A comparison of azacitidine and decitabine activities in acute myeloid leukemia cell lines

Abstract

Background: The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines.

Methodology/principal findings: Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 microM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping.

Conclusions/significance: These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65:35, RNA:DNA.

Figure 1. AZA and DAC differentially affect cell viability in AML cell lines.

Cell viability of AML cell lines, KG-1a, THP-1, OCI-AML3, and HL-60, was assessed after 72 hours of treatment with AZA (•) or DAC (□) (0–50 µM) using the CellTiter-Glo assay. Standard deviation was determined from 2 or 3 independent experiments, including triplicate wells per experiment. AML  =  acute myeloid leukemia; AZA  =  azacitidine; DAC  =  decitabine.

Figure 2. AZA incorporates into RNA and DNA of KG-1a cells.

KG-1a cells were treated with 0.3 µM radiolabeled AZA ([¹⁴C]-AZA) for 24 hours. The amount of AZA incorporated into total nucleic acid, DNA, and RNA was quantified as described previously. Standard error of the mean was determined from 3 independent experiments, including triplicate wells per experiment. AZA  =  azacitidine.

Figure 3. AZA inhibits protein synthesis in KG-1a and THP-1 cells.

Cells were treated daily with AZA or DAC (0–5 µM) for 24 or 48 hours prior to metabolic labeling with ³⁵S-methionine and ³⁵S-cysteine. Protein synthesis was quantified as described previously. AZA  =  azacitidine; DAC  =  decitabine.

Figure 4. AZA and DAC cause DNMT1 depletion and induction of DNA damage in KG-1a and THP-1 cells.

Cells were treated daily with AZA or DAC (0–3 µM in KG-1a; 0–10 µM in THP-1) for 48 and 72 hours. Protein lysates were analyzed by Western analysis for DNMT1 and phospho-H2AX (Ser 139) proteins. α-Tubulin is shown as a protein loading control. AZA  =  azacitidine; DAC  =  decitabine; DNMT  =  DNA methyltransferase.

Figure 5. AZA and DAC reduce DNA methylation in KG-1a and THP-1 cells.

Cells were treated daily with AZA (•) or DAC (□) (0–3 µM) for 48 hours. DNA methylation was measured using (A) pyrosequencing of LINE-1 DNA elements in bisulfite-converted DNA and (B) Illumina GoldenGate platform. AZA  =  azacitidine; DAC  =  decitabine.

Figure 6. Effects of AZA and DAC on cell cycle and apoptosis in KG-1a cells.

KG-1a cells were treated daily with AZA or DAC (0–3 µM) for 48 hours. (A) Cell cycle effects of AZA and DAC. Cells were stained with NIM-DAPI and quantification by flow cytometry for percentage of cells in sub-G1, G0/G1, S, and G2-M phases (normalized to 100%). (B) AZA and DAC induce apoptosis in KG-1a cells. Apoptosis was detected with flow cytometry by positive staining for Annexin V (early apoptosis) and 7-AAD (late apoptosis). (C) Protein lysates were analyzed by Western analysis for detection of PARP cleavage. α-Tubulin is shown as a protein loading control. AZA  =  azacitidine; DAC  =  decitabine.

Figure 7. AZA and DAC regulate different genes in KG-1a cells.

Venn diagrams reveal the number of genes that are distinctly and commonly regulated by daily treatment with AZA (1 µM) or DAC (0.3 µM or 1 µM) in KG-1a cells at 24 and 48 hours. AZA  =  azacitidine; DAC  =  decitabine.