Lated and activated by the tyrosine kinase, BCR-ABL. As shown in Supplementary Fig. S1B, imatinib therapy remarkably lowered the phosphorylation of STAT5 and ERK1/2 in K562 cells, whereas, the alterations in K562R cells were insignificant. These results suggested that K562R cells were resistant to imatinibinduced apoptosis and BCR-ABL downstream signaling pathway inhibition. To investigate the anticancer possible of CTD against CML, the cytotoxicity of CTD toward standard PBMCs, imatinibsensitive CML cell line, K562, and imatinib-resistant cell line, K562R, was tested working with CCK-8 assay. The results demonstrated that CTD suppressed the viability of both CML cell types (Figs. 1A and 1B) with small effect on normal blood cells (Fig. 1C). The IC50 value of CTD for PBMCs (one hundred M) was significantly higher than that for K562 and K562R cells (28.23 and 54.42 M, respectively) at 24 h. The IC50 values for PBMCs, K562, and K562R cells at 48 h were 102.69, 27.63 and 31.34 M, respectively. Trypan blue exclusion assay showed that remedy of CTD induced cell death in K562 and K562R cells at the concentration of five to 80 M (Figs. 1D and 1E).CTD induced mitotic arrest in CML cells Morphologic adjustments from the cells have been examined under phase contrast microscope. The standard spherical shape of K562 and K562R cells changed into unusual ellipsoid or spindle shape, with considerable enlargement, after exposure to CTD (5-20 M) for 24 h (Fig. 2A). This result suggests that CTD treatment could result in a failure of cytokinesis in CML cells. The cell cycle is usually divided into two distinct stages: the interphase stage and mitotic stage. In the second stage, or M-phase, chromatin condenses and cell division requires location. Earlier research have shown that Histone H3 phosphorylated (pH3) at Ser10 could be a dependable and distinct mitotic marker (Crosio et al., 2002). To examine no matter whether CTD could trigger mitotic arrest in CML cells, we analyzed CTD-treated cells by flow cytometry immediately after anti-pHistone H3/propidium iodide double staining. The results showed that CTD-treatment induced a significant enhance in mitotic phase inK562 and K562R cells (Fig. 2B). As shown in Fig. 2C, immediately after 24 h of CTD treatment19.2 to 24.five of K562 cells have been in mitotic phase, when compared with only 1.six in the manage cells in mitotic phase; and ten.eight to 13.0 of K562R cells had been in mitotic phase, compared to 3.11 from the handle cells in mitotic phase. These results indicate that CTD induced mitotic failure in CML cells. Effects of CTD on cell cycle regulating proteins To further verify that CTD induced mitotic perturbation, we studied the adjustments in nuclear morphology soon after exposure to CTD. The cells L-Gulose supplier underwent pronounced modifications in nuclear morphology, like chromatin condensation (Fig. 3A). K562 cells with the abnormal mitotic nuclei accounted for about 1.05 , 17 , 24 and 36 after remedy with CTD at the concentration of 0 M, 5 M, ten M, and 20 M, respectively (Fig. 3B). We next investigated the mechanism of CTD triggered mitotic arrest. Activation of cyclin B1/Cdc2 complex, a heterodimerhttp://molcells.orgMol. CellsCantharidin Overcomes Imatinib Resistance in CML Xiaoyan Sun et al.AABB C DC E FFig. two. CTD induced mitotic arrest in CML cells. (A) K562 and K562R cells have been treated with indicated concentrations of CTD for 24 h, and also the morphological adjustments were observed via microscopy. (B) K562 and K562R cells had been incubated with indicated concentrations of CTD for 24 h, then stained with Anti-pho.
