Neuroblastoma (NBL) originates from undifferentiated cells of the sympathetic nervous system. Chemotherapy is judged to be suitable for successful treatment of this disease. Here, the influence of histone deacetylase (HDAC) inhibitor valproate (VPA) combined with DNA-damaging chemotherapeutic, ellipticine, on UKF-NB-4 and SH-SY5Y neuroblastoma cells was investigated. Treatment of these cells with ellipticine in combination with VPA led to the synergism of their anticancer efficacy. The effect is more pronounced in the UKF-NB-4 cell line, the line with N-myc amplification, than in SH-SY5Y cells. This was associated with caspase-3-dependent induction of apoptosis in UKF-NB-4 cells. The increase in cytotoxicity of ellipticine in UKF-NB-4 by VPA is dictated by the sequence of drug administration; the increased cytotoxicity was seen only after either simultaneous exposure to these drugs or after pretreatment of cells with ellipticine before their treatment with VPA. The synergism of treatment of cells with VPA and ellipticine seems to be connected with increased acetylation of histones H3 and H4. Further, co-treatment of cells with ellipticine and VPA increased the formation of ellipticine-derived DNA adducts, which indicates an easier accessibility of ellipticine to DNA in cells by its co-treatment with VPA and also resulted in higher ellipticine cytotoxicity. The results are promising for in vivo studies and perhaps later for clinical studies of combined treatment of children suffering from high-risk NBL.

Analytical and Environmental Sciences Division MRC PHE Centre for Environment and Health King's College London London WC2R 2LS UK

Department of Biochemistry Faculty of Science Charles University Albertov 2030 CZ 128 43 Prague 2 Czech Republic

Department of Pediatric Hematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 1 CZ 150 06 Prague 5 Czech Republic

Division of Radiopharmaceutical Chemistry German Cancer Research Center Im Neuenheimer Feld 280 69120 Heidelberg Germany

NIHR Health Protection Research Unit in Health Impact of Environmental Hazards at King's College London in Partnership with Public Health England London WC2R 2LS UK

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Brodeur G.M. Neuroblastoma: Biological insights into a clinical enigma. Nat. Rev. Cancer. 2003;3:203–216. doi: 10.1038/nrc1014. PubMed DOI

Schwab M., Westermann F., Hero B., Berthold F. Neuroblastoma: Biology and molecular and chromosomal pathology. Lancet Oncol. 2003;4:472–480. doi: 10.1016/S1470-2045(03)01166-5. PubMed DOI

Maris J.M., Hogarty M.D., Bagatell R., Cohn S.L. Neuroblastoma. Lancet. 2007;369:2106–2120. doi: 10.1016/S0140-6736(07)60983-0. PubMed DOI

Furchert S.E., Lanvers-Kaminsky C., Jürgens H., Jung M., Loidl A., Frühwald M.C. Inhibitors of histone deacetylases as potential therapeutic tools for high-risk embryonal tumors of the nervous system of childhood. Int. J. Cancer. 2007;120:1787–1794. doi: 10.1002/ijc.22401. PubMed DOI

Decock A., Ongenaert M., Vandesompele J., Speleman F. Neuroblastoma epigenetics: From candidate gene approaches to genome-wide screenings. Epigenetics. 2011;6:962–970. doi: 10.4161/epi.6.8.16516. PubMed DOI

Santini V., Gozzini A., Ferrari G. Histone deacetylase inhibitors: Molecular and biological activity as a premise to clinical application. Curr. Drug Metab. 2007;8:383–393. doi: 10.2174/138920007780655397. PubMed DOI

Portela A., Esteller M. Epigenetic modifications and human disease. Nat. Biotechnol. 2010;28:1057–1068. doi: 10.1038/nbt.1685. PubMed DOI

Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705. doi: 10.1016/j.cell.2007.02.005. PubMed DOI

Perri F., Longo F., Giuliano M., Sabbatino F., Favia G., Ionna F., Addeo R., Della Vittoria Scarpati G., Di Lorenzo G., Pisconti S. Epigenetic control of gene expression: Potential implications for cancer treatment. Crit. Rev. Oncol. Hematol. 2017;111:166–172. doi: 10.1016/j.critrevonc.2017.01.020. PubMed DOI

Glozak M.A., Sengupta N., Zhang X., Seto E. Acetylation and deacetylation of non-histone proteins. Gene. 2005;363:15–23. doi: 10.1016/j.gene.2005.09.010. PubMed DOI

Kretsovali A., Hadjimichael C., Charmpilas N. Histone deacetylase inhibitors in cell pluripotency, differentiation, and reprogramming. Stem Cells Int. 2012;2012:1841154. doi: 10.1155/2012/184154. PubMed DOI PMC

Eckschlager T., Plch J., Stiborova M., Hrabeta J. Histone deacetylase inhibitors as anticancer drugs. Int. J. Mol. Sci. 2017;18:1414. doi: 10.3390/ijms18071414. PubMed DOI PMC

Chen C.L., Sung J., Cohen M., Chowdhury W.H., Sachs M.D., Li Y., Lakshmanan Y., Yung B.Y., Lupold S.E., Rodriguez R. Valproic acid inhibits invasiveness in bladder cancer but not in prostate cancer cells. J. Pharmacol. Exp. Ther. 2006;319:533–542. doi: 10.1124/jpet.106.106658. PubMed DOI

Stockhausen M.T., Sjölund J., Manetopoulos C., Axelson H. Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells. Br. J. Cancer. 2005;92:751–759. doi: 10.1038/sj.bjc.6602309. PubMed DOI PMC

Stiborova M., Eckschlager T., Poljakova J., Hrabeta J., Adam V., Kizek R., Frei E. The Synergistic effects of DNA-targeted chemotherapeutics and histone deacetylase inhibitors as therapeutic strategies for cancer treatment. Curr. Med. Chem. 2012;19:4218–4238. doi: 10.2174/092986712802884286. PubMed DOI

Atmaca A., Al-Batran S.-E., Maurer A., Neumann A., Heinzel T., Hentsch B., Schwarz S.E., Hövelmann S., Göttlicher M., Knuth A., et al. Valproic acid (VPA) in patients with refractory advanced cancer: A dose escalating phase I clinical trial. Br. J. Cancer. 2007;97:177–182. doi: 10.1038/sj.bjc.6603851. PubMed DOI PMC

Munster P., Marchion D., Bicaku E., Lacevic M., Kim J., Centeno B., Daud A., Neuger A., Minton S., Sullivan D. Clinical and biological effects of valproic acid as a histone deacetylase inhibitor on tumor and surrogate tissues: Phase I/II trial of valproic acid and epirubicin/FEC. Clin. Cancer Res. 2009;15:2488–2496. doi: 10.1158/1078-0432.CCR-08-1930. PubMed DOI

Rocca A., Minucci S., Tosti G., Croci D., Contegno F., Ballarini M., Nolè F., Munzone E., Salmaggi A., Goldhirsch A., et al. A phase I-II study of the histone deacetylase inhibitor valproic acid plus chemoimmunotherapy in patients with advanced melanoma. Br. J. Cancer. 2009;100:28–36. doi: 10.1038/sj.bjc.6604817. PubMed DOI PMC

Vandermeers F., Hubert P., Delvenne P., Mascaux C., Grigoriu B., Burny A., Scherpereel A., Willems L. Valproate, in combination with pemetrexed and cisplatin, provides additional efficacy to the treatment of malignant mesothelioma. Clin. Cancer Res. 2009;15:2818–2828. doi: 10.1158/1078-0432.CCR-08-1579. PubMed DOI

Kim M.S., Blake M., Baek J.H., Kohlhagen G., Pommier Y., Carrier F. Inhibition of Histone Deacetylase Increases Cytotoxicity to Anticancer Drugs Targeting DNA. Cancer Res. 2003;63:7291–7300. PubMed

Munshi A., Kurland J.F., Nishikawa T., Tanaka T., Hobbs M.L., Tucker S.L., Ismail S., Stevens C., Meyn R.E. Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity. Clin. Cancer Res. 2005;11:4912–4922. doi: 10.1158/1078-0432.CCR-04-2088. PubMed DOI

Das C.M., Zage P.E., Taylor P., Aguilera D., Wolff J.E., Lee D., Gopalakrishnan V. Chromatin remodelling at the topoisomerase II-beta promoter is associated with enhanced sensitivity to etoposide in human neuroblastoma cell lines. Eur. J. Cancer. 2010;46:2771–2780. doi: 10.1016/j.ejca.2010.05.010. PubMed DOI PMC

Poljakova J., Hrebackova J., Dvorakova M., Moserova M., Eckschlager T., Hrabeta J., Göttlicherova M., Kopejtkova B., Frei E., Kizek R., et al. Anticancer agent ellipticine combined with histone deacetylase inhibitors, valproic acid and trichostatin A, is an effective DNA damage strategy in human neuroblastoma. Neuroendocrinol. Lett. 2011;32:101–116. PubMed

Cipro Š., Hřebačková J., Hraběta J., Poljaková J., Eckschlager T. Valproic acid overcomes hypoxia-induced resistance to apoptosis. Oncol. Rep. 2012;27:1219–1226. doi: 10.3892/or.2011.1577. PubMed DOI PMC

Groh T., Hrabeta J., Poljakova J., Eckschlager T., Stiborova M. Impact of histone deacetylase inhibitor valproic acid on the anticancer effect of etoposide on neuroblastoma cells. Neuroendocrinol. Lett. 2012;33:16–24. PubMed

Groh T., Hrabeta J., Khalil M.A., Doktorova H., Eckschlager T., Stiborova M. The synergistic effects of DNA-damaging drugs cisplatin and etoposide with a histone deacetylase inhibitor valproate in high-risk neuroblastoma cells. Int. J. Oncol. 2015;4:343–352. doi: 10.3892/ijo.2015.2996. PubMed DOI

Wang G., Edwards H., Caldwell J.T., Buck S.A., Qing W.Y., Taub J.W., Ge Y., Wang Z. Panobinostat synergistically enhances the cytotoxic effects of cisplatin, dexorubicin or etoposide on high-risk neuroblastoma cells. PLoS ONE. 2013;8:e76662. PubMed PMC

Hrabeta J., Stiborova M., Adam V., Kizek R., Eckschlager T. Histone deacetylase inhibitors in cancer therapy. A review. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 2014:161–169. doi: 10.5507/bp.2013.085. PubMed DOI

New M., Olzscha H., La Thangue N.B. HDAC inhibitor-based therapies: Can we interpret the code? Mol. Oncol. 2012;6:637–656. doi: 10.1016/j.molonc.2012.09.003. PubMed DOI PMC

Poljaková J., Eckschlager T., Hraběta J., Hrebacková J., Smutný S., Frei E., Martínek V., Kizek R., Stiborová M. The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells. Biochem. Pharmacol. 2009;77:1466–1479. doi: 10.1016/j.bcp.2009.01.021. PubMed DOI

Stiborová M., Rupertová M., Frei E. Cytochrome P450- and peroxidase-mediated oxidation of anticancer alkaloid ellipticine dictates its anti-tumor efficiency. Biochim. Biophys. Acta. 2011;1814:175–185. doi: 10.1016/j.bbapap.2010.05.016. PubMed DOI

Procházka P., Libra A., Zemanová Z., Hřebačková J., Poljaková J., Hraběta J., Bunček M., Stiborová M., Eckschlager T. Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells. Cancer Sci. 2012;103:334–341. doi: 10.1111/j.1349-7006.2011.02137.x. PubMed DOI

Stiborova M., Frei E. Ellipticines as DNA-targeted chemotherapeutics. Curr. Med. Chem. 2014;21:575–591. doi: 10.2174/09298673113206660272. PubMed DOI

Hrabeta J., Groh T., Khalil M.A., Poljakova J., Adam V., Kizek R., Uhlik J., Doktorova H., Cerna T., Frei E., et al. Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug resistance in neuroblastoma cells. Int. J. Oncol. 2015;47:971–980. doi: 10.3892/ijo.2015.3066. PubMed DOI

Auclair C. Multimodal action of antitumor agents on DNA: The ellipticine series. Arch. Biochem. Biophys. 1987;259:1–14. doi: 10.1016/0003-9861(87)90463-2. PubMed DOI

Garbett N.C., Graves D.E. Extending nature's leads: The anticancer agent ellipticine. Curr. Med. Chem. Anti-Cancer Agents. 2004;4:149–172. doi: 10.2174/1568011043482070. PubMed DOI

Tmejova K., Krejcova L., Hynek D., Adam V., Babula P., Trnková L., Stiborova M., Eckschlager T., Kizek R. Electrochemical study of ellipticine interaction with single and double stranded oligonucleotides. Anti-Cancer Agents Med. Chem. 2014;14:331–340. doi: 10.2174/18715206113139990316. PubMed DOI

Zwelling L.A., Michaels S., Kerrigan D., Pommier Y., Kohn K.W. Protein-associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl-9-hydroxyellipticinium. Biochem. Pharm. 1982;31:3261–3267. doi: 10.1016/0006-2952(82)90560-3. PubMed DOI

Stiborová M., Sejbal J., Borek-Dohalská L., Aimová D., Poljaková J., Forsterová K., Rupertová M., Wiesner J., Hudecek J., Wiessler M., et al. The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide. Cancer Res. 2004;64:8374–8380. doi: 10.1158/0008-5472.CAN-04-2202. PubMed DOI

Stiborová M., Poljaková J., Ryslavá H., Dracínský M., Eckschlager T., Frei E. Mammalian peroxidases activate anticancer drug ellipticine to intermediates forming deoxyguanosine adducts in DNA identical to those found in vivo and generated from 12-hydroxyellipticine and 13-hydroxyellipticine. Int. J. Cancer. 2007;120:243–251. doi: 10.1002/ijc.22247. PubMed DOI

Stiborová M., Rupertová M., Aimová D., Ryslavá H., Frei E. Formation and persistence of DNA adducts of anticancer drug ellipticine in rats. Toxicology. 2007;236:50–60. doi: 10.1016/j.tox.2007.03.026. PubMed DOI

Stiborová M., Indra R., Moserová M., Cerná V., Rupertová M., Martínek V., Eckschlager T., Kizek R., Frei E. Cytochrome b5 increases cytochrome P450 3A4-mediated activation of anticancer drug ellipticine to 13-hydroxyellipticine whose covalent binding to DNA is elevated by sulfotransferases and N,O-acetyltransferases. Chem. Res. Toxicol. 2012;25:1075–1085. doi: 10.1021/tx3000335. PubMed DOI

Stiborová M., Poljaková J., Martínková E., Ulrichová J., Simánek V., Dvořák Z., Frei E. Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5. Toxicology. 2012;302:233–241. doi: 10.1016/j.tox.2012.08.004. PubMed DOI

Kizek R., Adam V., Hrabeta J., Eckschlager T., Smutny S., Burda J.V., Frei E., Stiborova M. Anthracyclines and ellipticines as DNA-damaging anticancer drugs: Recent advances. Pharmacol. Ther. 2012;133:26–39. doi: 10.1016/j.pharmthera.2011.07.006. PubMed DOI

Kotrbová V., Mrázová B., Moserová M., Martínek V., Hodek P., Hudeček J., Frei E., Stiborová M. Cytochrome b5 shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy. Biochem. Pharmacol. 2012;82:669–680. doi: 10.1016/j.bcp.2011.06.003. PubMed DOI

Li T., Wang L., Ke X.X., Gong X.Y., Wan J.H., Hao X.W., Xu M., Xiang Z., Cui Z.B., Cui H. DNA-damaging drug-induced apoptosis sensitized by N-myc in neuroblastoma cells. Cell Biol. Int. 2012;36:331–337. doi: 10.1042/CBI20110231. PubMed DOI

Khalil M.A., Hrabeta J., Groh T., Prochazka P., Doktorova H., Eckschlager T. Valproic acid increases CD133 positive cells that show low sensitivity to cytostatics in neuroblastoma. PLoS ONE. 2016;11:e0162916. doi: 10.1371/journal.pone.0162916. PubMed DOI PMC

Chou T.C., Talalay P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984;22:27–55. doi: 10.1016/0065-2571(84)90007-4. PubMed DOI

Chou T.C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 2006;58:621–681. doi: 10.1124/pr.58.3.10. PubMed DOI

Earnshaw W.C., Martins L.M., Kaufmann S.H. Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 1999;68:383–424. doi: 10.1146/annurev.biochem.68.1.383. PubMed DOI

Porter A.G., Jänicke R.U. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999;6:99–104. doi: 10.1038/sj.cdd.4400476. PubMed DOI

Slee E.A., Adrain C., Martin S.J. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J. Biol. Chem. 2001;276:7320–7326. doi: 10.1074/jbc.M008363200. PubMed DOI

Nakamura A.J., Rao V.A., Pommier Y., Bonner W.M. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle. 2010;9:389–397. doi: 10.4161/cc.9.2.10475. PubMed DOI PMC

Sharma A., Singh K., Almasan A. Histone H2AX phosphorylation: A marker for DNA damage. Methods Mol. Biol. 2012;920:613–626. PubMed

Stiborová M., Indra R., Frei E., Kopečková K., Schmeiser H.H., Eckschlager T., Adam V., Heger Z., Arlt V.M., et al. Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine. Monatsh. Chem. 2017;148:1983–1991. doi: 10.1007/s00706-017-1986-9. PubMed DOI PMC

Luchenko V.L., Salcido C.D., Zhang Y., Agama K., Komlodi-Pasztor E., Murphy R.F., Giaccone G., Pommier Y., Bates S.E., et al. Schedule-dependent synergy of histone deacetylase inhibitors with DNA damaging agents in small cell lung cancer. Cell Cycle. 2011;10:3119–3128. doi: 10.4161/cc.10.18.17190. PubMed DOI PMC

Lutz W., Fulda S., Jeremias I., Debatin K.M., Schwab M. MycN and IFN cooperate in apoptosis of human neuroblastoma cells. Oncogene. 1998;17:339–346. doi: 10.1038/sj.onc.1200201. PubMed DOI

Petroni M., Veschi V., Prodosmo A., Rinaldo C., Massimi I., Carbonari M., Dominici C., McDowell H.P., Rinaldi C., et al. MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response. Mol. Cancer Res. 2011;9:67–77. doi: 10.1158/1541-7786.MCR-10-0227. PubMed DOI

El-Sayed M.I., Ali A.M., Sayed H.A., Zaky E.M. Treatment results and prognostic factors of pediatric neuroblastoma: A retrospective study. Int. Arch. Med. 2010;3:37. doi: 10.1186/1755-7682-3-37. PubMed DOI PMC

Poljaková J., Groh T., Gudino Z.O., Hraběta J., Bořek-Dohalská L., Kizek R., Doktorová H., Eckschlager T., Stiborová M. Hypoxia-mediated histone acetylation and expression of N-myc transcription factor dictate aggressiveness of neuroblastoma cells. Oncol. Rep. 2014;31:1928–1934. doi: 10.3892/or.2014.2999. PubMed DOI

Cinatl J., Jr., Cinatl J., Driever P.H., Kotchetkov R., Pouckova P., Kornhuber B., Schwabe D. Sodium valproate inhibits in vivo growth of human neuroblastoma cells. Anticancer Drugs. 1997;8:958–963. doi: 10.1097/00001813-199711000-00007. PubMed DOI

Ke N., Wang X., Xu X., Abassi Y.A. The xCELLigence system for real-time and label-free monitoring of cell viability. Methods Mol. Biol. 2011;740:33–43. PubMed

Shechter D., Dormann H.L., Allis C.D., Hake S.B. Extraction, purification and analysis of histones. Nat. Protoc. 2007;2:1445–1457. doi: 10.1038/nprot.2007.202. PubMed DOI

Lowry O.H., Rosenbrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275. PubMed

Frei E., Bieler C.A., Arlt V.M., Wiessler M., Stiborová M. Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes. Biochem. Pharmacol. 2002;64:289–295. doi: 10.1016/S0006-2952(02)01072-9. PubMed DOI

Poljaková J., Frei E., Gomez J.E., Aimová D., Eckschlager T., Hrabeta J., Stiborová M. DNA adduct formation by the anticancer drug ellipticine in human leukemia HL-60 and CCRF-CEM cells. Cancer Lett. 2007;252:270–279. doi: 10.1016/j.canlet.2006.12.037. PubMed DOI