Sequential combination of decitabine and idarubicin synergistically enhances anti-leukemia effect followed by demethylating Wnt pathway inhibitor promoters and downregulating Wnt pathway nuclear target

Journal of Translational Medicine

Hongyan Tong³  Jie Jin³  Zhengping Zhuang²  Juan Carlos Vera²  Zhigang Ren⁴  Chen Mei¹  Chao Hu³  Kongfei Li³ 

[1] Department of Hematology, Sir Run Run Shaw Hospital of Zhejiang University, Hangzhou 310016, Zhejiang Province, People’s Republic of China;Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5D37, 9000, Rockvillle Pike, Bethesda, MD 20892, USA;Institute of Hematology, Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang Province, People’s Republic of China;Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhejiang University, Hangzhou 310003, Zhejiang Province, People’s Republic of China

关键词: Myelodysplastic syndromes;    Acute myeloid leukemia;    Wnt;    Idarubicin;    Decitabine;   

Others  :  804251
DOI  :  10.1186/1479-5876-12-167

 received in 2014-04-05, accepted in 2014-06-05,  发布年份 2014

【 摘 要 】

Background

The methylation inhibitor 5-Aza-2′-deoxycytidine (decitabine, DAC) has a great therapeutic value for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). But decitabine monotherapy was associated with a relatively low rate of complete remission in AML and MDS. We aimed to investigate the effect of several anti-leukemia drugs in combination with decitabine on the proliferation of myeloid leukemia cells, to select the most efficient combination group and explore the associated mechanisms of these combination therapies.

Methods

Cell proliferation was tested by MTT assay and CFU-GM assay. Cell apoptosis was evaluated by Annexin V and PI staining in cell culture, TUNEL assay and transmission electron microscopy in animal study. MicroPET was used to imaging the tumor in mouse model. Molecular studies were conducted using microarray expression analysis, which was used to explore associated pathways, and real-time quantitative reverse transcription-PCR, western blot and immunohistochemistry, used to assess regulation of Wnt/β-catenin pathway. Statistical significance among groups was determined by one-way ANOVA analysis followed by post hoc Bonferroni’s multiple comparison test.

Results

Among five anti-leukemia agents in combining with decitabine, the sequential combination of decitabine and idarubicin induced synergistic cell death in U937 cells, and this effect was verified in HEL, SKM-1 cells and AML cells isolated from AML patients. Importantly, tumor growth inhibition in this sequential combination was found to be higher than in single agent or controls in vivo. Moreover, sequential combination of the two agents induced apoptosis and depression of the Wnt/β-catenin pathway in both AML cell culture and animal studies.

Conclusions

The findings demonstrated that sequentially combination of decitabine and idarubicin had synergistic anti-leukemia effects. These effects were mainly attributed to demethylation of Wnt/β-catenin pathway inhibitors and downregulation of Wnt/β-catenin pathway nuclear targets.

【 授权许可】

   
2014 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Jones PA, Taylor SM: Cellular differentiation, cytidine analogs and DNA methylation. Cell 1980, 20:85-93.
• [2]Toyota M, Kopecky KJ, Toyota MO, Jair KW, Willman CL, Issa JP: Methylation profiling in acute myeloid leukemia. Blood 2001, 97:2823-2829.
• [3]Santi DV, Norment A, Garrett CE: Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A 1984, 81:6993-6997.
• [4]Qin T, Youssef EM, Jelinek J, Chen R, Yang AS, Garcia-Manero G, Issa JP: Effect of cytarabine and decitabine in combination in human leukemic cell lines. Clin Cancer Res 2007, 13:4225-4232.
• [5]Creusot F, Acs G, Christman JK: Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2′-deoxycytidine. J Biol Chem 1982, 257:2041-2048.
• [6]Yang AS, Doshi KD, Choi SW, Mason JB, Mannari RK, Gharybian V, Luna R, Rashid A, Shen L, Estecio MR, Kantarjian HM, Garcia-Manero G, Issa JP: DNA methylation changes after 5-aza-2′-deoxycytidine therapy in patients with leukemia. Cancer Res 2006, 66:5495-5503.
• [7]Kantarjian H, Issa JP, Rosenfeld CS, Bennett JM, Albitar M, DiPersio J, Klimek V, Slack J, de Castro C, Ravandi F, Helmer R 3rd, Shen L, Nimer SD, Leavitt R, Raza A, Saba H: Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 2006, 106:1794-1803.
• [8]Issa JP, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, Bayar E, Lyons J, Rosenfeld CS, Cortes J, Kantarjian HM: Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 2004, 103:1635-1640.
• [9]Gore SD, Baylin S, Sugar E, Carraway H, Miller CB, Carducci M, Grever M, Galm O, Dauses T, Karp JE, Rudek MA, Zhao M, Smith BD, Manning J, Jiemjit A, Dover G, Mays A, Zwiebel J, Murgo A, Weng LJ, Herman JG: Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms. Cancer Res 2006, 66:6361-6369.
• [10]Blum W, Klisovic RB, Hackanson B, Liu Z, Liu S, Devine H, Vukosavljevic T, Huynh L, Lozanski G, Kefauver C, Plass C, Devine SM, Heerema NA, Murgo A, Chan KK, Grever MR, Byrd JC, Marcucci G: Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia. J Clin Oncol 2007, 25:3884-3891.
• [11]Sundstrom C, Nilsson K: Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer 1976, 17:565-577.
• [12]Martin P, Papayannopoulou T: HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression. Science 1982, 216:1233-1235.
• [13]Nakagawa T, Matozaki S: The SKM-1 leukemic cell line established from a patient with progression to myelomonocytic leukemia in myelodysplastic syndrome (MDS)-contribution to better understanding of MDS. Leuk Lymphoma 1995, 17:335-339.
• [14]Griffiths EA, Gore SD, Hooker C, McDevitt MA, Karp JE, Smith BD, Mohammad HP, Ye Y, Herman JG, Carraway HE: Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation. Leuk Lymphoma 2010, 51:1711-1719.
• [15]Ren Z, Cui G, Lu H, Chen X, Jiang J, Liu H, He Y, Ding S, Hu Z, Wang W, Zheng S: Liver Ischemic Preconditioning (IPC) improves intestinal microbiota following liver transplantation in rats through 16 s rDNA-based analysis of microbial structure shift. PLoS One 2013, 8:e75950.
• [16]Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984, 22:27-55.
• [17]Issa JP: Decitabine. Curr Opin Oncol 2003, 15:446-451.
• [18]Decitabine: 2′-deoxy-5-azacytidine, Aza dC, DAC, dezocitidine, NSC 127716 Drugs R D 2003, 4:352-358.
• [19]Hu Z, Negrotto S, Gu X, Mahfouz R, Ng KP, Ebrahem Q, Copelan E, Singh H, Maciejewski JP, Saunthararajah Y: Decitabine maintains hematopoietic precursor self-renewal by preventing repression of stem cell genes by a differentiation-inducing stimulus. Mol Cancer Ther 2010, 9:1536-1543.
• [20]Suzuki M, Harashima A, Okochi A, Yamamoto M, Nakamura S, Motoda R, Yamasaki F, Orita K: 5-Azacytidine supports the long-term repopulating activity of cord blood CD34(+) cells. Am J Hematol 2004, 77:313-315.
• [21]Pinto A, Attadia V, Fusco A, Ferrara F, Spada OA, Di Fiore PP: 5-Aza-2′-deoxycytidine induces terminal differentiation of leukemic blasts from patients with acute myeloid leukemias. Blood 1984, 64:922-929.
• [22]Esteller M, Guo M, Moreno V, Peinado MA, Capella G, Galm O, Baylin SB, Herman JG: Hypermethylation-associated inactivation of the cellular retinol-binding-protein 1 gene in human cancer. Cancer Res 2002, 62:5902-5905.
• [23]Peng CY, Jiang J, Zheng HT, Liu XS: Growth-inhibiting effects of arsenic trioxide plus epigenetic therapeutic agents on leukemia cell lines. Leuk Lymphoma 2010, 51:297-303.
• [24]Anzai H, Frost P, Abbruzzese JL: Synergistic cytotoxicity with 2′-deoxy-5-azacytidine and topotecan in vitro and in vivo. Cancer Res 1992, 52:2180-2185.
• [25]Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B, Yang H, Rosner G, Verstovsek S, Rytting M, Wierda WG, Ravandi F, Koller C, Xiao L, Faderl S, Estrov Z, Cortes J, O’Brien S, Estey E, Bueso-Ramos C, Fiorentino J, Jabbour E, Issa JP: Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia. Blood 2006, 108:3271-3279.
• [26]Sekeres MA, List AF, Cuthbertson D, Paquette R, Ganetzky R, Latham D, Paulic K, Afable M, Saba HI, Loughran TP Jr, Maciejewski JP: Phase I combination trial of lenalidomide and azacitidine in patients with higher-risk myelodysplastic syndromes. J Clin Oncol 2010, 28:2253-2258.
• [27]Willemze R, Archimbaud E, Muus P: Preliminary results with 5-aza-2′-deoxycytidine (DAC)-containing chemotherapy in patients with relapsed or refractory acute leukemia. The EORTC Leukemia Cooperative Group. Leukemia 1993, 7(Suppl 1):49-50.
• [28]Willemze R, Suciu S, Archimbaud E, Muus P, Stryckmans P, Louwagie EA, Berneman Z, Tjean M, Wijermans P, Dohner H, Jehn U, Labar B, Jaksic B, Dardenne M, Zittoun R: A randomized phase II study on the effects of 5-Aza-2′-deoxycytidine combined with either amsacrine or idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group phase II study (06893). Leukemia 1997, 11(Suppl 1):S24-S27.
• [29]Bogason A, Bhuiyan H, Masquelier M, Paul C, Gruber A, Vitols S: Uptake of anthracyclines in vitro and in vivo in acute myeloid leukemia cells in relation to apoptosis and clinical response. Eur J Clin Pharmacol 2009, 65:1179-1186.
• [30]Scandura JM, Roboz GJ, Moh M, Morawa E, Brenet F, Bose JR, Villegas L, Gergis US, Mayer SA, Ippoliti CM, Curcio TJ, Ritchie EK, Feldman EJ: Phase 1 study of epigenetic priming with decitabine prior to standard induction chemotherapy for patients with AML. Blood 2011, 118:1472-1480.
• [31]Clevers H: Wnt/beta-catenin signaling in development and disease. Cell 2006, 127:469-480.
• [32]Reya T, Clevers H: Wnt signalling in stem cells and cancer. Nature 2005, 434:843-850.
• [33]Nelson WJ, Nusse R: Convergence of Wnt, beta-catenin, and cadherin pathways. Science 2004, 303:1483-1487.
• [34]Eaves CJ, Humphries RK: Acute myeloid leukemia and the Wnt pathway. N Engl J Med 2010, 362:2326-2327.
• [35]Li Y, Gao Q, Yin G, Ding X, Hao J: WNT/beta-catenin-signaling pathway stimulates the proliferation of cultured adult human Sertoli cells via upregulation of C-myc expression. Reprod Sci 2012, 19:1232-1240.
• [36]Udhayakumar G, Jayanthi V, Devaraj N, Devaraj H: Interaction of MUC1 with beta-catenin modulates the Wnt target gene cyclinD1 in H. pylori-induced gastric cancer. Mol Carcinog 2007, 46:807-817.
• [37]Valencia A, Roman-Gomez J, Cervera J, Such E, Barragan E, Bolufer P, Moscardo F, Sanz GF, Sanz MA: Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia. Leukemia 2009, 23:1658-1666.
• [38]Brown R, Plumb JA: Demethylation of DNA by decitabine in cancer chemotherapy. Expert Rev Anticancer Ther 2004, 4:501-510.