The software GraphPad Prism (version 5

The software GraphPad Prism (version 5.0 for Windows, GraphPad Software Inc., San Diego, CA, of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also exhibited that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition SB 239063 of proteasome pathways by bortezomib suppresses colony formation ability of CML cells. Conclusions Altogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0823-y) contains supplementary material, which is available to authorized users. from mitochondria, we performed the assay as reported earlier [34]. K562 cells were treated with 10, 25 and 50?nm bortezomib for 24?h, cells were harvested and resuspended in hypotonic buffer (1?mM TrisCHCl, pH 7.4, 0.13?M NaCl, 5?mM KCl, 7.5?mM MgCl2). Cells were Rabbit polyclonal to HGD homogenized and centrifuged to obtain the cytosolic as well as mitochondrial fractions. Twenty to twenty-five microgram of protein from cytosolic and mitochondrial fractions of each sample were analyzed by immunoblotting using an anti-cytochrome c and tubulin antibody. Clonogenic leukemic assays using methylcellulose K562, AR230 and LAMA84 (1??104) cells were treated with and without bortezomib as described in the figure legends and mixed with 1.0?mL of MethoCult H4034 Optimum (Stem Cell Technologies). Colonies were counted based on morphology after 10?days. Statistical analysis Comparisons between groups were made using the paired Students test. SB 239063 The software GraphPad Prism (version 5.0 for Windows, GraphPad Software Inc., San Diego, CA, Values of * p? ?0.05 were considered statistically significant. Results Bortezomib SB 239063 is usually antiproliferative and induces apoptosis in CML cells To assess the effect of bortezomib on cell viability, a panel of human CML cell lines (AR230, LAMA-84, and K562) were treated with increasing concentrations (10, 25 and 50?nm) of bortezomib for 24?h. A dose-dependent decrease in cell proliferation was observed in all the treated cell lines (Fig.?1a). Bortezomib-mediated inhibition of cell viability was also observed in a time-dependent manner (data not shown). Open in a separate window Fig.?1 Effects of Bortezomib on proliferation, cell cycle progression, and apoptosis in CML cells. a Bortezomib inhibits the cell viability of CML cells. AR230, LAMA-84 and K562 cells were incubated with 10, 25, 50 and 100?nm bortezomib for 24?h. Cell proliferation assays were performed using MTT as described in Methods section. Thegraphdisplays the mean??SD (standard deviation) of three independent experiments with replicates of six wells for all the doses. **p? ?0.01, ***p? ?0.001 b Bortezomib induces the increase of subG0 population of CML cells. K562 and AR230 cells were treated with 10, 25 and 50?nm of bortezomib for 24?h. Thereafter, the cells were washed, fixed and stained with propidium iodide, and analyzed for DNA content by flow cytometry as described in Methods section. c Bortezomib induces apoptosis in CML cells. K562 and AR230 cells were treated with 10, 25 and 50?nm of bortezomib for 24?h and cells were subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry. d Bortezomib treatment of CML cells induces DNA fragmentation. K562 and AR230 cells were treated with 10, 25 and 50?nm bortezomib as indicated for 24?h and DNA was extracted and separated by electrophoresis on 1.5?% agarose gel To investigate whether the inhibition of cell viability induced by bortezomib is due to cell cycle arrest or apoptosis K562 and AR230 cells were treated with different doses of bortezomib for 24?h as indicated. An increase in subG0 population was observed in a dose-dependent manner with the cell lines, K562, and AR230 (Fig.?1b). The sub-G0 population of cells was found to increase from 6.48?% in control cells to 19.5, 33.8 and 49.8?% at 10, 25 and 50?nm bortezomib-treated K562 cells respectively. Comparable results were obtained in AR230 cells with an increase of sub-G0 population from 6.56?% in control cells to 16.2, 27.6 and 38.4?% in cells treated with 10, 25 and 50?nm of bortezomib respectively. The increase in sub-G0 population was accompanied by decreased G0/G1 and G2/M phases in bortezomib-treated CML cells. To investigate whether the increased sub-G0 population in response to bortezomib treatment in CML cells was a resultant of induction of apoptosis, K562, and.