These results hold great promise in improving the clinical outcomes of TAM-resistant breast cancer patients. Acknowledgements Not applicable. Glossary AbbreviationsTAMtamoxifenCAMchorioallantoic membraneDAPI46-diamidino-2-phenylindoleDMEMDulbecco’s modified Eagle’s mediumEDTAethylenediamine tetraacetic acidEGFRepidermal growth factorEMTepithelial to mesenchymal transitionERestrogen receptorFBSfetal bovine serumFGFRfibroblast growth factor receptorIGFRinsulin-like growth factorIL-6interleukin-6JAKJanus kinaseMMP2matrix metalloproteinase-2MMP9matrix metalloproteinase-9MPNmyeloproliferative neoplasmPBSphosphate-buffered salinePDGFRplatelet-derived growth factor receptorRTroom temperatureSDSsodium dodecyl sulfateSTATsignal transducer and activator of transcriptionVEGFvascular endothelial growth factor Funding The present study was supported by the National Research Foundation of Korea (NRF) grant (grant nos. representative target gene of the JAK2-STAT3 pathway, functions as a key regulator of invasion and angiogenesis. Ruxolitinib significantly inhibited VEGF mRNA expression and transcriptional activity. The present study also performed a chick embryo chorioallantoic membrane assay to assess tumor growth and angiogenesis in TAMR-MCF-7 cells. Ruxolitinib reduced tumor weight and the number of blood vessels produced by TAMR-MCF-7 cells in a concentration-dependent manner. These results indicated that JAK2 could be a new therapeutic target for TAM-resistant breast cancer. migratory ability than MCF-7 cells (8). TAMR-MCF-7 cell migration was significantly suppressed under treatment with 10 M ruxolitinib (Fig. 3A). Because TAMR-MCF-7 cells acquire the migratory phenotype via EMT progression (7), we then examined whether JAK2-STAT3 inhibition by ruxolitinib affects the expression of EMT markers in TAMR-MCF-7 cells. Representative biochemical markers of EMT include loss of the epithelial adherence protein E-cadherin and upregulation of the mesenchymal protein N-cadherin (9). Immunocytochemistry for E-cadherin, N-cadherin, and phalloidin in MCF-7 and TAMR-MCF-7 cells showed that E-cadherin downregulation and N-cadherin upregulation in TAMR-MCF-7 cells were partially reversed by treatment with 10 M ruxolitinib (Fig. 3B). Western blot analyses confirmed that higher expression of mesenchymal marker proteins, such as N-cadherin, vimentin, snail, or twist, was suppressed in TAMR-MCF-7 cells by ruxolitinib in a concentration-dependent manner (Fig. 3C). Although E-Cadherin was slightly detected in TAMR-MCF-7 cells by immunocytochemistry, E-cadherin was not detectable by immunoblottings in TAMR-MCF7 cells (Fig. 3C) (8). It may result from the difference in detection sensitivity between immunoblotting and immunocytochemistry. These results demonstrate that ruxolitinib may inhibit cell migration in TAMR-MCF-7 cells, presumably by blocking EMT. Open in a separate window Figure 3. Effects of ruxolitinib on cell migration and EMT phenotype changes in EMD638683 R-Form TAMR-MCF-7 cells. (A) Effect of ruxolitinib on TAMR-MCF-7 cell migration. Transwell migration assays were performed in TAMR-MCF-7 cells 18 h post-treatment with ruxolitinib (0.1C10 M). Representative pictures of migrated cells (red circles) were shown (left panel). The relative cell numbers of migrated cells were counted (right panel). Data are presented as the mean standard deviation (n=4). EMD638683 R-Form *P 0.05 vs. vehicle-treated control. (B) Immunofluorescence staining of EMT markers in MCF-7 and TAMR-MCF-7 cells. TAMR-MCF-7 cells cultured on coverslips were incubated with or without 10 M ruxolitinib for 24 h, and then the fixed cells were stained with specific antibodies against phalloidin, E-cadherin or N-cadherin. Representative images were captured using a CELENAS Digital Imaging System. Magnification, 40. (C) Expression of EMT markers in TAMR-MCF-7 cells. EMT phenotype markers were determined following the exposure of TAMR-MCF-7 cells to 0.1C10 M ruxolitinib for 24 h. EMT, epithelial mesenchymal transition; TAMR, tamoxifen resistant. Ruxolitinib inhibits angiogenesis and tumor growth A clinical feature of TAM resistance in human breast cancer is an increase in microvessel counts (31). We previously reported that angiogenic potential was enhanced in TAMR-MCF-7 cells by VEGF upregulation (10). In the current study, high basal VEGF mRNA expression was observed in TAMR-MCF-7 cells, but not GINGF in MCF-7 cells (Fig. 4A). Consistent with the EMD638683 R-Form inhibitory effects of ruxolitinib on cell migration and EMT progression of TAMR-MCF-7 cells, ruxolitinib (0.1C10 M) reduced VEGF mRNA levels in TAMR-MCF-7 cells (Fig. 4A). Moreover, a VEGF-luc reporter gene assay further revealed that VEGF promoter binding activity was significantly diminished by treatment with 0.3C1 M ruxolitinib (Fig. 4B). As ruxolitinib above 3 M causes a significant cell death in the reporter gene analysis condition because of the lipid carrier-based membrane damage (32), MCF-7 and TAMR-MCF-7 cells were exposed to 0.3C1 M ruxolitinib. These results suggest that.