Background: DNA methyltransferases (DNMTs) are key epigenetic regulatory enzymes involved in the expression of many genes and are considered as an attractive target for cancer treatment, especially hematological malignancies. Therefore, promising DNMT inhibitors characterized by low toxicity, target activity and high selectivity are crucial for the development of new cancer therapy and research on the inhibitory mechanism. We had previously demonstrated that the novel 2’-fluoro-2’-deoxy-arabinofuranosyl 5-azacytosine nucleoside (2’F-araAC) showed high antiproliferative activity invitro and increased hydrolytic stability compared to the known agents like azacitidine and decitabine. Objective: The objective of the present study was to investigate the effect of novel 2’F-araAC as potent anti-leukemia agent and DNMTs inhibitor on nuclear extract of the HCT-116 human colorectal cell line and P388 and L1210 mouse leukemia cell lines. Methods: The DNMTs activity was evaluated using the fluorometric DNMT Activity Quantification Kit (Abcam) and were reported as the percentage of control. Nuclear proteins were extracted from HCT-116 cell line using the Nuclear Extraction Kit (Abcam). To explore the mechanism of anti-leukemic activity of 2’F-araAC, cell cycle and apoptosis analyses were performed on P388 and L1210 cell lines. Results: It has been shown that the DNMTs activity was significantly reduced at 1 and 10 μM of 2’F-araAC compared to controls. Moreover, 2’F-araAC can induce G2/M cell cycle arrest and apoptosis in P388 and L1210 mouse leukemia cell lines as shown by flow cytometry method. Apoptosis was 54.53% and 43.35% for 2’F-araAC vs. 2.88% and 5.25% for the control P388 and L1210 cell lines, respectively. Conclusions: Thus, our study presents a new and promising compound to further develop new epigenetic regulators to be used as antitumor agents.
References
[1]
Corey, S.J., Minden, M.D., Barber, D.L., Kantarjian, H., Wang, J.C.Y. and Schimmer, A.D. (2007) Myelodysplastic Syndromes: The Complexity of Stem-Cell Diseases. Nature Reviews Cancer, 7, 118-129. https://doi.org/10.1038/nrc2047
[2]
Steensma, D.P. and Bennett, J.M. (2006) The Myelodysplastic Syndromes: Diagnosis and Treatment. Mayo Clinic Proceedings, 81, 104-130. https://doi.org/10.4065/81.1.104
[3]
Ehrlich, M. (2002) DNA Methylation in Cancer: Too Much, but Also Too Little. Oncogene, 21, 5400-5413. https://doi.org/10.1038/sj.onc.1205651
[4]
Feinberg, A.P. (2018) The Key Role of Epigenetics in Human Disease Prevention and Mitigation. New England Journal of Medicine, 378, 1323-1334. https://doi.org/10.1056/nejmra1402513
[5]
Kiziltepe, T., Hideshima, T., Catley, L., Raje, N., Yasui, H., Shiraishi, N., et al. (2007) 5-Azacytidine, a DNA Methyltransferase Inhibitor, Induces ATR-Mediated DNA Double-Strand Break Responses, Apoptosis, and Synergistic Cytotoxicity with Doxorubicin and Bortezomib against Multiple Myeloma Cells. Molecular Cancer Therapeutics, 6, 1718-1727. https://doi.org/10.1158/1535-7163.mct-07-0010
[6]
Karahoca, M. and Momparler, R.L. (2013) Pharmacokinetic and Pharmacodynamic Analysis of 5-Aza-2’-Deoxycytidine (Decitabine) in the Design of Its Dose-Schedule for Cancer Therapy. Clinical Epigenetics, 5, Article No. 3. https://doi.org/10.1186/1868-7083-5-3
[7]
Seelan, R.S., Mukhopadhyay, P., Pisano, M.M. and Greene, R.M. (2018) Effects of 5-Aza-2’-Deoxycytidine (Decitabine) on Gene Expression. Drug Metabolism Reviews, 50, 193-207. https://doi.org/10.1080/03602532.2018.1437446
[8]
Okano, M., Bell, D.W., Haber, D.A. and Li, E. (1999) DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development. Cell, 99, 247-257. https://doi.org/10.1016/s0092-8674(00)81656-6
[9]
Yagi, M., Kabata, M., Tanaka, A., Ukai, T., Ohta, S., Nakabayashi, K., et al. (2020) Identification of Distinct Loci for De Novo DNA Methylation by DNMT3A and DNMT3B during Mammalian Development. Nature Communications, 11, Article No. 3199. https://doi.org/10.1038/s41467-020-16989-w
[10]
Gershman, A., Sauria, M.E.G., Guitart, X., Vollger, M.R., Hook, P.W., Hoyt, S.J., et al. (2022) Epigenetic Patterns in a Complete Human Genome. Science, 376, eabj5089. https://doi.org/10.1126/science.abj5089
[11]
Kato, Y., Kaneda, M., Hata, K., Kumaki, K., Hisano, M., Kohara, Y., et al. (2007) Role of the Dnmt3 Family in De Novo Methylation of Imprinted and Repetitive Sequences during Male Germ Cell Development in the Mouse. Human Molecular Genetics, 16, 2272-2280. https://doi.org/10.1093/hmg/ddm179
[12]
Sun, J., Yang, J., Miao, X., Loh, H.H., Pei, D. and Zheng, H. (2021) Proteins in DNA Methylation and Their Role in Neural Stem Cell Proliferation and Differentiation. Cell Regeneration, 10, Article No. 7. https://doi.org/10.1186/s13619-020-00070-4
[13]
Jurkowska, R.Z., Jurkowski, T.P. and Jeltsch, A. (2010) Structure and Function of Mammalian DNA Methyltransferases. ChemBioChem, 12, 206-222. https://doi.org/10.1002/cbic.201000195
[14]
Lyko, F. (2017) The DNA Methyltransferase Family: A Versatile Toolkit for Epigenetic Regulation. Nature Reviews Genetics, 19, 81-92. https://doi.org/10.1038/nrg.2017.80
[15]
Herman, J.G. and Baylin, S.B. (2003) Gene Silencing in Cancer in Association with Promoter Hypermethylation. New England Journal of Medicine, 349, 2042-2054. https://doi.org/10.1056/nejmra023075
[16]
Ye, F. and Li, N. (2019) Role of P15(INK4B) Methylation in Patients with Myelodysplastic Syndromes: A Systematic Meta-Analysis. Clinical Lymphoma Myeloma and Leukemia, 19, e259-e265.
[17]
Chen, H. and Wu, S. (2002) Hypermethylation of the p15(INK4B) Gene in Acute Leukemia and Myelodysplastic Syndromes. Chinese Medical Journal, 115, 987-990.
[18]
Chim, C.S., Tam, C.Y.Y., Liang, R. and Kwong, Y.L. (2001) Methylation of P15 and P16 Genes in Adult Acute Leukemia. Cancer, 91, 2222-2229. https://doi.org/10.1002/1097-0142(20010615)91:12<2222::aid-cncr1252>3.3.co;2-i
[19]
Aggerholm, A., Holm, M.S., Guldberg, P., Olesen, L.H. and Hokland, P. (2005) Promoter Hypermethylation of p15INK4B, HIC1, CDH1, and ER Is Frequent in Myelodysplastic Syndrome and Predicts Poor Prognosis in Early-Stage Patients. European Journal of Haematology, 76, 23-32. https://doi.org/10.1111/j.1600-0609.2005.00559.x
[20]
Brenner, C. and Fuks, F. (2006) DNA Methyltransferases: Facts, Clues, Mysteries. In: Current Topics in Microbiology and Immunology, Springer, 45-66. https://doi.org/10.1007/3-540-31390-7_3
[21]
Kar, S., Deb, M., Sengupta, D., Shilpi, A., Parbin, S., Torrisani, J., et al. (2012) An Insight into the Various Regulatory Mechanisms Modulating Human DNA Methyltransferase 1 Stability and Function. Epigenetics, 7, 994-1007. https://doi.org/10.4161/epi.21568
[22]
Derissen, E.J.B., Beijnen, J.H. and Schellens, J.H.M. (2013) Concise Drug Review: Azacitidine and Decitabine. The Oncologist, 18, 619-624. https://doi.org/10.1634/theoncologist.2012-0465
[23]
Hu, C., Liu, X., Zeng, Y., Liu, J. and Wu, F. (2021) DNA Methyltransferase Inhibitors Combination Therapy for the Treatment of Solid Tumor: Mechanism and Clinical Application. Clinical Epigenetics, 13, Article No. 166. https://doi.org/10.1186/s13148-021-01154-x
[24]
Krečmerová, M. and Otmar, M. (2012) 5-Azacytosine Compounds in Medicinal Chemistry: Current Stage and Future Perspectives. Future Medicinal Chemistry, 4, 991-1005. https://doi.org/10.4155/fmc.12.36
[25]
Ghavifekr Fakhr, M., Farshdousti Hagh, M., Shanehbandi, D. and Baradaran, B. (2013) DNA Methylation Pattern as Important Epigenetic Criterion in Cancer. Genetics Research International, 2013, 1-9. https://doi.org/10.1155/2013/317569
[26]
Cheng, J.C., Matsen, C.B., Gonzales, F.A., Ye, W., Greer, S., Marquez, V.E., et al. (2003) Inhibition of DNA Methylation and Reactivation of Silenced Genes by Zebularine. Journal of the National Cancer Institute, 95, 399-409. https://doi.org/10.1093/jnci/95.5.399
[27]
Zhou, L., Cheng, X., Connolly, B.A., Dickman, M.J., Hurd, P.J. and Hornby, D.P. (2002) Zebularine: A Novel DNA Methylation Inhibitor That Forms a Covalent Complex with DNA Methyltransferases. Journal of Molecular Biology, 321, 591-599. https://doi.org/10.1016/s0022-2836(02)00676-9
[28]
Gillis, E.P., Eastman, K.J., Hill, M.D., Donnelly, D.J. and Meanwell, N.A. (2015) Applications of Fluorine in Medicinal Chemistry. Journal of Medicinal Chemistry, 58, 8315-8359. https://doi.org/10.1021/acs.jmedchem.5b00258
[29]
Van Den Boogaart, J.E., Kalinichenko, E.N., Podkopaeva, T.L., Mikhailopulo, I.A. and Altona, C. (1994) Conformational Analysis of 3’-Fluorinated A(2’‐5’) A(2’-5’) A Fragments. European Journal of Biochemistry, 221, 759-768. https://doi.org/10.1111/j.1432-1033.1994.tb18789.x
[30]
Bozhok, T.S., Sivets, G.G., Baranovsky, A.V. and Kalinichenko, E.N. (2016) Synthesis of Novel 6-Substituted Thymine Ribonucleosides and Their 3’-Fluorinated Analogues. Tetrahedron, 72, 6518-6527. https://doi.org/10.1016/j.tet.2016.08.065
[31]
Gu, D., Dong, K., Jiang, A., Jiang, S., Fu, Z., Bao, Y., et al. (2022) PBRM1 Deficiency Sensitizes Renal Cancer Cells to DNMT Inhibitor 5-Fluoro-2’-Deoxycytidine. Frontiers in Oncology, 12, Article 870229. https://doi.org/10.3389/fonc.2022.870229
[32]
Bozhok, T.S., Kalinichenko, E.N., Kuz’mitskii, B.B. and Golubeva, M.B. (2016) Synthesis, Hydrolytic Stability, and Antileukemic Activity of Azacytidine Nucleoside Analogs. Pharmaceutical Chemistry Journal, 49, 804-809. https://doi.org/10.1007/s11094-016-1375-4
[33]
Zilberman, R., Bozhok, T., Panibrat, A., Savin, A., Vinnikova, O. and Kalinichenko, E. (2021) In vivo Study of Antileukemic Activity of New Fluorine Substituted Decitabine Analogue on Male P388D1 Tumor-Bearing Mice. In: Book of Abstracts VII International Conference “Chemistry, Structure and Function of Biomolecules”, Institute of Bioorganic Chemistry NAS of Belarus, 64.
[34]
Zilberman, R.D., Bozhok, T.S., Panibrat, O.V., Savin, A.O., Vinnikova, O.V. and Kalinichenko, E.N. (2021) Antitumor Activity of the 2’-Fluoro-Substituted Derivative of Decitabine in In-Vivo Experiments on the Model of Transplantable Hemoblastosis P388D1. In: Proceedings of the International Scientific and Practical Conference “Modern Technologies in Medical Education”, Belarusian State Medical University, 2090-2093.
[35]
Bozhok, Т.S., Golubeva, M.B., Zilberman, R.D. and Kalinichenko, Е.N. (2015) New Fluorine-Containing Analogue of Azacitidine: Cytotoxic Activity and Acute Toxicity. https://doi.org/10.13140/RG.2.1.1031.7843
[36]
Freshney, R.I. (2010) Culture of Animal Cells. Wiley. https://doi.org/10.1002/9780470649367
[37]
Riccardi, C. and Nicoletti, I. (2006) Analysis of Apoptosis by Propidium Iodide Staining and Flow Cytometry. Nature Protocols, 1, 1458-1461. https://doi.org/10.1038/nprot.2006.238
[38]
Rieger, A.M., Nelson, K.L., Konowalchuk, J.D. and Barreda, D.R. (2011) Modified Annexin V/propidium Iodide Apoptosis Assay for Accurate Assessment of Cell Death. Journal of Visualized Experiments, 50, Article ID: 2597. https://doi.org/10.3791/2597
[39]
Sanaei, M. and Kavoosi, F. (2022) Effect of 5’-Fluoro-2’-Deoxycytidine, 5-Azacytidine, and 5-Aza-2’–Deoxycytidine on DNA Methyltransferase 1, CIP/KIP Family, and INK4A/ARF in Colon Cancer HCT-116 Cell Line. International Journal of Cancer Management, 14, e110419. https://doi.org/10.5812/ijcm.110419
[40]
Qin, T., Jelinek, J., Si, J., Shu, J. and Issa, J.J. (2009) Mechanisms of Resistance to 5-Aza-2’-Deoxycytidine in Human Cancer Cell Lines. Blood, 113, 659-667. https://doi.org/10.1182/blood-2008-02-140038
[41]
Yang, X., Lay, F., Han, H. and Jones, P.A. (2010) Targeting DNA Methylation for Epigenetic Therapy. Trends in Pharmacological Sciences, 31, 536-546. https://doi.org/10.1016/j.tips.2010.08.001
[42]
Elmore, S. (2007) Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathology, 35, 495-516. https://doi.org/10.1080/01926230701320337
