Abstract
5-Aza-deoxycytidine (5-aza-dC) confers neuroprotection in ischemic mice by inhibiting DNA methylation. Zebularine is another DNA methylation inhibitor, less toxic and more stable in aqueous solutions and, therefore more biologically suitable. We investigated Zebularine’s effects on brain ischemia in a rat middle cerebral artery occlusion (MCAo) model in order to elucidate its therapeutic potential. Male Wistar wild-type (WT) rats were randomly allocated to three treatment groups, vehicle, Zebularine 100 μg, and Zebularine 500 μg. Saline (10 μL) or Zebularine (10 μL) was administered intracerebroventricularly 20 min before 45-min occlusion of the middle cerebral artery. Reperfusion was allowed after 45-min occlusion, and the rats were sacrificed at 24-h reperfusion. The brains were removed, sliced, and stained with 2 % 2,3,5-triphenyltetrazolium chloride (TTC) before measuring infarct size. Zebularine (500 μg) reduced infarct volumes significantly (p < 0.05) by 61 % from 20.7 ± 4.2 % in the vehicle treated to 8.1 ± 1.6 % in the Zebularine treated. Zebularine (100 μg) also reduced infarct volumes dramatically by 55 to 9.4 ± 1.2 %. The mechanisms behind this neuroprotection is not yet known, but the results agree with previous studies and support the notion that Zebularine-induced inhibition of DNA methyltransferase ameliorates ischemic brain injury in rats.
Similar content being viewed by others
References
Liu R, Yang SH. Window of opportunity: estrogen as a treatment for ischemic stroke. Brain Res. 2013;1514:83–90.
Writing Group Members, Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, et al. Heart disease and stroke statistics—2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2009;119(3):e21–181.
WHO Stroke, Cerebrovascular accident. World Health Organization. 2011.Available: http://www.who.int/topics/cerebrovascular accident/en/.
Wainer D. Strokes killing younger people as unhealthy habits rise. 2013. Available: http://www.bloomberg.com/news/2013-10-23/strokes-killing-younger-people-as-unhealthy-habits-rise.html.
Endre M, Meisel A, Biniszkiewicz D, Namura S, Prass K, Ruscher K, et al. DNA methyltransferase contributes to delayed ischemic brain injury. J Neurosci. 2000;20(9):3175–81.
Brown SE, Weaver IG, Meaney MJ, Szyf M. Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus. Neurosci Lett. 2008;440(1):49–53.
Bouchard J, Momparler RL. Incorporation of 5-Aza-2 deoxycytidine-5-triphosphate into DNA. Interactions with mammalian DNApolymerase α and DNA methylase. Mol Pharmacol. 1983;24(1):109–14.
Santi DV, Garrett CE, Barr PJ. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell. 1983;33(1):9–10.
Kogure K, Kato H. Altered gene expression in cerebral ischemia. Stroke. 1993;24(10):2121–7.
Lubbert M. DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr Top Microbiol Immunol. 2000;249:135–64.
Kim CH, Marquez VE, Mao DT, Haines DR, McCormack JJ. Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase. J Med Chem. 1986;29(8):1374–80.
Laliberte J, Marquez VE, Momparler RL. Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2-deoxycytidine by human cytidine deaminase. Cancer Chemother Pharmacol. 1992;30(1):7–11.
Gonzalgo ML, Hayashida T, Bender CM, Pao MM, Tsai YC, Gonzales FA, et al. The role of DNA methylation in expression of the p19/p16 locusin human bladder cancer cell lines. Cancer Res. 1998;58(6):1245–52.
Driscoll JS, Marquez VE, Plowman J, Liu PS, Kelley JA, Barchi Jr JJ. Antitumor properties of 2(1H)-pyrimidinone riboside (zebularine) and its fluorinated analogues. J Med Chem. 1991;34(11):3280–4.
Beisler JA. Isolation, characterization, and properties of a liable hydrolysis product of the antitumor nucleoside, 5-azacytidine. J Med Chem. 1978;21(2):204–8.
Holm L, Theodorsson E, Hökfelt T, Theodorsson A. Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats. Neuropeptides. 2011;45(1):17–23.
Lyko F, Brown B. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst. 2005;97(20):1498–506.
Ström JO, Ingberg E, Theodorsson E, Theodorsson A. Effects of high and low 17ß-estradiol doses on focal cerebral ischemia: negative results. Sci Rep. 2013;3:3111. doi:10.1038/srep03111.
Champion C, Guianvarch D, Senamaud-Beaufort C, Jurkowska RZ, Jeltsch A, Ponger L, et al. Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine. PLoS One. 2010;5(8):e12388.
Yoshida M, Horinouchi S, Beppu T. Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function. Bioassays. 1995;17(5):423–30.
Eden S, Hashimshony T, Keshet I, Cedar H, Thorne AW. DNA methylation models histone acetylation. Nature. 1998;394(6696):842.
Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet. 1998;19(2):187–91.
Nan X, Ng HH, Johnson CA, Laherty CD, Turner BT, Eisenmann RN, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature. 1998;393(6683):386–9.
Smallwood A, Esteve PO, Pradhan S, Carey M. Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes Dev. 2007;21(10):1169–78.
Faraco G, Pancani T, Formentini L, Mascaqni P, Fossati G, Leoni F, et al. Pharmacological inhibition of histone deacetylases by suberoylanilide hydroxamic acid specifically alters gene expression and reduces ischemic injury in the mouse brain. Mol Pharmacol. 2006;70(6):1876–84.
Saha RN, Pahan K. HATs and HDACs in neurodegeneration: a tale of disconcerted acetylation homeostasis. Cell Death Differ. 2006;13(4):539–50.
Lanzillotta A, Pignataro G, Branca C, Cuomo O, Sarnico I, Benarese M, et al. Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window. Neurobiol Dis. 2013;49:177–89.
Wozniak RJ, Klimecki WT, Lau SS, Feinstein Y, Futscher BW. 5-Aza-2-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation. Oncogene. 2007;26(1):77–90.
Krupinski J, Slevin M. Emerging molecular targets for brain repair after stroke. Stroke Res Treat. 2013;2013:473416.
Acknowledgments
The authors thank Jakob Ström for the discussion and critical reading of the manuscript. This study was supported by the County Council of Östergötland, Sweden.
Conflict of Interest
Hua Dock, Annette Theodorsson, and Elvar Theodorsson declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Dock, H., Theodorsson, A. & Theodorsson, E. DNA Methylation Inhibitor Zebularine Confers Stroke Protection in Ischemic Rats. Transl. Stroke Res. 6, 296–300 (2015). https://doi.org/10.1007/s12975-015-0397-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12975-015-0397-7