Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9

Abstract

Background: The 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.

Methods: Arsenic-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.

Results: As2O3-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.

Conclusions: Azacytidine 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.

Figures

Figure 1

Sensitization of leukemia cells to As2O3-induced cytotoxicity with azacytidine (5′Aza) pre-treatment. (A) Dose-dependent cytotoxicity of As2O3 with or without 5′Aza treatment in the human AML cell lines, HL-60, K562, NB4, and OCI-AML3. As determined by MTT assay, all four cell lines pre-treated with 5′Aza exhibited significant decrease in cell viability as compared with their untreated controls. Data were acquired from three independent experiments, and relative survival of each individual cell line was normalized to its respective control. (B) Representative plot of flow cytometric analysis, showing a significant increase in As2O3-mediated cytotoxicity (annexin V-positive cells) in K562 cells pre-treatment with 5′Aza, as compared with control cells not treated with 5′Aza. (C) Bar charts showing the fold changes of As2O3-induced cell death (annexin V-positive cells) in each AML lines with or without 5′Aza pre-treatment. Quantitative analysis by flow cytometry further confirmed a significant increase in As2O3-induced apoptotic cell death after 5′Aza pre-treatment.

Figure 2

Azacytidine-mediated up-regulation of AQP9 expression and intracellular arsenic uptake. (A) AQP9 mRNA expression was significantly up-regulated with 5′Aza treatment in HL-60, K562, NB4, and OCI-AML3 cells, as determined by RT-PCR (upper panel) and quantitative RT-PCR (lower panel). Sample data were normalized to the control of each cell line individually by the ΔΔCT method. (B) Up-regulation of membrane AQP9 protein expression was also confirmed by flow cytometric analysis. (C) Intracellular elemental arsenic concentration is significantly increased in K562 cells after pre-treatment with 5′Aza. (D) Treatment of K562 cells with HgCl2 completely abrogated the cytotoxicity induced by As2O3, either alone (left panel) or in cells pre-treatment with 5′Aza (right panel).

Figure 3

Quantitative RT-PCR analysis of the expression of potential transcription factors for the AQP9 gene after 5′Aza treatment in HL-60 and K562 cells. The expressions of five transcription factors, including HNF1A, CEBPα, CEBPγ, NF-1, and JUN, with or without 5′Aza treatment in HL-60 and K562 cells, were determined by quantitative RT-PCR. Sample data were normalized to the control gene GAPDH for each cell line individually.

Figure 4

Transcription factor HNF1A was involved in azacytidine-induced up-regulation of AQP9. (A). HNF1A mRNA and protein expressions were markedly up-regulated after treatment of 5′Aza as examined by semi-quantitative and quantitative RT-PCR (upper panel) and western immunoblotting (lower panel). (B) Diagram depicting the regions of the HNF1A promoter analyzed by Methylation-specific PCR (MSP) and Combined Bisulfite Restriction Analysis (COBRA). (C) The methylation status of HNF1A gene promoter was extensively studied and determined using combined bisulfite restriction analysis. 5′Aza treatment resulted in demethylation of the HNF1A gene promoter. (C: control; IVD: universal methylated DNA). (D) Methylation-specific PCR (MSP) was performed using PCR primers specific for methylated or unmethylated HNF1A gene promoter. The results showed that the HNF1A gene promoter was highly methylated, and 5′Aza treatment led to demethylation of the HNF1A gene promoter. (E) Specific HNF1A siRNA abrogated 5′Aza-induced AQP9 up-regulation as determined by RT-PCR (upper panel) and quantitative RT-PCR (lower panel). (F) In K562 cells treated with 5′Aza, specific HNF1A siRNA almost completely blocked the intracellular entry of As2O3 as compared with the control siRNA-treated cells. The results showed that HNF1A was the mediator for up-regulation of AQP9 after 5′Aza treatment.

Figure 5

Methylation status of HNF1A promoter and correlation between the expressions of AQP9 and HNF1A in primary AML samples. (A) MSP showing highly methylated HNF1A promoter in 16 human AML samples. (B) Quantification of AQP9 and HNF1A expressions in 16 human AML samples. Result was analyzed using Pearson test. AQP9 level was found to correlate positively with HNF1A level (p = 0.0045).

Figure 6

Pre-treatment with azacytidine sensitized primary AML cells to subsequent treatment with As2O3. (A) Treatment with 5′Aza resulted in significant up-regulation of HNF1A (left panel) and AQP9 (right panel) as determined by quantitative RT-PCR. (B) As2O3-mediated cytotoxicity in primary AML cells with or without 5′Aza pre-treatment as determined by MTT analysis. Sample data were normalized to the controls of each patient individually. Results represented triplicates of seven samples of primary AML cells.

Figure 7

Azacytidine did not enhance hepatotoxicity of As2O3in vitro. (A) Basal expression of AQP9 was much higher in MIHA compared with those of AML cells. (B) 5′Aza treatment did not increase AQP9 expression significantly in MIHA. (C) HNF1A promoter was hypomethylated in MIHA (upper panel), and 5′Aza treatment did not further increase HNF1A expression (lower panel). Sample data were normalized to the respective control. (D) Pre-treatment with 5′Aza did not significantly enhance the cytotoxicity of As2O3 in MIHA cells. Sample data were normalized to the respective control individually.