Figure 1.
C646 inhibited proliferation of AE-positive AML cell lines through inducing cell cycle arrest and apoptosis.
The cultured AE-positive AML cell lines Kasumi-1 and SKNO-1 cells were treated with given doses of C646 or 0.1% DMSO for 24 h before being subjected to the following assays. (A) C646-induced growth inhibition in both cell lines was detected by Cell Counting Kit-8 at the indicated times; means ± SD of 3 independent experiments. (B) C646-evoked ablation of leukemia colony-forming units in both cell lines was performed by colony formation assay. (C) Dose-dependent retardance of C646 on cell cycle distribution in both cell lines. The cells were stained with propidium iodide and measured by flow cytometry. (D) Dose- and time-dependent effects of C646 on apoptosis in both cell lines. The cells were stained with Annexin V-FITC and measured by flow cytometry. Histograms showed means ± SD of 3 independent experiments. * P<0.05. (E) C646 triggered caspase cleavage in Kasumi-1 cells. The Kasumi-1 cells treated with given doses of C646 in the presence or absence of 50 µM pan-caspase inhibitor Q-VD-OPH were collected and lysed at the indicated time points, and western blotting performed with the indicated antibodies. Equalization of protein loading was verified on the same membrane by reprobing after stripping. Data shown were representative of 2 independent experiments.
Figure 2.
Effects of C646 on cell cycle distribution and apoptosis in AE-negative AML cell lines.
Four AE-negative AML cell lines were respectively treated with given doses of C646 or 0.1% DMSO for 24 h before being subjected to the cell cycle distribution (A) and apoptosis (B) assays, as described in Figure 1. Histograms showed means ± SD of 3 independent experiments.
Figure 3.
Selectivity of C646 for AE-positive AML cell lines.
(A) AE expression in U937, U937-AE cell lines. U937-AE cells were treated in the absence or the presence of 100 µM ZnSO4 for 16 h. The cells were lysed and western blotting performed with the indicated antibodies. Equalization of protein loading was verified on the same membrane by reprobing after stripping. Data shown were representative of 2 independent experiments. Cells treated as in (A) were incubated further with given doses of C646 or 0.1% DMSO for 24 h before being subjected to the cell cycle distribution (B) and apoptosis (C) assays, as described in Figure 1. Histograms showed means ± SD of 3 independent experiments. * P<0.05.
Figure 4.
C646 inhibited in vivo proliferation of primary AML blasts isolated from AE9a leukemia mice.
The AML blasts were isolated from the spleen of transplanted AE9a mice and cultured with 10 µM C646 or 0.1% DMSO for 24 h before being subjected to the cell cycle distribution (A), apoptosis (B) and colony formation (C) assays. Histograms showed means ± SD of 3 independent experiments. * P<0.05. (D) Primary AML blasts isolated from the spleen of transplanted AE9a leukemia mice were treated with C646 or DMSO and injected into the tail vein of C57BL/6J mice at a dose of 1×106 cells/mouse, respectively, and the survival time of each mouse were recorded.
Figure 5.
Selectivity of C646 for primary AE-positive AML blasts.
The AML blasts were respectively isolated from the bone marrow samples of 2 t(8;21)(q22;q22) AML patients and a normal karyotype AML patient. The normal hematopoietic stem cells were isolated from granulocyte colony-stimulating factor-mobilized PBSCs of 2 healthy donors. The cells were cultured with given doses of C646 or 0.1% DMSO for 24 h before being subjected to the cell cycle distribution (A), apoptosis (B) or colony formation (C) assays. Histogram showed means ± SD for 2 independent experiments with triplicate cultures. * P<0.05.
Figure 6.
C646 reduced expression of acetylated histone H3, c-kit and bcl-2 in AE-positive AML cell lines.
Western blot analysis of (A) acetylated H3, total histone H3, (B) c-kit and bcl-2 proteins in Kasumi-1 and SKNO-1 cells after 24 h treatment with C646 or DMSO. The cells were lysed and western blotting performed with the indicated antibodies. Equalization of protein loading was verified on the same membrane by reprobing after stripping. Data shown were representative of 2 independent experiments. (C) qRT-PCR analysis of c-kit and bcl-2 mRNA levels in the cells after 24 h treatment with C646 or DMSO. Histograms show relative mRNA levels normalized to control ABL gene; means ± SD of 3 independent experiments. * P<0.05.