cell lysates were prepared from 107 cells according to a method described previously. Then 20 mg of lysates was separated electrophoretically using one hundred thousand polyacrylamide gel. Detection and immunoblotting by enhanced chemiluminescence were done as described previously. As an internal control a mouse monoclonal PF 573228 antibody against glyceraldehyde 3 phosphate dehydrogenase, which was used, was bought from Chemicon International. Rabbit polyclonal antibodies against anti cleaved caspase 3, anti cleaved caspase 7, anti cleaved caspase 9, anti cleaved PARP, anti Phospho Chk2, anti Phospho p53, anti ERK1/2, anti Phospho ERK1/2, anti STAT5, anti Phospho STAT5, anti JNK/SAPK and anti Phospho JNK/SAPK were obtained from Cell Signaling Technology. Ki values of VE 465 against Aurora A, Aurora B and Aurora C were all low, suggesting that VE 465 efficiently inhibits activity of Aurora family kinases. We first examined the cytotoxic ramifications of VE 465 in conjunction with conventional anti leukemia agencies, including cytosine arabinoside, daunorubicin, idarubicin, mitoxantron, doxorubicin, vincristine and etoposide, by Steel and Peckham isobologram analysis. As shown in Dining table 1, IC50 values of VE 465 against leukemia cells are very nearly exactly the same in various human leukemia cell lines. Isobolograms were then developed on the Cellular differentiation basis of the results of the dose?response shapes of VE 465 and various traditional anti leukemia providers. The outcome of isobologram studies are summarized in Table 2. Representative isobolograms showing the cytotoxic ramifications of VE 465 in conjunction with vincristine or cytosine arabinoside on THP 1, HL60, KY821 and KCL22 cells are shown in Fig. 1A. One of the agents tested, only vincristine confirmed an additive/synergistic inhibitory effect on the development of cells when it absolutely was combined with VE 465. Combined therapy GW0742 with VE 465 and other old-fashioned drugs triggered no complete inhibition but rather had an antagonistic impact on cell growth. Consistent with these results, treatment of THP 1, KY821 and KCL22 cells with the mixture of VE 465 and vincristine resulted in significant inhibition of cell growth compared to the effectation of VE 465 or vincristine alone. This inhibitory effect was almost the same when VE 465 or vincristine was added to the medium just before the addition of still another reagent, suggesting that the order of addition of the reagents didn’t influence the mix mediated inhibitory effect. To show the mechanisms underlying the inhibitory effect of the combination of VE 465 and vincristine on development of leukemia cells, we performed flow cytometric analysis using THP 1 cells.
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