- Research article
Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells
Cellular & Molecular Biology Letters volume 17, pages 501–525 (2012)
DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.
acetylated histone H4
acute promyelocytic leukemia
trimethyl histone H4 methylated at lysine 4 (K4)
nitro blue tetrazolium
sodium phenyl butyrate
phorbol myristate acetate
all-trans retinoic acid
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl) propanoic acid
Baylin, S.B., Herman, J.G., Graff, J.R., Vertino, P.M. and Issa, J.P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res. 72 (1998) 141–196.
Toyota, M., Kopecky, K.J., Toyota, M.O., Jair, K.W., Willman, C.L. and Issa, J.P. Methylation profiling in acute myeloid leukemia. Blood 9 (2001) 2823–2829.
Paz, M.F., Fraga, M.F., Avila, S., Avila, S., Guo, M., Pollan, M., Herman, J.G. and Esteller, M. A systematic profile of DNA methylation in human cancer cell lines. Cancer Res. 63 (2003) 1114–1121.
Teofilini, L., Martini, M., Luongo, M., Diverio, D., Capelli, G., Breccia, M., Lo Coco, F., Leone, G. and Larocca, L.M. Hypermethylation of CpG islands in the promoter region of p15 (INK4b) in acute promyelocytic leukemia represses p15 (INK4b) expression and correlates with poor prognosis. Leukemia 17 (2003) 919–924.
Ekmekci, C.G., Gutiérrez, M.I., Siraj, A.K., Ozbek, U. and Bhatia, K. Aberrant methylation of multiple tumor suppressor genes in acute myeloid leukemia. Am. J. Hematol. 77 (2004) 233–240.
Mizuno, S., Chijiwa, T., Okamura, T., Akashi, K., Fukumaki, Y., Niho Y. and Sasaki, H. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood 97 (2001) 1172–1179.
Silverman, L.R., Holland, J.F., Weinberg, R.S., Alter, B.P., Davi, R.B., Ellison, R.R., Demakos, E.P., Cornell, C.J. Jr., Carey, R.W. and Schiffer, C. Effect of treatment with 5-aza cytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes. Leukemia 7 (1993) 21–29.
Issa, J.P., Garcia-Manero, G., Giles, F.J., Mannari, R., Thomas, D., Faderl, S., Bayar, E., Lyons, J., Rosenfeld, C., Cortes, J. and Kantarjian, H.M. Phase I study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood 103 (2004) 1635–1640.
Bug, G. and Ottmann, O.G. The HDAC system and association with acute leukemias and myelodysplastic syndromes. Invest. New Drugs 28Suppl 1 (2010) S36–49.
Cashen, A.F., Schiller, G.J., O’Donnell, M.R. and DiPersio, J.F. Multicenter, phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia. J. Clin. Oncol. 28 (2010) 556–561.
Müller, S. and Krämer, O.H. Inhibitors of HDACs-effective drugs against cancer? Curr. Cancer Drug Targets 10 (2010) 210–228.
Kim, C.H., Marquez, V.E., Mao, D.T., Haines, D.R. and McCormack, J.J. Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase. J. Med. Chem. 29 (1986) 1374–1380.
Hurd, P.J., Whitmarsh, A.J., Baldwin, G.S., Kelly, S.M., Waltho, J.P., Price, N.C., Connolly, B.A. and Hornby, D.P. Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidone. J. Mol. Biol. 286 (1999) 389–401.
Zhou, L., Cheng, X., Connolly, B.A., Dickman, M.J., Hurd, P.J. and Hornby, D.P. Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. J. Mol. Biol. 23 (2002) 581–599.
Cheng, J.C., Matsen, C.B., Gonzales, F.A., Ye, W., Greer, S., Marquez, V.E., Jones, P.A. and Selker, E.U. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J. Natl. Cancer Inst. 95 (2003) 399–409.
Marquez, V.E., Kelly, J.A., Agbaria, R., Ben-Kasus, T., Cheng, J.C., Yoo, C.B. and Jones, P.A. Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. Ann. N. Y. Acad. Sci. 1008 (2005) 246–254.
Cheng, J.C., Weisenberger, D.J., Gonzales, F.A., Liang, G., Xu, G.L., Hu, Y.G., Marquez, V.E. and Jones P.A. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol. Cell Biol. 24 (2004) 1270–1278.
Scott, S.A., Lakshimikuttysamma, A., Sheridan, D.P., Sanche, S.E., Geyer, C.R. and DeCoteau, J.F. Zebularine inhibits human myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression. Exp. Hematol. 35 (2007) 263–273.
Veerla, S., Panagopoulos, I., Jin, Y., Lindgren, D. and Höglund, M. Promoter analysis of epigenetically controlled genes in bladder cancer. Genes Chromosomes Cancer 47 (2008) 368–378.
Brueckner, B., Boy, R.G., Siedlecki, P., Musch, T., Kliem, H.C., Zielenkiewicz, P., Suhai, S., Wiessler, M. and Lyko, F. Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. Cancer Res. 65 (2005) 6305–6311.
Tsai, H.-C. and Baylin, S.B. Cancer epigenetics: linking basic biology to clinical medicine. Cell. Res. 21 (2011) 502–517.
Cameron, E.E., Bachman, K.E., Myohanen, S., Herman, J.G. and Baylin, S.B. Synergy of demethylation and histone deacetylase inhibition in the reexpression of genes silenced in cancer. Nat. Genet. 21 (1999) 103–107.
Gore, S.D., Baylin, S., Sugar, E., Carraway, H., Miller, C.B., Carducci, M., Grever, M., Galm, O., Dauses, T., Karp, J.E., Rudek, M.A., Zhao, M., Smith, B.D., Manning, J., Jiemjit, A., Dover, G., Mays, A., Zwiebel, J., Murgo, A., Weng, L.J. and Herman, J.G. Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms. Cancer Res. 66 (2006) 6361–6369.
Blum, W., Klisovic., R.B., Hackanson., B., Liu, Z., Liu, S., Devine, H., Vukosavljevic, T., Huynh, L., Lozanski, G., Kefauver, C., Plass, C., Devine, S.M., Heerema, N.A., Murgo, A., Chan, K.K., Grever, M.R., Byrd, J.C. and Marcucci, G. Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia. J. Clin. Oncol. 25 (2007) 3884–3891.
Herman, J., Merlo, J.J. and Baylin, S.B. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15 INK4b. Cancer Res. 56 (1996) 722–727.
Herman, J.G., Civin, C.I., Issa, J.P., Collector, M.I., Sharkis, S.J. and Baylin, S.B. Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies. Cancer Res. 57 (1997) 837–841.
Cameron, E.E., Baylin, S.B. and Herman, J.G. p15INK4B CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing. Blood 94 (1999) 2445–2451.
Oki, Y. and Issa, J.P. Epigenetic mechanisms in AML-a target for therapy. Cancer Treat. Res. 145 (2010) 19–40.
Ribeiro-Filho, L.A., Franks, J., Sasaki, M., Shiina, H., Li, L.C., Nojima, D., Arap, S., Carroll, P., Enokida, H., Nakagawa, M., Yonezawa, S. and Dahiya, R. CpG hypermethylation of promoter region and inactivation of E-cadherin gene in human bladder cancer. Mol. Carcinog. 34 (2002) 187–198.
Corn, P.G., Smith, B.D., Ruckdeschel, E.S., Douglas, D., Baylin, S.B. and Herman, J.G. E-cadherin expression is silenced by 5′CpG island methylation in acute myeloid leukemia. Clin. Cancer Res. 6 (2000) 4243–4248.
Melki, J.R., Vincent, P.C., Brown, R.D. and Clark, S.J. Hypermethylation of E-cadherin in leukemia. Blood 95 (2000) 3208–3213.
Shimamoto, T., Ohyashiki, J.H. and Ohyashiki, K. Methylation of p15 (INK4b) and E-cadherin genes is independently correlated with poor prognosis in acute myeloid leukemia. Leuk. Res. 6 (2005) 653–659.
Farinha, N.J., Shaker, S., Lemaire, M., Momparler, L., Bernstein, M. and Momparler, R.L. Activation of expression of p15, p73 and E-cadherin in leukemic cells by different concentrations of 5-aza-2′-deoxycytidine (Decitabine). Anticancer Res. 24 (2004) 75–78.
Collins, S. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation and cellular oncogene expression. Blood 70 (1987) 1233–1244.
Hurley, C.K. Electrophoresis of histones: a modified Panyim and Chalkley system for slab gels. Anal. Biochem. 80 (1977) 624–626.
Nayera, H., El-Shakankiry, M.D., Ghada, I. and Mossallam, M.D. p15 (INK4B) and E-cadherin CpG Island methylation is frequent in Egyptian acute myeloid leukemia. J. Egypt. Nat. Cancer Inst. 18 (2006) 227–232.
Chuang, J.C., Yoo, C.B., Kwan, J.M., Li, T.W., Liang, G., Yang, A.S. and Jones, P.A. Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2′-deoxycytidine. Mol. Cancer Ther. 4 (2005) 1515–1520.
Stressemann, C., Brueckner, B., Musch, T., Stopper, H. and Lyko, F. Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res. 66 (2006) 2794–2800.
Flotho, C., Claus, R., Batz, C., Schneider, M., Sandrock, I., Inhde, S., Plass, C., Niemeyer, C.M. and Lubbert, M. The DNA methyltransferase inhibitors azacytidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells. Leukemia 23 (2009) 1019–1028.
Ben-Kausus, T., Ben-Zvi, Z., Marquez, V.E., Kelly, J.A. and Agbaria, R. Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Biochem. Pharmacol. 70 (2005) 121–133.
Lemaire, M., Momparlier, L.F., Bernstein, M.L., Marquez, V.E. and Momparlier, R.L. Enhancement of antineoplastic action of 5-aza-2′-deoxycytidine by zebularine on L1210 leukemia. Anticancer Drugs 16 (2005) 301–308.
Cheng, C.C., Yoo, C.B., Weisenberg, D.J., Chuang, J., Wozniak, C., Liang, G., Marquez, V.E., Greer, S., Orntoft, T.F., Thykjaer, T. and Jones, P.A. Preferential response of cancer cells to zebularine. Cancer Cell 6 (2004) 151–158.
Gotz, C., Wagner, P., Issinger, O.G. and Montenarh, M. p21WAF1/CIP 1 interacts with protein kinase CK2. Oncogene 13 (1996) 391–398.
Waga, S., Hannon, G.J., Beach, D. and Stillman, B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369 (1994) 1766–1771.
Chuang, L.S., Ian, H.I., Koh, T.W., Ng, H.H., Xu, G. and Li, B.F. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21 Waf1. Science 277 (1996) 2000.
Velicesku, M., Weisenberger, D.J., Gonzales, F.A., Tsai, Y.C., Nguen, C.T. and Jones, P.A. Cell division is required for de novo methylation of CpG islands in bladder cancer. Cancer Res. 62 (2002) 2378–2384.
Millutinovic, S., Zhuang, Q., Niveleu, A. and Szyf, M. Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes. J. Biol. Chem. 278 (2003) 14985–14995.
Billam, M., Sobolewski, M.D. and Davidson, N.E. Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Res. Treat. 120 (2010) 581–592.
Nieto, M., Sampler, E., Fraga, M.F., Gonzales de Buitrago, G., Esteller, M. and Serrano, M. The absence of p53 is critical for the induction of apoptosis by 5-aza-2′-cytidine. Oncogene 3 (2004) 735–743.
Neureither, D., Zopf, S., Leu, T., Dietze, O., Hauser-Kronberger, C., Hahn, E.G., Herold, C. and Ocker, M. Apoptosis, proliferation and differentiation patterns are influenced by zebularine and SAHA in pancreatic cancer models. Scand. J. Gastroenterol. 42 (2007) 103–116.
Savickiene, J., Treigyte, G., Jonusiene, V., Bruzaite, R., Borutinskaite, V.-V. and Navakauskiene, R. Epigenetic changes by zebularine leading to enhanced differentiation of human promyelocytic leukemia NB4 and KG1 cells. Mol. Cell. Biochem. 359 (2012) 245–261.
Martin, S.J., Bradley, G.J. and Cotter, T.G. HL-60 cells induced differentiate towards neutrophils subsequently die via apoptosis. Clin. Exp. Immunol. 79 (1990) 448–453.
Nagy, I., Thomazy, V.A., Shipley, G.L., Fesus, L., Lamph, W., Heyman, R.A., Chandraratana, R.A. and Davies, P.I. Activation of retinoid X receptors induces apoptosis in HL-60 cell line. Mol. Cell Biol. 15 (1995) 3440–3451.
Watson, R.W., Rostein, O.D., Parodo, J., Bitar, R., Hackman, D. and Marshall, J.C. Granulocytic differentiation of HL-60 cells result in spontaneous apoptosis mediated by increased caspase expression. FEBS Letters 412 (1997) 603–609.
Ozeki, M. and Shively, J.E. Differential cell fates induced by all-trans retinoic acid-treated HL-60 human leukemia cells. J. Leuk. Biol. 84 (2008) 769–779.
Ueno, H., Kizaki, M., Matsushita, H., Muto, A., Yamato, K., Nishihara, T., Hid, T., Yoshimura, H., Koeffler, H.P., Ikeda, Y. A novel retinoic acid receptor (RAR)-selective antagonist inhibits differentiation and apoptosis of HL-60 cells: implications of RARα-mediated signals in myeloid leukemic cells. Leuk. Res. 6 (1998) 517–525.
De The, H., Lavau, C., Marchio, A., Chomienne, C., Degos, L. and Dejean, A. A PML-RARα fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 66 (1991) 675–684.
Altucci, L., Rossin, A., Raffelsberger, W., Reitmair, A., Chomienne, C. and Gronemeyer, H. Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL. Nat. Med. 7 (2001) 680–686.
Benoit, G.R., Flexor, M., Besançon, F., Altucci, L., Rossin, A., Hillion, J., Balajthy, Z., Legres, L., Ségal-Bendirdjian, E., Gronemeyer, H. and Lanotte, M. Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells. Mol. Endocrinol. 15 (2011) 1154–1189.
Rao, S.P., Rechhsteiner, M.P., Berger, C., Sigrist, J.A., Nadal, D. and Bernaqsconi, M. Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkit lymphoma Akata cells. Mol. Cancer 6 (2007) 3–9.
Fazi, F., Travaglini, L., Carotti, D., Palitti, F., Diverio, D., Alcalay, M., McNamara, S., Miller, W.H.Jr., Lo Coco, F., Pelicci, P.G. and Nervi, C. Retinoic acid targets DNA-methyltransferases and histone deacetylases during APL blast differentiation in vitro and in vivo. Oncogene 24 (2005) 1820–1830.
Griffiths, E.A. and Gore, S.D. DNA methyltransferase and histone deacetyase inhibitors in the treatment of myelodysplastic syndromes. Semin. Hematol. 45 (2008) 23–30.
Savickiene, J., Borutinskaite, V.-V., Treigyte, G., Magnusson, K.-E. and Navakauskiene, R. The novel deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines. Europ. J. Pharmacol. 549 (2006) 9–18.
Bradbury, C.A., Khanim, F.L., Hayden, R., Bunce, C.M., White, D.A., Drayson, M.T., Craddock, C. and Turner, B.M. Histone deacetylases in acute myeloid leukemia show a distinct pattern of expression that changes selectively in response to deacetylase inhibitors. Leukemia 19 (2005) 1751–1759.
Wada, T., Kikuchi, J., Nishimura, N., Shimizu, R., Kitamura, T. and Furukawa, Y. Expression levels of histone deacetylases determine the cell fate of hematopoietic progenitors. J. Biol. Chem. 28 (2009) 3073–3069.
Krämer, O.H., Zhu, P., Ostendorff, H.P., Golebiewski, M., Tiefenbach, J., Peters, M.A., Brill, B., Groner, B., Bach, I., Heinzel, T. and Göttlicher, M. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. EMBO J. 22 (2003) 3411–3420.
Gozzini, A., Rovida, E., Sbarba, P.D., Galimbert, S. and Santini, V. Butyrates, as a single drug, induce histone acetylation and granulocytic maturation: possible selectivity on core binding factor-acute myeloid leukemia blasts. Cancer Res. 15 (2003) 8955–8961.
Peinado, H., Ballestar, E., Esteller, M. and Cano, A. Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex. Mol. Cell Biol. 24 (2004) 306–319.
Von Burstin, J., Eser, S., Paul, M.C., Seidler, B., Brandl, M., Messer, M., von Waerder, A., Schmit, A., Mages, J., Pagel, P., Schnieke, R.M., Schneider, G. and Saur, D. E-cadherin regulates metastasis of pancreatic cancer in vivo and is suppressed by a SNAIL/HDAC1/HDAC2 repressor complex. Gastroenterology 137 (2009) 361–371.
Grunstein, M. Histone acetylation in chromatin structure and transcription. Nature 389 (1997) 349–352.
Strahl, B.D. and Allis, D. The language of covalent histone modifications. Nature 403 (2000) 41–45.
Kouzarides, T. Histone methylation in transcriptional control. Curr. Opin. Genet. Dev. 12 (2002) 198–209.
Thomson, S., Clayton, A. L. and Mahadevan, L.C. Independent dynamic regulation of histone phosphorylation and acetylation during immediateearly gene induction. Mol. Cell 8 (2001) 1231–1241.
Nightingale, K.P., Gendreizig, S., White, D.A., Bradbury, C., Hollfelder, F. and Turner, B.M. Cross-talk between histone modifications in response to histone deacetylase inhibitors. J. Biol. Chem. 282 (2007) 4408–4416.
El-Osta, A., Kandharidis, P., Zalcberg, J.R. and Wolffe, A.P. Precipitous release of methyl-CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene (MDR1) on activation. Mol. Cell Biol. 22 (2002) 1844–1857.
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Savickiene, J., Treigyte, G., Borutinskaite, VV. et al. Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells. Cell Mol Biol Lett 17, 501–525 (2012). https://doi.org/10.2478/s11658-012-0024-5
- HDAC inhibitors