E. both cells ( 100, scale bar: 100 m). D. The protein expression of EMT markers was analyzed by WES analysis. The values above each lane indicate the relative intensity of bands as normalized by the intensity of -Tubulin. E. The motilities of cells were measured by the wound healing assay. The left Kinetin panels show the phase-contrast microscopy images ( 100, scale bar: 100 m) at the beginning of the experiment (0 hour) and the end point (18 hour). The right graph shows the percentage of wound Kinetin closure as mean SD (= 3). F. The mRNA expression of the ligands or receptors of TGF- signaling in breast cancer patients, Booser dataset from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). The statistical values were calculated by student’s t-test (between two groups) or ANOVA with Dunnett’s multiple comparison test (among groups more than three). *, **, and *** indicate < 0.05, < 0.01, and < 0.005, respectively). As shown in Physique ?Physique1C,1C, MDA-MB-231-P had an IC50 of 16 nM for paclitaxel, whereas MDA-MB-231 had an IC50 of 3 nM. MDA-MB-231-P cells are resistant to cytotoxic effect of 3 nM paclitaxel based on cell viability assays (Physique ?(Figure1C)1C) and cell cycle analysis (Supplementary Figure 1A). Moreover, the morphology of MDA-MB-231-P cells had changed into a more spindle shape. In accordance with the morphological changes, the expression of the mesenchymal proteins, Vimentin and Fibronectin, showed 2.5-fold and 1.5-fold increases, respectively, whereas the expression of the epithelial protein, Zo-1, showed a 0.3-fold decrease in MDA-MB-231-P Kinetin cells when compared to those of MDA-MB-231 cells (Figure ?(Figure1D).1D). We compared the motility of the MDA-MB-231-P cells with that of the MDA-MB-231 cells using wound healing assays (Physique ?(Figure1E).1E). The percentage of wound closure was significantly increased in the MDA-MB-231-P cells by 4.6 fold compared to that of MDA-MB-231 cells showing the similar growth rate as that of the parental MDA-MB-231 cells in paclitaxel-free media (Supplementary Physique 1B). These results suggest that the mesenchymal traits are correlated with taxane-resistance in patients as well as in cells was increased by paclitaxel as previously reported [30] (Supplementary Physique 2A). The treatment of paclitaxel reduced the cancer burden starting from the 2nd week (after 2 cycles of paclitaxel) until the 5th week (Physique ?(Physique2B2B and Supplementary Physique 2B). During this period, the TGF- inhibitor, EW-7197 could not reduce primary cancer burden in alone treatment and the combinatorial EW-7197 treatment could not enhance the cytotoxic effect of paclitaxel (Physique ?(Figure2B).2B). Notably, EW-7197 synergistically prolonged the survival time (Physique ?(Figure2C).2C). As paclitaxel reduced the burden of the primary tumor, it dramatically prolonged the median-survival time to 66 days, whereas that of the control group was 33.5 days. However, the survival of the paclitaxel-group decreased rapidly once the first death started. Even though the effect of treatment with EW-7197 alone on survival was minimal (the median survival time = 36 days), the combinatorial treatment of EW-7197 with paclitaxel extended the survival time over that of paclitaxel alone (Physique ?(Figure2C2C). Open in a separate window Physique 2 A. The schematic of the experimental breast cancer mouse model for the combinatorial treatment of EW-7197 and paclitaxel (MDA-MB-231-xenografted mice). Mice were inoculated with MDA-MB-231 cells and the mice, of which the tumor sizes were around 70 mm3, were randomly grouped and the treatment started as described in the Material and Methods section. Mice were treated with paclitaxel once a week for a total 4 cycles and EW-7197 was treated for 5 consecutive days a week for 7 weeks (for efficacy test) or 10 weeks (for survival test). (B-E) Analyses of tumor growth and metastasis in MDA-MB-231-xenografted mice B. The primary tumor volume was calculated once a week by the Rabbit polyclonal to beta defensin131 formula: volume (mm3) = (length (mm)) (width (mm))2 0.5 (= 5~6/group). C. The Kaplan-Meier analysis of survival (= 6~7/group) B. and C. Black line or black dotted line represents the control group, red line represents the paclitaxel-treated group, blue line represents the EW-7197-treated group, and green line represents the combinatorial EW-7197 and paclitaxel-treated group. D. The representative phase-contrast microscopy images of H&E-stained lungs and the mRNA expressions of human ((= 5~6/group). E. The protein expression of EMT markers in primary tumors of the MDA-MB-231-xenografted mice (= 5~6/ group). Lysates were obtained directly from primary tumor tissues. In the box.