation was detected using PI and FACS analysis. PC3- CCL20 clones that secrete moderate levels of CCL20, PC3- CCL20.10 and PC3-CCL20.30, demonstrated 1.7-fold and 2.5- fold increases in the number of viable cells in culture, respectively (Fig. 3B). The growth of PC3-CCL20 clones that secrete high CCL20 and CXCR4 Interaction levels of CCL20 (PC3-CCL20.7 and PC3-CCL20.8) was similar to parental control PC3 cells (Fig. 3B). This may suggest that autocrine secretion of CCL20 may drive tumor growth. To further explore the effect of CCL20 overexpression on prostate cancer cell behavior, we assessed the adhesion of PC3 clones that overexpress CCL20 to ECM proteins fibronectin and collagen I. CCL20-overexpressing PC3 cells were grown to confluence, harvested, and allowed to adhere to fibronectin or collagen I-coated plates. Over-expression of CCL20 significantly increased the adhesion of all four CCL20-expressing clones to the collagen I. Comparing to mock-transfected PC3 cells, clones 7, 8, 10 and 30 demonstrated 2.1-fold (p,0.04), 3.8-fold (p,0.01), 2.2-fold (p,0.03) and 2.8-fold (p,0.004) increases in their adhesion to collagen I, respectively (Fig. 3C). No significant change was observed in the adhesion of these cells to the fibronectin (data not shown). CXCR4 and CCL20 expression has been described in a variety of human neoplasms, including colorectal, lung, pancreatic and breast human adenocarcinomas, malignant glioma, leukemia, lymphoma and melanoma as well as by normal keratinocytes. We therefore next tested by ELISA the expression of CCL20 in a range of human cancer cell lines. We found that in addition to PC3 cells, CCL20 was secreted by promyelocytic leukemia (APL) cell lines, NB4 and HL60, by primary blasts of patients with acute myelocytic leukemia, human HT-29 colon carcinoma cells as well as and by normal keratinocytes (Fig. 3D). Furthermore, in NB4, HL60, primary AML blasts, HT-29 cells, and normal keratino- cytes, the secretion of CCL20 was increased following stimulation with CXCL12, in a dose-dependent manner (Fig. 3D). Next, we characterized the expression of the CXCR4 receptor on NB4, HL60 cells, AML blasts and HT-29 cells. Leukemic lines NB4 and HL60, primary human AML blasts and HT-29 cells demonstrated high cell-surface expression levels of the CXCR4 receptor (data not shown). These results suggest a more general role for CXCR4 in regulating CCL20 expression in various cancer cells. To confirm the role of CCL20 in autocrine stimulation of cancer cells from a different origin, we tested the expression of CCR6 in CCL20-secreting NB4, HL60, and HT-29 cells. We found that NB4, HL60, and HT-29 cells expressed CCR6 at the mRNA level (Fig. 3E), however HL60 possessed higher levels of cell-surface CCR6 than NB4 and HT-29 cells (data not shown). Next, we tested the effect of CCL20 stimulation on NB4, HL60, and HT-29 cell proliferation and adhesion to the ECM components, collagen I and fibronectin. We found a 1.6-fold increase in adhesion of HL60 cells to collagen I upon simulation with CCL20 250 ng/ml (p,0.006) and a 2-fold increase in adhesion to fibronectin upon stimulation with 500 ng/ml (p,0.015) (Fig. 3F, data not shown). Stimulation of HT-29 resulted in a dose-dependent increased adhesion to collagen type I, but not fibronectin (Fig. 3F, data not shown). In contrast to HL60 and HT-29, NB4 cells that expressed low surface level of CCR6 did not proliferate or adhere to fibronectin or collagen in response to CCL20 (data not shown). Overexpression of CCL20 increases the growth, invasion and vascularization of PC3 cells in vivo The in vivo role of CCL20 in cancer development is not clear. To determine the role of CCL20 in CXCR4-dependent and - independent tumor development in vivo, a tumor xenograft model was used. Human mock-transfected and CCL20-overexpressing PC3 cells were injected subcutaneously into SCID/bg mice. For in vivo experiments, we chose to use PC3-CCL20 clones 10 and 30, since these clones demonstrated an increased proliferation rate in culture, and produced either comparable levels of CCL20 (100, and 320 pg/ml) to PC3-CXCR4.5. Mice injected with PC3-CCL20.30 PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5125