Introduction
Arsenic trioxide (As
2O
3) is an important and highly efficacious drug in the management of acute promyelocytic leukemia (APL) [
1,
2]. It was first successfully used as a salvage therapy for relapsed APL [
3]. More recently, As
2O
3 has also been shown to be effective as first line induction therapy in newly diagnosed APL [
4,
5] and as a maintenance therapy [
6]. The unique sensitivity of APL cells to As
2O
3 is likely to be related to As
2O
3-mediated degradation of the PML-RARA chimeric protein, which is an oncogenic protein produced as a result of the specific chromosomal translocation t(15;17)(q22;q12) [
7,
8]. In addition, As
2O
3 has also been shown to induce apoptosis through the generation of superoxides and reactive oxygen species [
9,
10], disruption of the mitochondrial transmembrane potential with release of cytochrome c and caspase activation [
11], and inhibition of DNA methyltransferase DNMT leading to demethylation of tumor suppressor genes [
12].
Because As
2O
3 must enter cells before exerting its cytotoxic activity, the control of arsenic trafficking through the plasma membrane could conceivably modulate arsenic sensitivity. Aquaglyceroporin 9 (AQP9) is a transmembrane solute transporting protein and is expressed in human leukocytes, liver, lung, and spleen [
13]. Unlike the typical aquaporin water channel, it also facilitates the passage of glycerol and many other non-charged solutes. We and others have shown that AQP9 controls the transmembrane transport of As
2O
3, thereby playing a critical role in determining the sensitivity of cells towards As
2O
3-induced cytotoxicity [
14,
15]. In myeloid leukemia cells, expression levels of AQP9 are directly proportional to intracellular arsenic concentrations upon As
2O
3 treatment, which translate into increased arsenic sensitivity [
14]. Of the different subtypes of acute myeloid leukemia (AML), APL cells express the highest concentration of AQP9, which might in part explain their exquisite sensitivity to As
2O
3 [
14]. Furthermore, all-trans retinoic acid (ATRA) up-regulates AQP9 expression, contributing to its synergistic cytotoxic effect with As
2O
3 [
14]. However, the regulation of AQP9 expression remains unclear. Understanding the mechanisms controlling AQP9 expression may enable pharmacological strategies to be designed to up-regulate AQP9 in leukemia cells, hence constituting a potential method to expand the therapeutic spectrum of As
2O
3 in the treatment of AML.
Demethylating agents, including azacitidine and decitabine, are a standard medication for the management of myelodysplastic syndrome and elderly subjects with AML [
16-
19]. Recently, demethylating agents have been shown to synergize with As
2O
3 in the treatment of AML
in vitro and
in vivo [
20,
21]. However, the biological basis of the synergism between demethylating agents and As
2O
3 has not been defined.
In this study, we proposed that one of the mechanisms of synergism between demethylating agents and As2O3 might be through modulation of AQP9 expression. To test this hypothesis, we examined the effect of azacytidine treatment on AQP9 expression and plasma membrane arsenic trafficking in AML cell lines and primary AML samples.
Materials and methods
Cells and reagents
The human myeloid leukemia cell lines HL-60 and K562 (purchased from ATCC, Manassas, VA, USA) and the APL cell line NB4 (a kind gift from Dr. Shen ZX, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai, China) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 5% CO
2 at 37°C. They have been characterized and tested as described previously [
14]. The human leukemia line OCI-AML3 (purchased from DSMZ, Braunschweig, Germany) was cultured in α-MEM with 20% FBS in similar conditions. The immortalized human liver cell line MIHA (a kind gift from Dr. J Roy-Chowdhury, Albert Einstein College of Medicine, New York, USA) was cultured in DMEM with 10% FBS. MIHA has been characterized and tested as described previously [
22]. Primary AML samples from peripheral blood (PB) and/or bone marrow (BM) were obtained with informed consent from patients treated at Queen Mary Hospital, Hong Kong. Primary cells were cultured in StemSpan H3000 supplemented with StemSpan CC100 cytokine cocktail (StemCell Technologies, Vancouver, Canada). Archival samples were obtained from marrow mononuclear cells of AML patients stored at −80°C. Procurement of these samples was approved by the institute review board according to the Declaration of Helsinki. The demethylating drug azacytidine (5-aza-2′deoxycytidine; 5′Aza) and As
2O
3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The polyclonal phycoerythrin (PE)-conjugated anti-AQP9 and PE-conjugated isotypic control antibodies were purchased from Bioss Antibodies (Bioss Inc., Woburn, MA, USA).
As2O3 cytotoxicity
Cells pre-treated with or without azacytidine (5 μM for 3 days) were washed twice with phosphate-buffered saline (PBS), re-suspended in fresh RPMI-1640 supplemented with 10% FBS, and treated with various concentration of As2O3 (0.0, 0.3125, and 0.625 μM for NB4; 0.0, 2.5, 5.0, and 10.0 μM in other cells). For experiments where AQP9 blockade was involved, cells were incubated in addition with mercury chloride (HgCl2) at 10 μM for 2 hours. For 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, 100 μL of each cell suspension was incubated for 48 hours in 96-well plates, followed by the addition of MTT reagents (10 μL for 4 hours) and the solubilizing buffer (100 μL overnight), and absorbance measurement at 560 nm. All experiments were performed in triplicates.
Flow cytometric analysis
For apoptosis assay, cells treated with or without azacytidine were analyzed for apoptotic cells using a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA), using an annexin V: phycoerythrin and 7-AAD apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Cells were incubated with respective antibodies for 15 min and subjected to flow cytometric analysis. At least 10,000 events were collected. All flow cytometry plots and data were acquired from at least three independent experiments.
Quantification of gene expression
Total RNA was extracted using Trizol reagent (Life technologies, Carlsbad, CA, USA), and 1 μg of RNA was reversely transcribed (SuperScript III First Strand Synthesis system, Life technologies, Carlsbad, CA, USA). The resulting cDNAs were used for semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (q-RT-PCR). Primers for quantification of target and control genes were designed by the Primer Express software (Applied Biosystems, Life technologies, Carlsbad, CA, USA) (primer sequences and reaction conditions were listed in Additional file
1). Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) and a StepOnePlus Real Time PCR system (Applied Biosystems, Carlsbad, CA, USA). The expressions of target genes with respect to the internal control gene were analyzed with the comparative C
T (ΔΔC
T) method. Experiments were performed in triplicates.
Western immunoblotting
Cells were washed twice with PBS and were then lysed in RIPA buffer [RIPA: 50 mM of Tris-HCl buffer pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, and 0.25% (w/v) sodium deoxycholate, with the addition of a protease inhibitor cocktail (Roche Diagnostic, Sulzfeld, Germany), 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Total protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. For Western blot analysis, antibodies against HNF1A (Cell Signaling, Danvers, MA, USA) and β-actin (Sigma, St. Louis, MO, USA) were used. For detection of bound antibodies, HRP-conjugated secondary antibodies were used (Life Technologies (Carlsbad, CA, USA)).
RNA interference and transfection
Gene knockdown experiments were performed with siRNA (sequences of siRNA in Additional file
1), with negative controls (AllStars Negative Controls, Qiagen, Venlo, Netherlands). K562 cells (2 × 10
5 cells in 100 μL) were transfected with siRNA (200 ng diluted in RPMI-1640 medium, incubated at room temperature for 10 min with 6 μL HiPerfect transfection reagent, Qiagen, Germantown, MD, USA) for 6 h in 5% CO
2 at 37°C, after which 600 μL of medium with FBS and antibiotics was added, followed by another 72 h of incubation before analysis. Experiments were performed in triplicates.
Measurement of intracellular arsenic concentration
Cells were harvested, washed twice in ice-cold PBS, pelleted, and lysed in 0.9 mL of double-distilled water by sonication for 10 min. Yttrium (Chem Service, West Chester, PA, USA) dissolved in 2% nitric acid to ten parts per billion was then added as an internal standard to the lysate, which was vortexed and centrifuged at 3000 ×
g for 10 min. The supernatant was collected and assayed for arsenic concentration by inductively coupled plasma-mass spectrometry [
23]. Experiments were performed in triplicates.
Bisulfite modification of DNA, methylation-specific PCR and combined bisulfite restriction analysis
Genomic DNA was isolated with the QIAamp DNA Blood Mini Kit (Qiagen, Germantown, MD, USA). Bisulfite modification of DNA was performed with the Zymo DNA modification kit (Zymo Research, Irvine, CA, USA). Methylation-specific PCR (MSP) specific primers for the methylated and unmethylated alleles were designed using the OLIGO 6 primer analysis software (Molecular Biology Insights, Cascade, CO, USA) (primer sequences in Additional file
1: Supplementary Information), and the PCR products were analyzed by gel electrophoresis. CpGenome universal methylated DNA (Chemicon International Inc, Billerica, MA, USA) was used as a positive control. For combined bisulfite restriction analysis (COBRA), bisulfite-modified DNA before and after treatment was amplified by PCR with primers spanning the CG-rich region of target gene promoter (primer sequences in Additional file
1: Supplementary Information) and digested with the restriction endonuclease
BstUI (New England Biolabs, Ipswich, MA, USA). PCR products were analyzed by gel electrophoresis to determine the respective methylation status.
Statistical analysis
Comparative analysis of cell viability and AQP9 expression were performed by the Student’s t-test, whereas correlation analysis on AQP9 and HNF1A expression was performed using the Pearson’s χ
2-test. All values were presented as mean ± standard deviation (SD). Differences were considered statistically significant when p value was <0.05.
Discussion
With the use of ATRA and chemotherapy, complete remission (CR) rates of over 90% can be achieved in newly diagnosed APL. In relapsed APL, As
2O
3 has been shown to be a highly effective salvage therapy [
3]. Combination therapy with As
2O
3 and ATRA as frontline treatment for APL also results a very high CR rate and improved long-term outcomes [
4,
5]. We have also shown that the use of As
2O
3 and ATRA as maintenance therapy is safe and may decrease relapse in APL patients in first CR [
6]. Our previous study showed that one of the mechanisms underlying the synergistic interaction between As
2O
3 and ATRA might be ATRA-mediated up-regulation of AQP9, resulting in increased cellular uptake and hence intracellular concentrations of arsenic [
14].
In this study, we tested the hypothesis that AQP9 might be involved in the observed synergistic interaction of azacytidine with As2O3. Because we wanted to minimize the cytotoxic action of azacytidine as a confounding factor in our experiments, azacytidine was used only as a pre-treatment and removed from the culture system during subsequent As2O3 treatment. In this way, we showed that azacytidine pre-treatment sensitized cells to As2O3-induced cytotoxicity.
Our data clearly showed that azacytidine up-regulated AQP9 through demethylation of the promoter and therefore increased transcription of HNF1A, itself a transcription activator of AQP9. The increase in AQP9 expression at the transcription and protein levels led to increase arsenic uptake and intracellular concentrations, thereby enhancing As2O3-induced cytotoxicity. Blockade of AQP9 by HgCl2 and siRNA knock-down of HNF1A both suppressed azacytidine-induced As2O3 sensitization. Because azacytidine was removed after pre-treatment, its intrinsic cytotoxicity did not contribute to the subsequent cytotoxicity observed with As2O3 treatment. However, in actual clinical practice, if azacytidine were to be combined with As2O3 in treating patients, the inherent cytotoxicity of azacytidine might further enhance cell-kill and hence the clinical efficacy of this combination.
One concern of combined azacytidine/As
2O
3 treatment would be potentiation of arsenic toxicity in other organs and tissues where AQP9 is also up-regulated. The liver is a key organ that is affected by As
2O
3 treatment, particularly when As
2O
3 is used in the oral formulation due to a first-pass effect. Reassuringly, our preliminary data in MIHA showed that liver cells already expressed a high level of AQP9, which was not further increased by azacytidine treatment. Interestingly, it had been shown that As
2O
3-related toxicities, especially in the heart and liver, were more pronounced in
AQP9-null mice when compared with wild-type mice, as a result of reduced As
2O
3 clearance [
25]. Finally, in a phase I clinical trial examining As
2O
3 in combination with another demethylating drug decitabine in AML, no excessive adverse effects were reported [
21]. Therefore, concomitant azacytidine and As
2O
3 treatment might not lead to increased toxicity in other organs.
The transcription regulation of
AQP9 has not been thoroughly investigated, and other pharmacological agents may also activate
AQP9 gene through various mechanisms to enhance the anti-tumor effects of As
2O
3 [
14,
15]. Previous investigations had shown that indirubin and tanshinone IIA up-regulated
AQP9 expression and augmented the anti-leukemia effect of As
2O
3 as present in the Realgar-Indigo naturalis formula [
26]. As AQP9 transmembrane protein can be detected and quantified by fluorochrome-conjugated antibody and flow cytometric analysis, a high-throughput screening is therefore feasible in the future to identify compounds that may up-regulate AQP9 and potentially enhance the therapeutic effect of As
2O
3.
HNF1A is a transcription factor and is highly expressed in liver, pancreas, and kidneys [
27]. It plays a critical role in transcriptional activation of differentiated hepatocyte-specific genes critical for liver function such as albumin [
28]. Epigenetic regulation is involved in tissue-specific expression of HNF1A, and hypomethylation of the
HNF1A gene promoter has been demonstrated in mature hepatocytes and renal tubular cells [
29]. In kidneys, HNF1A regulates the expression of several organic anion transporters but its role in aquaporin or aquaglyceroporin expression has not been reported previously [
29]. Furthermore, HNF1A level has been shown to be associated with differentiation of hepatocellular carcinoma (HCC) [
28]. Given that AQP9 expression may be associated with differentiation of myeloid cells [
14], it remains to be determined if HNF1A is also involved in myeloid differentiation. Finally, chronic exposure of the HCC cell line HepG2 to sub-toxic levels of arsenic leading to arsenic resistance has been shown to be associated with down-regulation of HNF1A expression [
30]. This is in agreement with our result, in that decreasing HNF1A level would result in down-regulation of AQP9, which led to decreased arsenic entry into cells, thereby contributing to arsenic resistance.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The University of Hong Kong holds the patent for the use of oral formulation of arsenic trioxide in USA and Japan. DC, YLK, and ET are employees of the University of Hong Kong. Other authors report no potential conflicts of interest.
Authors’ contributions
DC and KN contributed equally to this work, performed the experiments, and wrote the manuscript. TSYC managed the patients and collected the clinical data. YYC performed the experiments. BF and ST performed the intracellular arsenic trioxide concentration assays. YLK designed the experiments, managed the patients, and wrote the manuscript. ET conceived the project, designed the experiments, managed the patients, and wrote the manuscript. All authors read and approved the manuscript.