Figure 1.
Furazolidone (FZD) suppresses in vitro murine bone marrow cell transformation mediated by a series of leukemia fusion proteins.
(A) The flow chart of the in vitro retrovirus transduction/transformation assay (RTTA) with transduction of leukemia fusions for the initial drug screening. For the initial screening, drugs at 50 µM from the Prestwick chemical library were combined with cells at the 2nd round of plating and added into the methylcellulose medium for the third-round of colony formation. Colonies were counted and morphology was analyzed after 6–7 days and the drugs that had the ability to suppress colony formation of the third-round replating were selected. Next, these selected drugs were tested on c-Kit positive cells at the first-round of plating as a control test for drug toxicity. (B) The bar chart of corresponding absolute colony numbers after the third round of re-plating in methylcellulose culture medium. The concentrations of FZD (from 1 µM to 50 µM) used in bone marrow cells transformed with a panel leukemia fusion genes are indicated. Data are mean ± SD of 3 independent experiments. (C) The structure of FZD. (D) Typical third-round colonies of murine primary bone marrow cells transduced with retroviruses carrying various leukemia fusion genes, after treatment with increasing concentrations of FZD or DMSO control. The graph shows the relative colony numbers after the third-round of re-plating for cells expressing AML1-ETO, MLL-ENL, MLL-AF9, R1A-RAR-RIIa as well as non-transduced c-Kit+ murine hematopoietic cells, after treatment with increasing concentrations of FZD. The IC50 values (for inhibition of colony formation by RTTA) are shown in the right panel.
Figure 2.
Furazolidone inhibits the proliferation of AML cells.
(A) MTS assay in a panel of AML cell lines treated with FZD for 24 h, 48 h and 72 h at indicated concentrations. The data shown are from one representative experiment of three independent experiments. The IC50 values were measured when cells were treated with FZD for 72 hours. (B) MTS assay in AML cell lines after treatment with FZD for 72 hours. Data are mean ± SD of 3 independent experiments. (C) Colony formation assays in Kasumi-1, NB4 and MolM13 cells with DMSO control and FZD treatment at 24, 48, and 72 hours, respectively. Scale bars represent 1 mm. The bar chart represents the relative numbers of colonies in the left panel. Data are mean ± SD of 3 independent experiments.
Table 1.
IC50 values and relevant cytogenetic/molecular data for AML cell lines.
Figure 3.
Furazolidone induces apoptosis of AML cells.
The apoptosis was measured by Annexin V-PE/7-AAD kit using FACS in Kasumi-1, NB4, MV4-11 and MolM13 in the presence of DMSO control or the predetermined IC50 value of FZD for 72 hours.There are reports that FZD can inhibit cell proliferation and provokes apoptosis by inducing S-phase arrest in human hepatoma G2 cells [14]. We therefore tested whether FZD could also cause cell-cycle arrest in AML cells. We treated AML cells with FZD at the predetermined IC50 value and analyzed by flow cytometry. We showed that this compound did not modulate cell-cycle distribution (Figure S1), suggesting that it prolonged cell-doubling time rather than inducing cell-cycle arrest.
Figure 4.
Furazolidone induces the leukemic cellular differentiation.
(A) The myeloid differentiation antigen CD11b, was measured by FACS in Kasumi-1, NB4 and MV4-11 after FZD (predetermined concentration) and DMSO control treatment for 72 hours. The representative graph was from one of the three independent experiments. (B) The Giemsa staining of Kasumi-1, NB4 and MV4-11 cells treated with FZD (predetermined concentration) or DMSO control for 96 hours (magnification, 400×). Arrows indicate nuclear condensation and multilobulated nucleus. Scale bars represent 10 µm. (C) Photomicrographs and the relative histograms (bottom panel) of the NBT reduction assay with Kasumi-1, NB4 and MV4-11 cells in the DMSO control group (top panel) and FZD treatment group (middle panel) for 72 hours (Giemsa stain; magnification, 1,000×). There were many black particles (formazan) in the FZD treatment group of leukemia cells, so-called NBT-positive cells, which indicated that the AML cells had differentiated. Data are mean ± SD of 3 independent experiments (bottom panel). (D) The expression of p53 protein was measured by Western blot in Kasumi-1, NB4, MV4-11 and MolM13 cells after 72 hours treatment with the predetermined IC50 value for FZD or control (DMSO). Relative protein levels, normalized to the beta-actin control, were quantitated and listed below each panel. (E) The p53 mRNA expression was measured by RT-PCR in AML cell lines.