title: 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.
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. dhwchau@gmail.com.
authors.forename: David
authors.initials: D
authors.lastname: Chau
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. karen.nmt@gmail.com.
authors.forename: Karen
authors.initials: K
authors.lastname: Ng
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. thomas28@netvigator.com.
authors.forename: Thomas Sau-Yan
authors.initials: TS
authors.lastname: Chan
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. yuen_yee@yahoo.com.|Asbestos Diseases Research Institute, ADRI Bernie Banton Centre, University of Sydney, Concord Hospital, Sydney, Australia. yuen_yee@yahoo.com.
authors.forename: Yuen-Yee
authors.initials: YY
authors.lastname: Cheng
authors.affiliation: Department of Pathology and Clinical Biochemistry, Queen Mary Hospital, Pok Fu Lam, Hong Kong, Hong Kong. fongmw@ha.org.hk.
authors.forename: Bonnie
authors.initials: B
authors.lastname: Fong
authors.affiliation: Department of Pathology and Clinical Biochemistry, Queen Mary Hospital, Pok Fu Lam, Hong Kong, Hong Kong. sidneytam@hku.hk.
authors.forename: Sidney
authors.initials: S
authors.lastname: Tam
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. ylkwong@hku.hk.
authors.forename: Yok-Lam
authors.initials: YL
authors.lastname: Kwong
authors.affiliation: Division of Haematology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong. ewctse@hku.hk.
authors.forename: Eric
authors.initials: E
authors.lastname: Tse
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D020346:Aquaporins / Q000096:biosynthesis*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000008:administration & dosage*; D001374:Azacitidine / Q000008:administration & dosage*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D010087:Oxides / Q000008:administration & dosage*; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering; D014162:Transfection; D015854:Up-Regulation
chemical_list: C109669:AQP9 protein, human; D020346:Aquaporins; D001152:Arsenicals; D010087:Oxides; D034741:RNA, Small Interfering; D001374:Azacitidine; D000077237:Arsenic Trioxide