A novel organic arsenic derivative MZ2 remodels metabolism and triggers mtROS-mediated apoptosis in acute myeloid leukemia

Abstract

Purpose: Acute myeloid leukemia (AML) is one of the most common neoplasms in adults, and it is difficult to achieve satisfactory results with conventional drugs. Here, we synthesized a novel organic arsenic derivative MZ2 and evaluated its ability to remodel energy metabolism to achieve anti-leukemia.

Methods: MZ2 was characterized by the average 1-min full mass spectra analysis. Biological methods such as Western blot, qPCR, flow cytometry and confocal microscopy were used to assess the mode and mechanism of MZ2-induced death. The in vivo efficacy of MZ2 was assessed by constructing a patient-derived xenograft (PDX) AML model.

Results: Unlike the precursor organic arsenical Z2, MZ2 can effectively reduce the level of aerobic glycolysis. Our in-depth found that MZ2 inhibited the expression of PDK2 in a dose-dependent manner and did not affect the expression of LDHA, another key enzyme of the glycolytic pathway. MZ2 reconstituted energy metabolism to induce the generation of mitochondrial ROS (mtROS) and then triggerd intrinsic apoptosis pathway. We also assessed whether MZ2 generates autophagy and results showed that MZ2 can induce autophagy of AML cells, which may be associated with the precursor organic arsenic drug. In vivo, MZ2 effectively attenuated leukemia progression in mice, and immunohistochemical results suggested its PDK2 inhibitory effect.

Conclusion: In summary, the novel organic arsine derivative MZ2 exhibited excellent anti-tumor effects in acute myeloid leukemia, which may provide a potential strategy for the treatment of acute myeloid leukemia.

Keywords: Apoptosis; Leukemia; Organic arsine; Pyruvate dehydrogenase kinase 2; Warburg effect.

Fig. 1

Characterization of MZ2. A The chemical structure of MZ2 and the process of synthesis from Z2. B The average 1-min full mass spectra of MZ2 showed base ions at m/z 339.98. Characterization of MZ2 using ¹H (C) and ¹³C (D) NMR spectroscopy

Fig. 2

Cytotoxic effects of MZ2. A Molm-13, THP-1, U937 and C1498 were treated with MZ2 at various concentrations and times. The cell viability of MZ2 was measured by CCK-8 Assay. B IC50 represents the 50% concentration of inhibition. C AML cells were treated with 0.6 μM of MZ2 and Z2 for 24 h, and then cell viability was measured. D Healthy peripheral blood mononuclear cells (PBMC) and human embryonic kidney cells (293 T) were treated with 0.6 μM of MZ2, Z2, Ara-C and ATO for 24 h, and then cell viability was measured. The results are presented as mean ± standard deviation (SD) from three independent experiments. ***P < 0.001 and ****P < 0.0001

Fig. 3

PDK2 is highly expressed in AML, and MZ2 reduces lactate production by inhibiting PDK2 activity. A GEO13159-derived bioinformatics analysis of PDK2 expression in acute myeloid leukemia (AML). Overall survival curves obtained from OHSU (B), TARGET (C), and TCGA (D) for significant associations between PDK2 and AML survival (P < 0.05). The lactate content in culture medium (E) and Molm-13 cells (F) was determined using a commercial lactate kit. G Molm-13 cells were treated with different concentrations of MZ2 for 24 h, and cell lysates were used to measure LDHA activity. H Molm-13 cells were treated with different concentrations of MZ2 for 24 h and mitochondria were extracted to measure PDH activity. I Molm-13 cells were treated with different concentrations of MZ2 for 24 h and the levels of phosphorylated PDHA1, total PDHA1 and PDK2 were detected by Western blot. J Molm-13 cells were treated with MZ2 and Z2 for 24 h, the levels of phosphorylated PDHA1 and total PDHA1 were detected by Western blot. K The RNA changes to PDK2 after MZ2 and Z2 treatment for 24 h. The results are presented as mean ± standard deviation (SD) from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 4

MZ2 induces apoptosis AML cells. Molm-13 (A), U937 (B), C1498 (C) and AML patient primary cells (AML-PC) (D) were treated with different concentrations of MZ2 for 24 h and analyzed using Annexin V-FITC/PI by flow cytometry. Histograms (E) show the percentage of apoptosis from three independent experiments. F Mitochondrial membrane potential was measured with the fluorescent mitochondrial probe JC-1 and assessed by flow cytometry. The results are presented as mean ± standard deviation (SD) from three independent experiments. *P < 0.05, and ****P < 0.0001

Fig. 5

Cellular morphological changes observed using the intelligent whole-blood imaging flow cytometer and MZ2-induced cell cycle arrest in AML. A Molm-13 and U937 cells were treated with different concentrations of MZ2 for 24 h. Western blot was used to examine apoptosis-associated proteins such as PARP, cleaved PARP, caspase3, cleaved-caspase3, Bcl-2, and Bax. B Molm-13 and U937 cells were treated with various concentration of MZ2, yielding a library of AML images at a flow rate of 10 m/s. Scale bars: 10 μm. After treating Molm-13 (C) and U937 (D) cells with 0.6 μM MZ2 for 24 h, and the cell cycle was analyzed. The results are presented as mean ± standard deviation (SD) from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 6

MZ2 induces autophagy in AML cells, and inhibition of autophagy suppresses MZ2-induced apoptosis. A Cells treated with or without MZ2 (0.6 μM) for 24 h were collected and then incubated with 50 nM of LysoTracker Red. Red spots show lysosomes and autolysosomes. Scale bars: 5 μm. B Molm-13 and U937 cells were Transfected with mRFP-GFP-LC3 tandem construct lentivirus and then treated with or without MZ2 (0.6 μM) for 24 h. Autophagosome formation was observed using confocal microscopy. Scale bars: 5 μm. C The morphological characteristics of autophagy was observed TEM after Molm-13 cells were treated with 0.6 μM MZ2 for 24 h. Magnification: × 5000. D, F Molm-13 and U937 cells were treated with different concentrations of MZ2 for 24 h. Autophagy-associated proteins were analyzed by Western blot, ULK-1, P62/SQSTM1, Beclin-1 and LC3. E, G Molm-13 cells were pre-incubated with 3-MA (2.5 μM) for 4 h, then treated with MZ2 for 24 h and finally analyzed using Annexin V-FITC/PI by flow cytometry. H Molm-13 cells were pre-incubated with 3-MA (2.5 μM) for 4 h, then treated with different concentrations of MZ2 for 24 h, and finally cell viability was measured by CCK-8. The results are presented as mean ± standard deviation (SD) from three independent experiments. **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 7

MZ2 induces mitochondrial ROS (mtROS) production and blocks PI3K/AKT / mTOR signaling pathway. A Mitochondria-specific superoxide probe Mito SOX Red was used to detect mtROS production. Molm-13 cells were treated with different concentrations of MZ2 for 24 h and the mean fluorescence intensity was measured by flow cytometry. B Molm-13 cells treated with or without MZ2 (0.6 μM) for 24 h were collected and then incubated with 50 nM of MitoTracker Green. Mitochondrial green fluorescence intensity observed by flow cytometry. (C) AML cells were treated with 0.6 μM MZ2 for 24 h. Cell homogenates were harvested and ATP levels were measured by reading RLU. D, E Molm-13 cells were treated with MZ2 and NAC (ROS scavenger) for 24 h and finally analyzed using Annexin V-FITC/PI by flow cytometry. F Molm-13 cells were incubated with NAC and different concentrations of MZ2 for 24 h, and finally cell viability was measured by CCK-8. G–I AML cells were treated with various concentrations of MZ2 for 24 h. Levels of p-mTOR, mTOR, p-PI3K, PI3K, p-AKT and AKT, were analyzed by western blot. The results are presented as mean ± standard deviation (SD) from three independent experiments. **P < 0.01, ***P < 0.001 and ****P < 0.0001

Fig. 8

MZ2 inhibits leukemia progression in vivo. A Animal experiment protocol. B At day 7, one mouse was randomly executed and AML cells were observed in the peripheral blood (PB) and bone marrow (BM). C Weight change. D Survival analysis. E The remission state of bone marrow in the MZ2 group. F The immunophenotype of leukemic cells in bone marrow by flow cytometry analysis. G Statistics for the ratio of leukemic cells. H The tissue weight index. I The expression levels of p-PDHA1, PDHA1 and LC3 detected by IHC in the bone marrow biopsy. The results are presented as mean ± standard deviation (SD) from three independent experiments. *P < 0.05, ***P < 0.001 and ****P < 0.0001

Fig. 9

Schematic diagram showing the mechanism of MZ2-induced AML cell death