Bithionol eliminates acute myeloid leukaemia stem-like cells by suppressing NF-κB signalling and inducing oxidative stress, leading to apoptosis and ferroptosis

Abstract

Acute myeloid leukaemia (AML) is a lethal bone marrow neoplasm caused by genetic alterations in blood cell progenitors. Leukaemic stem cells (LSCs) are responsible for the development of AML, drug resistance and relapse. Bithionol is an old anthelmintic drug with potential antibacterial, antiviral, antifungal, anti-Alzheimer, and antitumour properties. In this work, we focused on the anti-AML LSC properties of bithionol. This compound inhibited the viability of both solid and haematological cancer cells, suppressed AML stem-like cells, and inhibited AML growth in NSG mice at a dosage of 50 mg/kg, with tolerable systemic toxicity. Bithionol significantly reduced the levels of phospho-NF-κB p65 (Ser529) and phospho-NF-κB p65 (Ser536) and nuclear NF-κB p65 translocation in AML cells, indicating that this molecule can suppress NF-κB signalling. DNA fragmentation, nuclear condensation, cell shrinkage, phosphatidylserine externalisation, loss of transmembrane mitochondrial potential, caspase-3 activation and PARP-(Asp 214) cleavage were detected in bithionol-treated AML cells, indicating the induction of apoptosis. Furthermore, this compound increased mitochondrial superoxide levels, and bithionol-induced cell death was partially prevented by cotreatment with the selective ferroptosis inhibitor ferrostatin-1, indicating the induction of ferroptosis. In addition, bithionol synergised with venetoclax in AML cells, indicating the translational potential of bithionol to enhance the effects of venetoclax in patients with AML. Taken together, these data indicate that bithionol is a potential new anti-AML drug.

Fig. 1. Bithionol is cytotoxic to solid and haematological cancer cells.

A Chemical structure of bithionol. B IC₅₀ values for the cytotoxicity of bithionol against haematological (red bars) and solid cancer cells (blue bars), as well as against three different populations of noncancerous cells (green bars). C Heatmap of selectivity indices calculated for bithionol.

Fig. 2. Bithionol reduces the number of AML stem-like cells.

Immunophenotypic analysis of the myeloid lineage markers CD13 (A) and CD33 (B) and the AML progenitor/stem markers CD34 (C), CD38 (D) and CD123 (E) in bithionol-treated KG-1a cells after 48 h of incubation. The vehicle (0.2% DMSO) was used as a negative control (CTL). The data are shown as the mean ± SEM of three biological replicates carried out in duplicate. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Fig. 3. Effect of bithionol on the growth of xenografts derived from KG-1a cells.

A A xenograft model was established in NSG mice. Two weeks after the inoculation of KG-1a cells, the mice were randomly divided into a bithionol (50 mg/kg) group and a control group (5% DMSO). The treatments were injected into the mice intraperitoneally every day for two weeks. hCD45-positive cells in the bone marrow (B) and peripheral blood (D) and mCD45-positive bone marrow (C) and peripheral blood (E) cells were quantified by flow cytometry. The data are shown as the mean ± SEM of 6 animals. *p < 0.05 compared with CTL by Student’s t test.

Fig. 4. Bithionol inhibits NF-κB signalling in AML cells.

A Western blot of phospho-IKKα/β (Ser176/180), IKKα, IKKβ, NF-κB p65, phospho-NF-κB p65 (Ser536), phospho-IκBα (Ser32) and IκBα in the AML KG-1a, KG-1, Kasumi-1 and HL-60 cell lines treated with bithionol for 72 h. GAPDH was used as an internal control. B, C Effect of bithionol (28 μM) on the levels of phospho-NF-κB p65 (Ser536) after 24 h of treatment in KG-1a cells, as assessed by flow cytometry. D, E Effect of bithionol (28 μM) on the levels of phospho-NF-κB p65 (Ser529) after 24 h of treatment in KG-1a cells, as assessed by flow cytometry. The vehicle (0.2% DMSO) was used as a negative control (CTL). The data are shown as the mean ± SEM of three biological replicates carried out in duplicate. * p < 0.05 compared with CTL by Student’s t test. MFI = mean fluorescence intensity. F Representative immunofluorescence images of NF-κB p65 in KG-1a cells after 24 h of incubation with 28 μM bithionol. Scale bar = 25 μm. G Up- and downregulated genes in KG-1a cells after 12 h of treatment with 28 µM bithionol. Genes that displayed RQ ≥ 2 (red bars) were upregulated, and those that displayed RQ ≤ 0.5 (green bars) were downregulated.

Fig. 5. Cell cycle progression in KG-1a cells after treatment with bithionol.

Representative histograms after 12 (A), 24 (B), 48 (C), and 72 (D) h of treatment. Percentages of cells in sub-G₀/G₁ (E), G₀/G₁ (F), S (G), and G₂/M (H) after different incubation periods with bithionol. Vehicle (0.2% DMSO) was used as a negative control (CTL), and doxorubicin (DOX, 1 µM) was used as a positive control. The data are shown as the mean ± S.E.M. of three biological replicates carried out in duplicate. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Fig. 6. Bithionol induced apoptotic cell death in KG-1a cells.

A Representative flow cytometry dot plots. B Apoptosis quantification in KG-1a cells after 12, 24, 48, and 72 h of treatment with bithionol. Quantification of live (YO-PRO-1/PI double negative cells), apoptotic (YO-PRO-1-positive/PI-negative cells), and dead (YO-PRO-1/PI double positive cells plus PI-positive/YO-PRO-1 negative cells) KG-1a cells. Vehicle (0.2% DMSO) was used as a negative control (CTL). The data are shown as the mean ± SEM of three biological replicates carried out in duplicate. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Fig. 7. Bithionol causes caspase-mediated apoptosis in AML cells.

A, B Effects of bithionol (28 μM) on the levels of active caspase 3 and (C, D) cleaved PARP (Asp214) after 24 h of treatment in KG-1a cells, as assessed by flow cytometry. *p < 0.05 compared with CTL by Student’s t test. MFI = mean fluorescence intensity. E Western blot of PARP from AML KG-1a, KG-1, Kasumi-1 and HL-60 cells treated with bithionol for 72 h. GAPDH was used as an internal control. F Effect of bithionol on mitochondrial activity in KG-1a cells after 24 h of incubation. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test. G, H Concentration‒response curves of WT SV40 MEFs and BAD KO SV40 MEFs upon treatment with 5-fluorouracil (5-FU, a positive control) and bithionol. The curves were obtained from at least three biological replicates carried out in duplicate via the Alamar blue assay after 72 h of incubation. I, J Induction of cell death in the WT SV40 MEFs and BAD KO SV40 MEFs after 48 h of incubation with 40 μM 5-FU and 28 μM bithionol. *p < 0.05 compared with CTL by Student’s t-test. Vehicle (0.2% DMSO) was used as a negative control (CTL). The data are shown as the mean ± SEM of three biological replicates carried out in duplicate.

Fig. 8. Bithionol causes ferroptosis in AML cells.

Mitochondrial ROS in KG-1a cells after 1 (A) and 24 (B) h of treatment with bithionol. C, D Effects of the selective ferroptosis inhibitor ferrostatin-1 on the death of KG-1a cells induced by bithionol. The cells were pretreated for 2 h with 1 μM ferrostatin-1 and then co-incubated with 28 μM bithionol for 72 h. Vehicle (0.2% DMSO) was used as a negative control (CTL), and hydrogen peroxide (H₂O₂, 100 µM) was used as a positive control. The data are shown as the mean ± SEM of three biological replicates carried out in duplicate. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test. ^#p < 0.05 compared with the respective treatment without inhibitor by one-way ANOVA followed by Dunnett’s multiple comparisons test. MFI mean fluorescence intensity.

Fig. 9. Bithionol synergises with venetoclax in AML cells.

A Heatmap of the results of the drug combination assay carried out in KG-1a, KG-1, Kasumi-1, and HL-60 cells after 72 h of incubation with bithionol (28 μM) and 48 selected drugs (2 nM) of different pharmacological classes, the majority of which are already used to treat myeloid malignancies. B Heatmap of the results of the drug combination assay carried out in KG-1a, KG-1, Kasumi-1, and HL-60 cells after 72 h of incubation with bithionol (28 μM) and 14 selected drugs (2 nM). C Combination index plot of the interaction between venetoclax and bithionol in KG-1a, KG-1, Kasumi-1, and HL-60 cells after 72 h of incubation. The synergistic, additive, and antagonistic effects of drugs are defined by combination index values of <1.0, 1.0, and >1.0, respectively. D Phosphatidylserine externalisation induced by bithionol combined with venetoclax in KG-1a, KG-1, Kasumi-1, and HL-60 cells after 72 h of incubation. The data are shown as the mean ± SEM of three biological replicates carried out in duplicate. *p < 0.05 compared with CTL by one-way ANOVA followed by Dunnett’s multiple comparisons test. E Western blot of the PARP, caspase-3, cleaved caspase-3, and BCL-2 proteins in KG-1a and KG-1 cells after treatment with bithionol combined with venetoclax. GAPDH was used as an internal control.