Lu Li, Hui-min Xi, Hao Lu, Xun Cai

Combination of Ethacrynic Acid and ATRA Triggers Differentiation and/or Apoptosis of Acute Myeloid Leukemia Cells through ROS

  • Cancer Research
  • Pharmacology
  • Molecular Medicine
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Background and objective: All-trans retinoic acid (ATRA), an effective differentiation inducer, has been applied clinically to treat acute promyelocytic leukemia (APL). Unfortunately, it is not as potent in other kinds of acute myeloid leukemia (AML). Ethacrynic acid (EA), a classical powerful diuretic, can increase reactive oxygen species (ROS) contents, which can assist ATRA in inducing differentiation in AML cells. Here, we investigated the effect of EA combined with ATRA (EA+RA) on some AML cells except APL. background: All-trans retinoic acid (ATRA) is an effective differentiation agent only for acute promyelocytic leukemia (APL), a subtype of AML. Ethacrynic acid (EA), a classical powerful diuretic, can increase the level of intracellular reactive oxygen species (ROS), which can enhance the differentiation-inducing effects of ATRA in AML cells. Methods: Apoptosis and differentiation were determined by morphology, cell viability, Annexin-V assay and CD11c expression. Western blot analysis and the detection of ROS and mitochondrial transmembrane potentials (MMP) were used to investigate the mechanisms. objective: To investigate the combined effect of EA and ATRA (EA+RA) in non-APL AML cells. Results: AML cells exhibited differentiation and/or apoptosis after EA+RA treatment. EA+RA increased the intracellular ROS contents. EA+RA-induced apoptosis was accompanied by MMP attenuation and caspase-3/7 activation. EA+RA-induced differentiation was along with MEK/ERK and Akt activation and increased expression of PU.1, CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPε. N-acetyl-L-cysteine (NAC), an antioxidant, thoroughly reduced EA+RA-increased ROS, and also inhibited MMP attenuation, the activation of caspase- 3/7, MEK/ERK and Akt pathways, the elevation of PU.1 and C/EBPs, and apoptosis and differentiation. However, MEK or PI3K specific inhibitors only suppressed EA+RA-triggered differentiation and the elevation of PU.1 and C/EBPs, but not ROS levels. Conclusion: EA+ATRA induced cell apoptosis through ROS dependent MMP attenuation and caspase 3/7 activation while inducing differentiation by ROS-MEK/ERK-PU.1/C/EBPs and ROS-Akt-PU.1/C/EBPs pathways. In summary, it may provide innovative ATRA-based combination therapy strategies for AML patients via ROS. result: EA+RA induced differentiation and/or apoptosis in AML cell lines and primary cells. EA+RA elevated the intracellular levels of ROS. EA+RA-induced apoptosis was accompanied with mitochondrial transmembrane potentials collapse and caspase-3/7 activation. EA+RA-induced differentiation was along with the activation of MEK/ERK and Akt, and up-regulation of CCAAT/enhancer-binding protein β (C/EBPβ), C/EBPε and PU.1. N-acetyl-L-cysteine (NAC), the ROS scavenger, completely reduced EA+RA-increased ROS, and also inhibited m collapse, the activation of caspase-3/7, MEK/ERK and Akt, the up-regulation of C/EBPs and PU.1, and apoptosis and differentiation. However, MEK or PI3K specific inhibitor only suppressed EA+RA-induced differentiation and up-regulation of C/EBPs and PU.1, but not ROS level.

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