Animal experiments The animal experiments were performed as described previousl

This signaling pathway, which is com posed of the chemokine CXCL12 and its receptors CXCR4 and CXCR7, play pivotal roles in the cell migration, angiogenesis, proliferation, and survival of many cancer cells, including breast cancer. ABT-888 分子量 CXCR4 is typically highly expressed in metastatic cells and supports the pri vileged homing of these metastatic cells to specific sites where the local secretion of CXCL12 is important, namely the bone, liver, brain, and lung. Indeed, reduction or the loss of the local secretion of CXCL12 at the tumor site can induce the emergence of metastatic cells that may spread in the organism toward endocrine sources of CXCL12. Although the pivotal role of the CXCL12 CXCR4 axis in cell motility and consequently in cancer metastasis in several tissues is well established, the contribution of CXCL12 via its receptor CXCR7 is less understood.<br><br> CXCL12 signaling may be connected to the pheno typic characteristics modified by COUP TFI, thus, we hypothesized that COUP TFI could target this signaling pathway in breast cancer cells. Furthermore, as the en tire CXCL12 CXCR4 signaling axis is an endogenous Afatinib 構造 target of E2 and is pivotal to hormonal induced MCF 7 cell growth, COUP TFI could achieve the loss of its estrogenic regulation. In the present study, we developed MCF 7 breast cancer cells overexpressing COUP TFI protein and examined the regulation of CXCL12 signal ing axis. We provide evidence that COUP TFI increases the motility of MCF 7 ER positive breast cancer cells by acting on CXCL12 CXCR4 signaling as an endogen ous target.<br><br> The modification of CXCL12 CXCR4 expres sion by COUP TFI is mediated by the activation AG-1478 溶解度 of epithelial growth factor and its receptor in MCF 7 cells. These results correlate with the expres sion profiles of COUP TFI, CXCL12, and CXCR4 in breast tumors compared to healthy samples. Methods Antibodies and reagents A goat polyclonal antibody against human CXCL12, rabbit polyclonal antibody against CXCR4, mouse monoclonal anti body against human CXCR7 RDC1, a rabbit polyclonal antibody against COUP TFI and a rabbit poly clonal antibody against HA epitope were used for the immunofluorescence and western blot assays. A mouse polyclonal antibody against phosphory lated ERK and rabbit polyclonal anti body against total ERK were used for the western blot assays. The reagents used for treatments, ICI182,780, and AMD3100 were purchased from Sigma Aldrich Co.<br><br> The recombinant CXCL12 used for the proliferation and migration assays was purchased from R D Systems. Cell culture and treatments MCF 7 cells were routinely maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics at 37 C in 5% CO2. Stably transfected MCF 7 clones were obtained as previ ously described. A pool of two independent control clones and two independent COUP TFI overexpressing clones were used for this study. When treatments with steroids were required, the cells were maintained for 24 h in DMEM without phenol red supplemented with 2. 5% dextran treated charcoal stripped FBS prior to the experiments. The treatments were then performed in phenol red free DMEM with 2. 5% dsFBS and E2, ICI, or both together for 48 hours, 0.