Shikonin Exerts an Antileukemia Effect against FLT3-ITD Mutated Acute Myeloid Leukemia Cells via Targeting FLT3 and Its Downstream Pathways

Abstract

Introduction: Acute myeloid leukemia (AML) with internal tandem duplication (ITD) mutations in Fms-like tyrosine kinase 3 (FLT3) has an unfavorable prognosis. Recently, using newly emerging inhibitors of FLT3 has led to improved outcomes of patients with FLT3-ITD mutations. However, drug resistance and relapse continue to be significant challenges in the treatment of patients with FLT3-ITD mutations. This study aimed to evaluate the antileukemic effects of shikonin (SHK) and its mechanisms of action against AML cells with FLT3-ITD mutations in vitro and in vivo.

Methods: The CCK-8 assay was used to analyze cell viability, and flow cytometry was used to detect cell apoptosis and differentiation. Western blotting and real-time polymerase chain reaction were used to examine the expression of certain proteins and genes. Leukemia mouse model was created to evaluate the antileukemia effect of SHK against FLT3-ITD mutated leukemia in vivo.

Results: After screening a series of leukemia cell lines, those with FLT3-ITD mutations were found to be more sensitive to SHK in terms of proliferation inhibition and apoptosis induction than those without FLT3-ITD mutation. SHK suppresses the expression and phosphorylation of FLT3 receptors and their downstream molecules. Inhibition of the NF-κB/miR-155 pathway is an important mechanism through which SHK kills FLT3-AML cells. Moreover, a low concentration of SHK promotes the differentiation of AML cells with FLT3-ITD mutations. Finally, SHK could significantly inhibit the growth of MV4-11 cells in leukemia bearing mice.

Conclusion: The findings of this study indicate that SHK may be a promising drug for the treatment of FLT3-ITD mutated AML.

Keywords: Acute myeloid leukemia; Apoptosis; Differentiation; FLT3-ITD mutations; Receptor tyrosine kinase.

Fig. 1.

Proliferation inhibition effects of SHK against different leukemia cell lines. Different leukemia cell lines were treated with SHK at concentrations of 125–8,000 nm, and cell viability was determined by CCK-8 assay 24–48 h after treatment. The value of half maximum inhibitory concentration (IC50) was calculated thereafter. Data are representative of three independent experiments or presented as the mean ± SEM of at least three independent experiments. *p < 0.0332; **p < 0.0021 (analysis of variance [ANOVA] with Dunnett multiple comparison tests as post hoc analyses).

Fig. 2.

Apoptosis induction effects of SHK against different leukemia cell lines. Different leukemia cell lines were treated without or with SHK at concentrations of 250–750 nm, and cell apoptosis was determined by flow cytometry 48 h after treatment. a Representative plots of flow cytometric analysis. Apoptosis rate of MV4-11 (b), MOLM-13 (c), Kasumi-1 (d), THP-1 (e), and NB4 (f) cells after SHK treatment. Data are representative of three independent experiments or presented as the mean ± SEM of at least three independent experiments. *p < 0.0332; ***p < 0.0002, ****p < 0.0001 (analysis of variance [ANOVA] with Dunnett multiple comparison tests as post hoc analyses).

Fig. 3.

SHK induce apoptosis of AML cells with FLT3-ITD mutations via caspase-3 dependent way. MV4-11 and MOLM-13 cells were treated without or with SHK at concentrations of 62.5–250 nm, and cleaved caspase-3 was determined by Western blotting 24 h after treatment. In some experiments, caspase-3 inhibitor was used, and apoptosis was determined by flow cytometry. Representative Western blotting plots of cleaved caspase-3 of MV4-11 (a) and MOLM-13 (b) cells after treatment. Levels of cleaved caspase-3 of MV4-11 (c) and MOLM-13 (d) cells after treatment. e Representative plot of apoptosis of MV4-11 cells after SHK treatment with (w) or without (w/o) caspase-3 inhibitor Z-DEVD-FMK. f Apoptosis rates of MV4-11 cells after SHK treatment with (w) or without (w/o) caspase-3 inhibitor Z-DEVD-FMK. Data are representative of three independent experiments or presented as the mean ± SEM of at least three independent experiments. *p < 0.0332; **p < 0.0021; ***p < 0.0002 (analysis of variance [ANOVA] with Dunnett multiple comparison tests as post hoc analyses).

Fig. 4.

Expression and phosphorylation of FLT3, ERK, AKT after SHK treatment. MV4-11 cells were treated without or with SHK at concentrations of 62.5–250 nm, and expression and phosphorylation of protein were determined by Western blotting 24 h after treatment. a Representative Western blotting plots of FLT3 and pFLT3 after treatment. Levels of FLT3 (b) and pFLT3 (d) in different groups. c Representative Western blotting plots of ERK, pERK, ATK, and pATK after treatment. Levels of ERK (e), pERK (f), AKT (g), and pAKT (h) in different groups. Data are representative of three independent experiments or presented as the mean ± SEM of at least three independent experiments. *p < 0.0332; **p < 0.0021; ***p < 0.0002 (analysis of variance [ANOVA] with Dunnett multiple comparison tests as post hoc analyses).

Fig. 5.

Molecule docking to predict the interaction between SHK, FLT3 inhibitors, and FLT3. The interactions between Midostaurin, Quizartinib, Gilteritinib, SHK were performed autoDock 4.2 software. a The results of molecule docking as indicated. b The binding site of SHK on FLT3 receptor (left) and the molecular pocket (right).

Fig. 6.

SHK impeded NF-κB/miR-155 pathways after treatment. MV4-11 cells were treated without or with SHK at concentrations of 62.5–1,404 nm, and location of NF-κB was determined by immunofluorescence analysis. Expression of miR-155 was analyzed by RT-PCR and expression of CEBPA, PU.1 and SHIP-1 was detected by Western blotting. a Distribution of NF-κB p65 subunit after SHK treatment. b Representative Western blotting plots of CEBPA, SHIP-1, and PU.1 after treatment. Levels of miR-155 (c), CEBPA (d), SHIP-1 (e), and PU.1 (f) in different groups. Data are representative of three independent experiments or presented as the mean ± SEM of at least three independent experiments. *p < 0.0332; **p < 0.0021; ***p < 0.0002 (analysis of variance [ANOVA] with Dunnett multiple comparison tests as post hoc analyses). RT-PCR, real-time polymerase chain reaction.

Fig. 7.

SHK induced the differentiation of MV4-1 cells. MV4-11 cells were treated without or with SHK at concentrations of 32.5–125 nm for 1 week, and expression of clusters of differentiation was determined by flow cytometry. Shown are the expression of CD11b, CD11c, CD14, CD15, CD36, CD123, and CD33 after SHK treatment.

Fig. 8.

SHK inhibited the growth of MV4-11 cells in vivo. Leukemia mouse model was created using MV4-11 cells (s.c.; 2 × 10⁶/mouse), and they were treated with SHK or control reagent once every other day for five times when the leukemia mass was palpable. The leukemia volume was determined every 2∼3 days post injection. a Leukemia growth curves of female mice in SHK and control groups (up) and leukemia tissue mass of female mice in SHK and control groups when they were sacrificed (bottom) (n = 5 mice per mouse). b Leukemia growth curves of male mice in SHK and control groups (up) and leukemia tissue mass of male mice in SHK and control groups when they were sacrificed (bottom) (n = 5 mice per group). c Bodyweight change of male mice in SHK and control groups (n = 5 mice per group). Two-way ANOVA followed by Sidak’s multiple comparison test (for ANOVA p value was defined as **p < 0.01 and ****p < 0.0001; for Sidak’s multiple comparison test p value was defined as *p < 0.0332, **p < 0.0021, and ***p < 0.0002).