Figure 7

Combination of SNS-032 with perifosine strongly inhibits phosphor-Akt and enhances cytotoxicity against AML cells. (A) KG-1 and NB4 cells were treated with a series of dilutions of each agent individually and with both compounds simultaneously in a fixed ratio (1:50) for 24 h and cell growth was determined by an MTT assay. Values represent the mean plus or minus s.d. of triplicate experiments. The combination index (CI) was calculated by Calcusyn software (lower panel). (B) KG-1 and NB4 cells were treated with SNS-032, perifosine, or combination, and then plated in triplicate, at a density of 2000 cells/ml in methylcellulose medium. Colony counts at 7 days are shown as the average of three independent experiments; bar, ± s.d. (C) The primary AML cells from 2 patients were treated with two agents alone or in combination followed by cultured in methylcellulose medium. The number of leukemic colony at 7 days was counted. The data are shown as the means of triplicate experiment. (D) AML cell lines were incubated with or without SNS-032, in the presence or absence of perifosine. At 6 h later, cell lysates were prepared, and activation of caspase-3 and cleavage of PARP were examined by Western blot analysis. The expression and phosphorylation of ERK1/2, Akt, mTOR, and its downstream targets were also determined. The difference in the level of protein expression was semi-quantitatively determined by densitometry and expressed as a ratio. Actin was used as protein loading control.