Antineoplastic effects and mechanisms of micheliolide in acute myelogenous leukemia stem cells

Abstract

Leukemic stem cells (LSCs) greatly contribute to the initiation, relapse, and multidrug resistance of leukemia. Current therapies targeting the cell cycle and rapidly growing leukemic cells, including conventional chemotherapy, have little effect due to the self-renewal and differentiated malignant cells replenishment ability of LSCs despite their scarce supply in the bone marrow. Micheliolide (MCL) is a natural guaianolide sesquiterpene lactone (GSL) which was discovered in michelia compressa and michelia champaca plants, and has been shown to exert selective cytotoxic effects on CD34+CD38- LSCs. In this study, we demonstrate that DMAMCL significantly prolongs the lifespan of a mouse model of human acute myelogenous leukemia (AML). Mechanistic investigations further revealed that MCL exerted its cytotoxic effects via inhibition of NF-κB expression and activity, and by generating intracellular reactive oxygen species (ROS). These results provide valuable insight into the mechanisms underlying MCL-induced cytotoxicity of LSCs, and support further preclinical investigations of MCL-related therapies for the treatment of AML.

Keywords: leukemic stem cells; micheliolide.

Figure 1. MCL selectively induces the apoptosis of AML leukemic stem cells, but not normal hematopoietic stem cells

(A) Percentage of apoptosis was assessed in KG1a cells treated with MCL. Control represents untreated cells. (B) Percentage of apoptosis was assessed in cells isolated from primary AML specimens (n = 20) and treated with MCL. (C) Representative flow cytometry scatter plots displaying apoptotic cell populations after treatment with MCL. (D) Percentage of apoptosis was assessed in KG1a cells treated with MCL at different time points over 24 h. (E) Percentage of viability was assessed in MNCs, LSCs (CD34⁺), and LSPCs (CD34⁺ CD38⁻) isolated from AML specimens and treated with MCL. (F) Percentage of apoptosis was assessed in MNCs and HSCs (CD34⁺CD38⁻) isolated from human umbilical cord blood and treated with MCL. (G) Colony-forming abilities of MNCs isolated from primary AML specimens and treated with MCL. (H). Colony-forming abilities of HSCs isolated from human umbilical cord blood and treated with MCL. Control represents untreated cells. Error bars represents the SEM. ***P < 0.001; **P < 0.01; *P < 0.05; ns, no significance.

Figure 2. MCL improves the survival of mice with human AML

(A) Survival plot representing the percentage of surviving NOD/SCID mice injected with human AML cells and treated with varying doses of DMAMCL. (B) Percentage of CD45⁺ cell engraftment in the bone marrow of mice after treatment with control, DMAMCL, or ADR. (C) Percentage of mice exhibiting varying degrees of leukemic cell engraftment after treatment with control, DMAMCL, or ADR. Administration of PBS is represented by Control and ADR was administered as a positive control.

Figure 3. Transcriptome analysis of MCL-treated cells

(A) Heat map analysis of microarray data showing hierarchical clustering of differentially expressed genes between untreated and MCL-treated KG1a cells. Three independent untreated and MCL-treated cell lines were analyzed. (B) Heat map demonstrating the differences in the anti-apoptotic transcriptional signature between control and MCL-treated cells. (C) Heat map demonstrating the differences in the pro-apoptotic transcriptional signature between control and MCL-treated cells. (D) Heap map demonstrating the differences in the NF-κB transcriptional signature between control and MCL-treated cells. (E) Heap map demonstrating the differences in the ROS transcriptional signature between control and MCL-treated cells. (F) Western blot analysis of apoptosis-associated proteins in Control and MCL-treated KG1a cells. GAPDH was used as a loading control.

Figure 4. MCL inhibits NF-κB expression and activity in leukemic cells

(A) Relative NF-κB expression in mononuclear cells isolated from human umbilical cord blood or primary AML specimens. Expression was normalized to GAPDH.(b) Relative NF-κB expression in mononuclear and HSPCs isolated from human umbilical cord blood. Expression was normalized to GAPDH. (B) Relative NF-κB expression in HSPCs isolated from umbilical cord blood and LSPCs isolated from primary AML specimens. Expression was normalized to GAPDH. (C) Relative NF-κB expression in AMI sample treated with 10 μM MCL after 2 h and 6 h. Control represents untreated cells. Expression was normalized to GAPDH. (D) Immunofluorescence assays detected cellular localization of NF-κB in bone marrow cells isolated from the AML mouse. The P65 subunit is shown in green and the nuclei are shown in red. (E) EMSA assay exhibiting NF-κB binding to DNA at various time points after primary AML cells were treated with 10 μM MCL. Control represents untreated cells. (F) Quantitative densitometry of gel shifts from (f) relative to control cells. (G) MNCs-mononuclear cells, UCB-umbilical cord blood, HSPCs-hematopoietic stem/progenitor cells, and LSPCs-leukemic stem/progenitor cells. (H) The gene expression of NF-κB in AML specimens compared to normal cells.

Figure 5. Generation of intracellular ROS promotes MCL-induced apoptosis

(A) ROS levels in primary AML cells treated with 10 μM MCL at various time points over 240 min as measured by DCF-DA fluorescence. (B) ROS levels in primary CD34⁺ LSPCs treated with MCL for 1 h. (C) ROS levels in primary AML cells co-treated with MCL and NAC. (D) Percentage of viable LSPCs pretreated with NAC and then exposed to MCL. (E) Relative HO-1 expression in primary AML cells treated with 10 μM MCL after 2 h and 6 h. Control represents untreated cells. Expression was normalized to GAPDH. (F) Western blot analysis of HO-1 protein expression in primary AML cells treated with 10 μM MCL for 6 h. GAPDH was used as a loading control. (G) Immunofluorescence assays that show cellular localization of HO-1 and Nrf2 in bone marrow cells isolated from AML mice. HO-1/Nrf2 proteins are shown in green and nuclei are shown in red. (H) Western blot analysis of p65 protein expression with or without ROS inhibitor NAC. (I) Mechanism of MCL in inducing cell apoptosis was summarized.