Decitabine Induces Delayed Reactive Oxygen Species (ROS) Accumulation in Leukemia Cells and Induces the Expression of ROS Generating Enzymes

Abstract

Purpose Azanucleoside DNA methyltransferase (DNMT) inhibitors are currently FDA-approved for treatment of myelodysplastic syndrome (MDS). The relative contributions of DNMT inhibition and other off-target effects to their clinical efficacy remain unclear. Data correlating DNA methylation reversal and clinical response have been conflicting. Consequently, it is necessary to investigate so-called off-target effects and their impact on cell survival and differentiation. Experimental Design Flow cytometry was used for cell cycle, apoptosis and reactive oxygen species (ROS) accumulation analysis. Gene expression analysis was performed using RealTime PCR. DNA methylation was detected by methylation specific PCR. Mitochondrial membrane potential was analyzed using JC-1 dye staining. Western blotting was used for quantitative protein expression analysis. Results 5-aza-2’-deoxycytidine (DAC) induced cell cycle arrest and apoptosis in leukemia cells. p53 expression was dispensable for DAC-induced apoptosis. DAC induced delayed ROS accumulation in leukemia cells but not in solid tumor cells and p53 expression was dispensable for ROS increase. ROS increase was deoxycytidine kinase-dependent, indicating that incorporation of DAC into nuclear DNA is required for ROS generation. ROS accumulation by DAC was caspase-independent and mediated the dissipation of the mitochondrial membrane potential. Concordantly, ROS scavengers diminished DAC-induced apoptosis. DAC induced the expression of different NADPH oxidase isoforms and upregulated Nox4 protein expression in an ATM-dependent manner, indicating the involvement of DNA damage signaling in Nox4 upregulation. Conclusion These data highlight the importance of mechanisms other than DNA cytosine demethylation in modulating gene expression and suggest investigating the relevance of ROS accumulation to the clinical activity of DAC.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

“…The weak correlation between AZA-induced promoter demethylation and increased gene expression implies that the mechanisms through which AZA potentiates RA are not exclusively dependent on its demethylation activity. Previous reports imply that off-target effects of AZA include the accumulation of reactive oxygen species (ROS) (29) and DNA damage (30). However, in the AZA+RA-treated tumors, there was rather a reduction of genes governing the response to oxidative phosphorylation and DNA repair in the AZA+RA-treated tumors (Fig.…”

Different Mechanisms of Drug Resistance in Myelodysplastic Syndromes and Acute Myeloid Leukemia

“…However, reactive oxygen species accumulation was not always present in the sample of AML patients after DAC treatment. Therefore, the relevance of reactive oxygen species generation in the mechanism of DAC pharmacological effectiveness should be studied more intensively in the future [76].…”

Computational fishing of new DNA methyltransferase inhibitors from natural products

“…DNMTis nucleosides, cytosine analogs, have been studied in several cancer types [22,23]. Most of these compounds that inhibit the activity of DNMT have been related with significant ¨off target¨ effects [24], a fact that has prevented their use with pharmacological purposes. However, two of them, azacitidine and decitabine, have been approved by FDA (Food and Drug Administration) for the treatment of myelodysplastic syndromes [25,26].…”