title: 5-azacytidine enhances efficacy of multiple chemotherapy drugs in AML and lung cancer with modulation of CpG methylation.
abstract: The DNA methyltransferase (DNMT) inhibitory drugs such as 5-azacytidine induce DNA hypomethylation by inhibiting DNA methyltransferases. While clinically effective, DNMT inhibitors are not curative. A combination with cytotoxic drugs might be beneficial, but this is largely unexplored. In the present study, we analyzed potential synergisms between cytotoxic drugs and 5-azacytidine in acute myeloid leukemia (AML) and non-small cell lung cancer (NSCLC) cells. Lung cancer and leukemia cell lines were exposed to low doses of 5-azacytidine with varying doses of cytarabine or etoposide for AML cells (U937 and HL60) as well as cisplatin or gemcitabine for NSCLC cells (A549 and HTB56) for 48 h. Drug interaction and potential synergism was analyzed according to the Chou-Talalay algorithm. Further analyses were based on soft agar colony formation assays, active caspase-3 staining and BrdU incorporation flow cytometry. To identify effects on DNA methylation patterns, we performed genome wide DNA methylation analysis using 450K bead arrays. Azacytidine at low doses was synergistic with cytotoxic drugs in NSCLC and in AML cell lines. Simultaneous exposure to 5-azacytidine with cytotoxic drugs showed strong synergistic activity. In colony formation assays these synergisms were repeatedly verified for 5-azacytidine (25 nM) with low doses of anticancer agents. 5-azacytidine neither affected the cell cycle nor increased apoptosis. 450K methylation bead arrays revealed 1,046 CpG sites in AML and 1,778 CpG sites in NSCLC cells with significant DNA hypomethylation (24-h exposure) to 5-azacytidine combined with the cytotoxic drugs. These CpG-sites were observed in the candidate tumor-suppressor genes MGMT and THRB. Additional incubation time after 24-h treatment led to a 4.1-fold increase of significant hypomethylated CpG-sites in NSCLC cells. These results suggest that the addition of DNA demethylating agents to cytotoxic anticancer drugs exhibits synergistic activity in AML and NSCLC. Dysregulation of an equilibrium of DNA methylation in cancer cells might increase the susceptibility for cytotoxic drugs.
authors.affiliation: Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Füller
authors.affiliation: Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Klein
authors.affiliation: Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Schmidt
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Rohde
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Göllner
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Isabell
authors.initials: I
authors.lastname: Schulze
authors.affiliation: Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qianli
authors.affiliation: Department of Medicine A, Hematology and Oncology, University of Münster, Münster, Germany.
authors.forename: Wolfgang E
authors.initials: WE
authors.lastname: Berdel
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Bayram
authors.initials: B
authors.lastname: Edemir
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Müller-Tidow
authors.affiliation: Department of Medicine IV, Hematology and Oncology, University of Halle, Halle, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Tschanter
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001374:Azacitidine / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics; D018899:CpG Islands / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects*; D004357:Drug Synergism; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000235:genetics; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics; D014407:Tumor Cells, Cultured; D020298:U937 Cells
chemical_list: D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine