Alantolactone selectively ablates acute myeloid leukemia stem and progenitor cells

Abstract

Background: The poor outcomes for patients diagnosed with acute myeloid leukemia (AML) are largely attributed to leukemia stem cells (LSCs) which are difficult to eliminate with conventional therapy and responsible for relapse. Thus, new therapeutic strategies which could selectively target LSCs in clinical leukemia treatment and avoid drug resistance are urgently needed. However, only a few small molecules have been reported to show anti-LSCs activity.

Methods: The aim of the present study was to identify alantolactone as novel agent that can ablate acute myeloid leukemia stem and progenitor cells from AML patient specimens and evaluate the anticancer activity of alantolactone in vitro and in vivo.

Results: The present study is the first to demonstrate that alantolactone, a prominent eudesmane-type sesquiterpene lactone, could specifically ablate LSCs from AML patient specimens. Furthermore, in comparison to the conventional chemotherapy drug, cytosine arabinoside (Ara-C), alantolactone showed superior effects of leukemia cytotoxicity while sparing normal hematopoietic cells. Alantolactone induced apoptosis with a dose-dependent manner by suppression of NF-kB and its downstream target proteins. DMA-alantolactone, a water-soluble prodrug of alantolactone, could suppress tumor growth in vivo.

Conclusions: Based on these results, we propose that alantolactone may represent a novel LSCs-targeted therapy and eudesmane-type sesquiterpene lactones offer a new scaffold for drug discovery towards anti-LSCs agents.

Keywords: Acute myeloid leukemia stem cells; Alantolactone; Apoptosis; KG1a.

Fig. 1

Cytotoxicity of alantolactone against different leukemia cell lines. Cytotoxicity was assayed by MTT. HL60, HL60/ADR, KG1a, K562, K562/A02, and THP-1 cells were treated with a range dose of alantolactone (1.25, 2.5, 5, 10, 20, and 50 μM) for 72 h, respectively. Numerical analysis data represented mean ± SD based on three independent experiments

Fig. 2

Alantolactone induced the apoptosis and differentiation of leukemia stem-like cell line KG1a and primary AML cells. a The expression of CD34⁺CD38^– on the surface of KG1a cells by flow cytometry. The χ axis represented CD34-APC, and the Y axis represented CD38-AF488. b Alantolactone induced the differentiation of leukemia stem cells after 3 days treatment. c Alantolactone induced apoptosis of primary AML cells with a dose-dependent manner after the treatment of alantolactone for 18 h. Each point represents an AML specimen. d Alantolactone induced apoptosis with a dose-dependent manner in KG1a cells with the treatment of alantolactone for 24 h. Analysis results represented mean ± SD based on three independent experiments, **P < 0.01, ***P < 0.0001

Fig. 3

Alantolactone selectively induced apoptosis of LSCs in primary AML cells. a Alantolactone induced the apoptosis of CD34⁺CD38^– cells with a dose-dependent manner in primary AML cells from AML specimens after treatment of alantolactone for 18 h. Each point represents an AML specimen. b Alantolactone reduced the percentage of CD34⁺CD38^– cells with a dose-dependent manner in primary AML cells after the treatment of alantolactone for 18 h. Each point represents an AML specimen. c Alantolactone selectively induced apoptosis of CD34⁺CD38^– cells comparing with total cells in primary AML cells after the treatment of alantolactone for 18 h by flow cytometry. d Ara-C showed negligible cytotoxicity to CD34⁺CD38^– and total cells in primary AML cells at the concentrations of 5 and 10 μM. e Alantolactone induced apoptosis of CD34⁺CD38^– cells in primary AML cells by flow cytometry at the concentrations of 2.5, 5, and 10 μM. f Ara-C showed almost no effect on inducing apoptosis of CD34⁺CD38^– cells in primary AML cells at the concentrations of 5 and 10 μM. g Alantolactone treatment demonstrated significantly less effect on inducing apoptosis of CD34⁺CD38^– cells in normal hematopoietic cells comparing to that in primary AML cells at a concentration of 10 μM. *P < 0.05, **P < 0.01, ***P < 0.0001

Fig. 4

Alantolactone suppressed primary AML but not normal colony formation after 14 days in MethoCult H4434. a Alantolactone dramatically reduced the number of CFUs in primary AML cells from eight AML specimens with a dose-dependent manner. b The representative microscopy images of CFUs in primary AML cells were shown. c Alantolactone showed little effect on the CFUs in normal hematopoietic cells and Ara-C treatment led to notable reduction of colonies from normal donors. d The representative microscopy images of CFUs in normal hematopoietic cells were shown. *P < 0.05, **P < 0.01, ***P < 0.0001

Fig. 5

DMA-alantolactone inhibited tumor growth effectively in vivo. a Synthesis of the pro-drug of alantolactone, i.e., DMA-alantolactone. b The body weight of the mice after the oral administration of alantolactone at a dose of 500 mg/kg. c KG1a cells were injected into 5-week-old female BALB/c nude mice. After 20 days, tumor was cut to the same volume (0.5 mm³). The small tumor pieces were implanted into mice. When tumor volume reached to 100 mm³, DMA-alantolactone was administrated by oral every 3 days with 100 mg/kg. Mice were sacrificed after 30 days. *P < 0.05, **P < 0.01, ***P < 0.0001

Fig. 6

Alantolactone induced apoptosis by the suppression of NF-kB and its downstream target proteins. The expression of p65, XIAP, FLIP, Bcl-2, Bax, and the cleavage of PARP, caspase-3, caspase-9 was detected by western blot assay after the treatment of alantolactone for 12 h in primary CD34⁺ cells, HL60, and KG1a cells