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Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia.

ABSTRACT: The incidence of acute myeloid leukemia (AML) is rising and the outcome of current therapy, which has not changed significantly in the last 40 years, is suboptimal. Cellular oxidative state is a credible target to selectively eradicate AML cells, because it is a fundamental property of each cell that is sufficiently different between leukemic and normal cells, yet its aberrancy shared among different AML cells. To this end, we tested whether a short-time treatment of AML cells, including cells with FLT3-ITD mutation, with sub-lethal dose of dichloroacetate (DCA) (priming) followed by pharmacologic dose of arsenic trioxide (ATO) in presence of low-dose DCA could produce insurmountable level of oxidative damage that kill AML cells. Using cellular cytotoxicity, apoptotic and metabolic assays with both established AML cell lines and primary AML cells, we found that priming with DCA significantly potentiated the cytotoxicity of ATO in AML cells in a synergistic manner. The combination decreased the mitochondrial membrane potential as well as expression of Mcl-1 and GPx in primary AML cells more than either drug alone. One patient with AML whose disease was refractory to several lines of prior treatments was treated with this combination, and tolerated it well. These data suggest that targeting cellular redox balance in leukemia may provide a therapeutic option for AML patients with relapsed/refractory disease.

SUBMITTER: Emadi A

PROVIDER: S-EPMC7310114 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia.

Emadi Ashkan A Sadowska Mariola M Carter-Cooper Brandon B Bhatnagar Vishal V van der Merwe Isabella I Levis Mark J MJ Sausville Edward A EA Lapidus Rena G RG

Leukemia research 20150430 7

The incidence of acute myeloid leukemia (AML) is rising and the outcome of current therapy, which has not changed significantly in the last 40 years, is suboptimal. Cellular oxidative state is a credible target to selectively eradicate AML cells, because it is a fundamental property of each cell that is sufficiently different between leukemic and normal cells, yet its aberrancy shared among different AML cells. To this end, we tested whether a short-time treatment of AML cells, including cells w ...[more]

PMID: 25982179

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Project description:BackgroundThe therapeutic efficacy of arsenic trioxide (As2O3) in acute myeloid leukemia (AML) is modest, which is partly related to its limited intracellular uptake into the leukemic cells. As2O3 enters cells via the transmembrane protein aquaglyceroporin 9 (AQP9). Azacytidine, a demethylating agent that is approved for the treatment of AML, has been shown to have synergistic effect with As2O3. We tested the hypothesis that azacytidine might up-regulate AQP9 and enhances As2O3-mediated cytotoxicity in AML.MethodsArsenic-induced cytotoxicity, the expression of AQP9, and the intracellular uptake of As2O3 were determined in AML cell lines and primary AML cells with or without azacytidine pre-treatment. The mechanism of AQP9 up-regulation was then investigated by examining the expression of transcription factors for AQP9 gene and the methylation status of their gene promoters.ResultsAs2O3-induced cytotoxicity in AML cell lines was significantly enhanced after azacytidine pre-treatment as a result of AQP9 up-regulation, leading to increased arsenic uptake and hence intracellular concentration. Blocking AQP9-mediated As2O3 uptake with mercury chloride abrogated the sensitization effect of azacytidine. AQP9 promoter does not contain CpG islands. Instead, azacytidine pre-treatment led to increased expression of HNF1A, a transcription activator of AQP9, through demethylation of HNF1A promoter. HNF1 knockdown abrogated azacytidine-induced AQP9 up-regulation and almost completely blocked intracellular As2O3 entry, confirming that azacytidine enhanced As2O3-mediated cell death via up-regulation of HNF1A and hence increased AQP9 and As2O3 intracellular concentration. Azacytidine sensitization to As2O3 treatment was re-capitulated also in primary AML samples. Finally, azacytidine did not enhance arsenic toxicity in a liver cell line, where HNF1A was largely unmethylated.ConclusionsAzacytidine sensitizes AML cells to As2O3 treatment, and our results provide proof-of-principle evidence that pharmacological up-regulation of AQP9 potentially expands the therapeutic spectrum of As2O3. Further clinical trial should evaluate the efficacy of azacytidine in combination with As2O3 in the treatment of AML.

Project description:BACKGROUND:At present, it is generally believed that leukemia stem cells are the source of AML, so the killing of leukemia stem cells has become important. Previous studies have suggested that HHT combined with ATO can synergistically kill U937 cells, and HHT has also demonstrated the ability to kill leukemia stem cells. We evaluated whether HHT combined with ATO can systematically kill leukemia stem cells (LSCs) and explored the synergistic effect and molecular mechanism. METHODS:CCK-8 was used to detect cell viability. The changes of cell cycle (PI staining), apoptosis (Annexin V/PI) and surface markers (CD34, CD38, CD96, CD45) were detected by flow cytometry. The cells of CD34+ primary leukemia and CD38- KG-1, and TF-1 were separated by flow cytometry. High-throughput mRNA sequencing was used to analysis mRNA level changes after the application of the two drugs. Western blot was used to verify the changes of pathway protein expression. NRG mice were used as the receptor of xenograft model. Histological H&E staining assess the invaded ability of leukemia cells, and laser scanning confocal microscopy evaluated the molecule markers change. RESULTS:HHT and ATO synergistically killed KG-1 (CD34+/CD96+/CD38+/-) and Kasumi-1 (CD34+/CD38-) cells. Their combination had a stronger effect of inducing apoptosis and blocking the cell cycle than HHT or ATO administrator alone, meanwhile significantly reducing the numbers of LSCs. Further, CD34+CD38- cells in KG-1, KG-1a, TF-1, and primary leukemia cells were more sensitive to HHT and ATO. High-throughput mRNA sequencing suggested that HHT alone could significantly upregulate molecules related to the Notch, P53, and NF-?B signaling pathways. When combined with ATO, HHT further upregulated P53, whereas HHT-induced NF-?B pathway activation was significantly suppressed. Western blot analysis verified the change of protein expression in the above pathways and further demonstrated that GSI, could eliminate these effects. In vivo, HHT combined with ATO significantly reduced the LSC burden, and weakened the expression of LSC markers. CONCLUSIONS:This is the first evidence that HHT combined with arsenic can synergistically kill LSCs in vitro and in vivo, along with identification of the underlying mechanism, highlighting a potentially effective treatment strategy.

Project description:Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25-200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.

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Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia.

Publications

Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia.

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