PFT-μ inhibits proliferation of leukemic cell lines and primary blasts

PFT-μ inhibits proliferation of leukemic cell lines and primary blasts Leukemic cell lines and primary cells from AML patients were exposed to different concentrations of PFT-μ (0. sensitivity to PFT-μ was observed in a sample derived from a patient with FLT3-internal tandem duplication; however no statistically significant associations between patients’ clinical or genetic features DCHS1 and IC50 values were found. Notably no difference was seen between pretreatment samples and relapsed patients regarding IC50 values in the small number of patient samples tested (Table 1). To evaluate cytotoxicity of PFT-μ in non-malignant cells we analyzed BMSC samples of four AML patients as well as PB MNC (n=6) and CD34-positive cell samples (n=5) from healthy donors. In one BMSC sample IC50 value was not reached with 100?μ PFT-μ. The remaining three BMSC samples showed a KP372-1 IC50 median IC50 value of 37.7?μ (range 36.3-44.1). Median IC50 values in PB MNC and CD34-positive cells were 17.6?μ (range 10.4-42.3) and 15.1?μ (range 8.0-20.0) respectively suggesting a higher resistance of normal hematopoietic and stromal cells to PFT-μ as compared with leukemic blasts. PFT-μ induces cell cycle arrest and apoptosis in leukemic cells To further evaluate the impact of PFT-μ on leukemic cells we performed cell cycle and apoptosis analyses with the cell lines NALM-6 and KG-1a. Cell routine analyses using BrdU/7-AAD staining revealed a lower life expectancy proportion of cells in S stage following 24 KP372-1 IC50 markedly?h incubation with PFT-μ in concentrations of 4 and 5?μ for NALM-6 and 40 and 60?μ for KG-1a (Shape 2a). NALM-6 cells shifted similarly to G0/1 and G2/M stages KG-1a mainly moved into G2/M stage arrest (Shape 2a). Oddly enough about 22% of NALM-6 cells had been in the sub-G0/1 small fraction after incubation with 5?μ PFT-μ (two-fold IC50) whereas just 2% of KG-1a cells had been observed within this small fraction after 60?μ PFT-μ (4.7-fold IC50; Shape 2a). The effect of PFT-μ on particular apoptosis was dependant on AnnexinV/7-AAD staining after incubation with different concentrations of KP372-1 IC50 PFT-μ for 48?h in NALM-6 KG-1a TOM-1 End up KP372-1 IC50 being-13 K562 and Jurkat cells. PFT-μ considerably induced particular apoptosis in every cell lines inside a dose-dependent fashion (Physique 2b; data only shown for NALM-6 and KG-1a). In accordance with the results from cell cycle analyses induction of apoptosis by PFT-μ was more pronounced in NALM-6 cells as compared with KG-1a. In NALM-6 incubation with PFT-μ at 4 5 and 6.5?μ resulted in 34 59 and 80% apoptotic cells above spontaneous apoptosis (11%) respectively. KG-1a cells showed a rate of specific apoptosis of 17% (20?μ PFT-μ) 18 (30?μ PFT-μ) and 25% (40?μ PFT-μ) above control (11% Physique 2b). Determination of caspase-3 activation revealed a dose-dependent increase of the cleaved active form of caspase-3 in NALM-6 cells after treatment with 3 4 and 5?μ PFT-μ for 24?h (Physique 2c). In KG-1a no caspase-3 activation was detected after incubation with 20 40 and KP372-1 IC50 60?μ PFT-μ. This observation was further strengthened by the actual fact that pre-incubation with skillet caspase inhibitor Z-VAD-FMK (50?μ for 1?h) significantly reduced apoptosis after PFT-μ in NALM-6 whereas KG-1a cells weren’t rescued by Z-VAD-FMK (data not shown). Hence PFT-μ exerted different impacts in cell apoptosis and cycle in both leukemic cell lineages. PFT-μ decreases intracellular concentrations of AKT and ERK1/2 in NALM-6 cells Following we performed intracellular staining and fluorescence-activated cell sorting analyses of AKT p-AKT ERK1/2 and p-ERK1/2 kinases in NALM-6 cells to judge whether PFT-μ impacts these two main signaling kinases or their phosphorylation position. After incubation with 10?μ PFT-μ for 10?h a reduction in AKT and ERK1/2 amounts was discovered (Body 3). Oddly enough concentrations from the phosphorylated forms p-AKT and p-ERK1/2 had been suprisingly low at baseline and didn’t modification after PFT-μ treatment (data not really.