Honokiol Induces Ferroptosis by Upregulating HMOX1 in Acute Myeloid Leukemia Cells

Figures

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  FIGURE 1. Effects of honokiol on the viability and apoptosis of THP-1, U-937, and SKM-1 cells. (A) Chemical structure of honokiol. (B) Effects of honokiol on the viability of THP-1, U-937, and SKM-1 cells. Cells were incubated with different concentrations of honokiol for 24, 48, 72 h, then CCK-8 was added, and the absorbance at 450 nm was determined. The points represent mean ± SEM from three independent experiments. (C) Cytotoxic effect of honokiol on human PBMCs. PBMCs were incubated with different concentrations of honokiol for 24, 48, 72 h, then CCK-8 reagent was added, and the absorbance at 450 nm was determined. The points represent mean ± SEM from three independent experiments.

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  FIGURE 2. Effects of honokiol on the apoptosis of THP-1, U-937, and SKM-1 cells. The cells were incubated with the indicated concentration of honokiol for 24 h, and Annexin-V-FITC/PI staining and flow cytometry analysis were performed. The experiments were performed in triplicate. Statistical analysis of cell apoptosis was shown as bar graphs (*, p < 0.05, **, p < 0.01, compared to the control group).

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  FIGURE 3. Honokiol arrests cell cycle of THP-1, U-937 and SKM-1 cells at G0/G1 Phases. The cells were incubated with the specified concentration of honokiol (THP-1: 20 μM; U-937: 35 μM; SKM-1: 30 μM) and then analyzed using flow cytometry with PI staining. Statistical analysis of G0/G1 phase cells was shown as a bar graph (*, p < 0.05, **, p < 0.01, compared to the control group). These data are from three separate replicates.

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  FIGURE 4. Honokiol inhibits colony formation of THP-1 and SKM-1 cells. (A) Cell colony results of THP-1 and SKM-1 cells. Cells were incubated with different concentrations of honokiol for 14 days and then observed under a light microscope. (B) Bar graphs show the statistical analysis of colony formation, presented as (*, p < 0.01; **, p < 0.001). These results represent three separate experiments.

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  FIGURE 5. The mechanism of the inhibitory effect of honokiol on THP-1 and U-937 cells. (A) Venn diagram. (B) KEGG pathway analyses of all differentially expressed genes. (C) Heatmap of the partial differentially expressed genes. (D) Protein-Protein interaction (PPI) Network diagram.

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  FIGURE 6. Effects of honokiol on lipid peroxidation in THP-1, U-937, and SKM-1 cells. The cells were incubated with different concentrations of honokiol for 24h, then liperfluo was added for flow cytometry analysis. Statistical analysis of lipid peroxide was presented as scatter plots (*, p < 0.05; **, p < 0.01).

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  FIGURE 7. HMOX1 is a necessary molecule to induce ferroptsis in AML cells induced by honokiol. (A) The mRNA expression of HMOX-1, SLC7A11, and GPX4 after honokiol treatment, showing Honokiol increased the expression of HO-1 and SLC7A11. The statistical analysis of mRNA expression was presented as bar graphs (*, p < 0.05; **, p < 0.01). (B) Western blot results on the protein expression of HMOX-1, SLC7A11, and GPX4 in 3 cell lines. (C) HMOX1 inhibitor ZnPP rescued the ferroptosis of AML cells induced by honokiol. Compared with honokiol treatment group, the cell viability of honokiol combined with ZnPP treated group was significantly increased (**, p < 0.01).