Decitabine Downregulates TIGAR to Induce Apoptosis and Autophagy in Myeloid Leukemia Cells

Abstract

Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to hypomethylation, DAC in AML is also involved in cell metabolism, apoptosis, and immunity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) functions to inhabit glycolysis and protect cancer cells from reactive oxygen species- (ROS-) associated apoptosis. Our previous study revealed that TIGAR is highly expressed in myeloid leukemia cell lines and AML primary cells and associated with poor prognosis in adult patients with cytogenetically normal AML. In the present study, it was found that in a time- and concentration-dependent manner, DAC downregulates the TIGAR expression, induces ROS production, and promotes apoptosis in HL-60 and K562 cells. However, blocking the glycolytic pathway partially reversed the combined effects of DAC and TIGAR knockdown on apoptosis, ROS production, and cell cycle arrest, indicating that DAC induced apoptosis through the glycolytic pathway. Furthermore, TIGAR also has a negative impact on autophagy, while DAC treatment upregulates autophagy-related proteins LC3, Beclin-1, ATG3, and ATG-5, downregulates p62, and promotes the formation of autophagosomes, indicating that DAC may activate autophagy by downregulating TIGAR. Taken together, DAC plays an unmethylated role in inducing apoptosis and activating autophagy in myeloid leukemia by downregulating TIGAR.

Figure 1

DAC inhibits the TIGAR mRNA and protein expression. The TIGAR mRNA (a) and protein (b) levels were observed when HL-60 cells were treated with different concentrations of DAC (0.25, 0.50, and 1.00 μM) for 24 hours. The TIGAR mRNA (c) and protein (d) levels were observed when HL-60 cells were treated with 0.5 μM of DAC for different lengths of time (1, 3, 7, and 24 hours). The TIGAR mRNA (e) and protein (f) levels were observed when HL-60 cells were treated with or without 0.5 μM of DAC for 24 hours after TIGAR with or without knockdown. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001, NS P > 0.05 compared to the control group; ^###P < 0.001 compared to DAC alone.

Figure 2

DAC induces intracellular ROS production by downregulating TIGAR. HL-60 and K562 cells were treated with different doses of DAC for 24 hours. (a, b) The production of ROS was assessed by FCM. (c, d) NADPH levels were detected by the NADPH detection kit. The NADPH (e) and ROS (f) levels in K562 cells, with or without TIGAR overexpression, and HL-60 cells, with or without TIGAR knockdown, when compared with CD34+ cells. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001, NS P > 0.05 compared to the control group.

Figure 3

DAC triggered the ROS-associated apoptosis by downregulating TIGAR. (a) FCM analysis of the apoptosis using the double staining of Annexin-V-R-PE and 7-AAD in HL-60 and K562 cells with the treatment of 0.25, 0.50, and 1.00 μM of DAC for 24 hours. (b) The levels of caspase-3, -8, and -9, and cytochrome c were detected by western blot. (c) The FCM analysis of the apoptosis of K562 cells, with or without TIGAR overexpression, and HL-60 cells, with or without TIGAR knockdown. The ROS levels (d) and apoptosis (e) of HL-60 cells after DAC treatment in the presence or absence of NADPH. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001, NS P > 0.05 compared to the control group.

Figure 4

DAC induces apoptosis by reducing p-Tyr and BCR-ABL. The 0.5 μM of DAC and 0.1 μM of Imatinib treatment of K562, K562/R, and K562/TIGAR cells is shown, and the level of p-Tyr was assessed by FCM. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗P < 0.05, ^∗∗∗P < 0.001, NS P > 0.05 compared to the control group.

Figure 5

DAC induces the apoptosis by reducing p-Tyr and BCR-ABL. The 0.5 μM of DAC and 0.1 μM of imatinib treatment of K562, K562/R, and K562/TIGAR cells is shown, and the BCR-ABL protein (a) and apoptosis (b) were assessed by FCM. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to the control group.

Figure 6

The inhibition of the glycolysis pathway attenuates the effects of DAC. The ROS levels (a), cell cycle distribution (b), and apoptosis (c) of HL-60 cells detected by FCM after TIGAR knockdown, and 0.5 μM of DAC treatment, in the presence or absence of 1 mg/ml of 2-DG are shown. The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, NS P > 0.05 compared to the control group.

Figure 7

The TIGAR knockdown enhanced the DAC-induced autophagy activation. HL-60 cells were treated with 0.25, 0.50, and 1.00 μM of DAC for 24 hours, with or without TIGAR knockdown. The levels of LC3, Beclin-1, and ATG5 were detected by western blot (a) and qPCR (b). The values were presented as the mean ± standard deviation (SD) of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared to the control group.

Figure 8

The TIGAR knockdown enhanced the DAC-induced autophagy activation. HL-60 cells were treated with 0.25, 0.50, and 1.00 μM of DAC for 24 hours, with or without TIGAR knockdown. The autophagosome formation was detected by transmission electron microscopy. Scale bar: 500 nm. Red arrows indicate autophagosomes.