Korean J Physiol Pharmacol.  2022 May;26(3):157-164. 10.4196/kjpp.2022.26.3.157.

Evaluation of the effects of disulfiram, an alcohol-aversive agent with anti-cancer activity, on mouse bone marrow cells

• Park SR
• Joo HG

• KMID: 2529398
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.157

Abstract

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor. DSF has potent anti-cancer activity for solid and hematological malignancies. Although the effects on cancer cells have been proven, there have been few studies on DSF toxicity in bone marrow cells (BMs). DSF reduces the metabolic activity and the mitochondrial membrane potential of BMs. In subset analyses, we confirmed that DSF does not affect the proportion of BMs. In addition, DSF significantly impaired the metabolic activity and differentiation of BMs treated with granulocyte macrophage-colony stimulating factor, an essential growth and differentiation factor for BMs. To measure DSF toxicity in BMs in vivo, mice were injected with 50 mg/kg, a dose used for anti-cancer effects. DSF did not significantly induce BM toxicity in mice and may be tolerated by antioxidant defense mechanisms. This is the first study on the effects of DSF on BMs in vitro and in vivo. DSF has been widely studied as an anti-cancer drug candidate, and many anti-cancer drugs lead to myelosuppression. In this regard, this study can provide useful information to basic science and clinical researchers.

Keyword

Bone marrow cells; Cell death; Disulfiram; GM-CSF; In vivo toxicity

Figure

• 

  Fig. 1 DSF decreases the metabolic activity of BMs. BMs (1 × 106 cells/ml) were incubated in 96-well culture plates and treated with DSF at the indicated concentrations (0–5 µM) in the absence or presence of 1 µg/ml LPS. After 3 days, the CCK-8 assay was performed. Data are presented as mean ± SD. DSF, disulfiram; BMs, bone marrow cells; LPS, lipopolysaccharide; CCK-8, Cell Counting Kit-8; O.D., optical density. ***, ###indicate p < 0.001 compared to BMs treated without LPS or LPS alone (DSF 0 µM), respectively.

• 

  Fig. 2 DSF diminishes the MMP and causes the death of BMs. BMs were cultured in 6-well culture plates (1 × 106 cells/ml) and treated with DSF at the indicated concentrations (0–5 µM). To measure MMP, treated BMs were stained with rhodamine 123 solution. The number in the histograms indicates mean fluorescence intensity (MFI) (A). The control group was set to 100% and the MFI of each histogram was calculated (B). The cells were treated for 2 days and stained with annexin V-FITC/PI. The cells in quadrants indicate necrosis (upper left), late apoptosis (upper right), early apoptosis (lower right), and viable (lower left) cells. The number in quadrants indicates the percentage of cells (C) and the percentage of PI-positive cells was presented (D). DSF, disulfiram; MMP, mitochondrial membrane potential; BMs, bone marrow cells; V-FITC, V-fluorescein isothiocyanate; PI, propidium iodide. *, **Indicate p < 0.05, 0.01 compared to the control (DSF concentration 0 μM), respectively.

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  Fig. 3 DSF does not affect the ratio of BM subsets. BMs (1 × 106 cells/ml) were seeded in 24-well culture plates and treated with DSF. The representative dot plots (A) and the percentage of positive cells (B) were presented. DSF, disulfiram; BMs, bone marrow cells.

• 

  Fig. 4 DSF impairs the metabolic activity and differentiation of GM-CSF-treated BMs. BMs (1 × 106 cells/ml) were incubated in 96-well culture plates and treated with DSF at the indicated concentrations (0–0.5 µM) in the absence or presence of 10 ng/ml GM-CSF for 3 days. The metabolic activity of BMs was measured using the CCK-8 assay (A). Data are expressed as the mean ± SD. To investigate the differentiation to DCs, BMs treated with GM-CSF and DSF were stained with DC-specific markers, CD11c. The percentage of CD11c-positive cells after gating a region based on forward scatter (FSC) and side scatter (SSC) was presented (B). DSF, disulfiram; GM-CSF, granulocyte-macrophage colony-stimulating factor; BMs, bone marrow cells; CCK-8, Cell Counting Kit-8; DC, dendritic cell; O.D., optical density. **, ##, ### indicate p < 0.01 and 0.001 compared to the control BMs in the absence or presence of GM-CSF, respectively.

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  Fig. 5 DSF does not significantly induce myelosuppression in vivo. The mice were intraperitoneally injected with 50 mg/kg DSF every 2 days, on day 0, 2, and 4 (A). Control mice were injected with DMSO alone. The mice were sacrificed at indicated times and BMs were harvested. The cellularity of BMs was calculated using the trypan blue exclusion test. The graph was generated using in vivo data of 12 mice in each group from more than three independent experiments (B). The representative subset proportion (C) and the percentage of positive cells (D) were presented. DSF, disulfiram; DMSO, dimethyl sulfoxide; BMs, bone marrow cells; ns, non-significant.

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