Human metallothionein-3 (hMT-3), also known as growth inhibitory factor, is predominantly expressed in the central nervous system. hMT-3 is presumed to participate in the processes of heavy metal detoxification, regulation of metabolism and protection against oxidative damage of free radicals in the central nervous system; thus, it could play important neuromodulatory and neuroprotective roles. However, the primary functions of hMT-3 and the mechanism underlying its multiple functions in neuroblastoma have not been elucidated so far. First, we confirmed relatively high expression of hMT-3 encoding mRNA in biopsies (n = 23) from high-risk neuroblastoma subjects. Therefore, we focused on investigation of the impact of hMT-3 up-regulation in N-Myc amplifying neuroblastoma cells. The differentially up-regulated genes involved in biological pathways related to cellular senescence and cell cycle were identified using electrochemical microarray with consequent bioinformatic processing. Further, as experimental verification of microarray data, the cytotoxicity of the cisplatin (CDDP) was examined in hMT-3 and mock cells by MTT and clonogenic assays. Overall, our data strongly suggest that up-regulation of hMT-3 positively correlates with the genes involved in oncogene-induced senescence (CDKN2B and ANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC50: 7.5 vs. 19.8 μg/ml), indicating its multipurpose biological significance.

Central European Institute of Technology Brno University of Technology CZ 616 00 Brno Czech Republic

Department of Biochemistry Faculty of Science Charles University CZ 128 40 Prague 2 Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno CZ 613 00 Brno Czech Republic

Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol CZ 150 06 Prague 5 Czech Republic

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Margoshes M, Vallee BL. A cadmium protein from equine kidney cortex. J Am Chem Soc. 1957;79:4813–14. https://doi.org/10.1021/ja01574a064 DOI

Faller P. Neuronal growth-inhibitory factor (metal-lothionein-3): reactivity and structure of metal-thiolate clusters. FEBS J. 2010;277:2921–30. https://doi.org/10.1111/j.1742-4658.2010.07717.x PubMed DOI

West AK, Hidalgo J, Eddins D, Levin ED, Aschner M. Metallothionein in the central nervous system: roles in protection, regeneration and cognition. Neurotoxicology. 2008;29:489–503. https://doi.org/10.1016/j.neuro.2007.12.006 PubMed DOI PMC

Zhou B, Yang W, Ji JG, Ru BG. Differential protein expression induced by transient transfection of metallothionein-3 gene in SH-SY5Y neuroblastoma cell line. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2003;35:522–28. PubMed

Adam P, Křížková S, Heger Z, Babula P, Pekařík V, Vaculovičoá M, Gomes CM, Kizek R, Adam V. Metallothioneins in prion- and amyloid-related diseases. J Alzheimers Dis. 2016;51:637–56. https://doi.org/10.3233/JAD-150984 PubMed DOI

Xue Z, Wu X, Chen X, Luo Q. MT3-MMP down-regulation promotes tumorigenesis and correlates to poor prognosis in esophageal squamous cell carcinoma. Cancer Med. 2016;5:2459–68. https://doi.org/10.1002/cam4.790 PubMed DOI PMC

Howman-Giles R, Shaw PJ, Uren RF, Chung DK. Neuroblastoma and other neuroendocrine tumors. Semin Nucl Med. 2007;37:286–302. https://doi.org/10.1053/j.semnuclmed.2007.02.009 PubMed DOI

Manor E, Kapelushnik J, Joshua BZ, Bodner L. Metastatic neuroblastoma of the mandible: a cytogenetic and molecular genetic study. Eur Arch Otorhinolaryngol. 2012;269:1967–71. https://doi.org/10.1007/s00405-011-1863-9 PubMed DOI

Nakamura Y, Suganami A, Fukuda M, Hasan MK, Yokochi T, Takatori A, Satoh S, Hoshino T, Tamura Y, Nakagawara A. Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma. Cancer Med. 2014;3:25–35. https://doi.org/10.1002/cam4.175 PubMed DOI PMC

Ratner N, Brodeur GM, Dale RC, Schor NF. The “neuro” of neuroblastoma: neuroblastoma as a neurodevelopmental disorder. Ann Neurol. 2016;80:13–23. https://doi.org/10.1002/ana.24659 PubMed DOI PMC

Mehrian-Shai R, Yalon M, Simon AJ, Eyal E, Pismenyuk T, Moshe I, Constantini S, Toren A. High metallothionein predicts poor survival in glioblastoma multiforme. BMC Med Genomics. 2015;8:68. https://doi.org/10.1186/s12920-015-0137-6 PubMed DOI PMC

Piskareva O, Harvey H, Nolan J, Conlon R, Alcock L, Buckley P, Dowling P, Henry M, O'Sullivan F, Bray I, Stallings RL. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett. 2015;364:142–55. https://doi.org/10.1016/j.canlet.2015.05.004 PubMed DOI

Donzelli E, Carfì M, Miloso M, Strada A, Galbiati S, Bayssas M, Griffon-Etienne G, Cavaletti G, Petruccioli MG, Tredici G. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. J Neurooncol. 2004;67:65–73. https://doi.org/10.1023/B:NEON.0000021787.70029.ce PubMed DOI

Groh T, Hrabeta J, Khalil MA, 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;47:343–52. https://doi.org/10.3892/ijo.2015.2996 PubMed DOI

Yu F, Megyesi J, Price PM. Cytoplasmic initiation of cisplatin cytotoxicity. Am J Physiol Renal Physiol. 2008;295:F44–52. https://doi.org/10.1152/ajprenal.00593.2007 PubMed DOI PMC

Fuertes MA, Castilla J, Alonso C, Pérez JM. Cisplatin biochemical mechanism of action: from cytotoxicity to induction of cell death through interconnections between apoptotic and necrotic pathways. Curr Med Chem. 2003;10:257–66. https://doi.org/10.2174/0929867033368484 PubMed DOI

Fabrik I, Krizkova S, Huska D, Adam V, Hubalek J, Trnkova L, Eckschlager T, Kukacka J, Prusa R, Kizek R. Employment of electrochemical techniques for metallothionein determination in tumor cell lines and patients with a tumor disease. Electroanalysis. 2008;20:1521–32. https://doi.org/10.1002/elan.200704215 DOI

Ruttkay-Nedecky B, Nejdl L, Gumulec J, Zitka O, Masarik M, Eckschlager T, Stiborova M, Adam V, Kizek R. The role of metallothionein in oxidative stress. Int J Mol Sci. 2013;14:6044–66. https://doi.org/10.3390/ijms14036044 PubMed DOI PMC

Marini P, MacLeod RA, Treuner C, Bruchelt G, Böhm W, Wolburg H, Schweizer P, Girgert R. SiMa, a new neuroblastoma cell line combining poor prognostic cytogenetic markers with high adrenergic differentiation. Cancer Genet Cytogenet. 1999;112:161–64. https://doi.org/10.1016/S0165-4608(98)00269-6 PMI. PubMed DOI

Felizola SJ, Nakamura Y, Arata Y, Ise K, Satoh F, Rainey WE, Midorikawa S, Suzuki S, Sasano H. Metallothionein-3 (MT-3) in the human adrenal cortex and its disorders. Endocr Pathol. 2014;25:229–35. https://doi.org/10.1007/s12022-013-9280-9 PubMed DOI

Chandeck C, Mooi WJ. Oncogene-induced cellular senescence. Adv Anat Pathol. 2010;17:42–48. PubMed

Ewald JA, Desotelle JA, Wilding G, Jarrard DF. Therapy-induced senescence in cancer. J Natl Cancer Inst. 2010;102:1536–46. https://doi.org/10.1093/jnci/djq364 PubMed DOI PMC

Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. Genes Dev. 2010;24:2463–79. https://doi.org/10.1101/gad.1971610 PubMed DOI PMC

Iolascon A, Giordani L, Moretti A, Tonini GP, Lo Cunsolo C, Mastropietro S, Borriello A, Ragione FD. Structural and functional analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in neuroblastoma. Pediatr Res. 1998;43:139–44. https://doi.org/10.1203/00006450-199801000-00021 PubMed DOI

Seçme M, Eroğlu C, Dodurga Y, Bağcı G. Investigation of anticancer mechanism of oleuropein via cell cycle and apoptotic pathways in SH-SY5Y neuroblastoma cells. Gene. 2016;585:93–99. https://doi.org/10.1016/j.gene.2016.03.038 PubMed DOI

Progressive Neuroblastoma: Innovation Novel Therapeutic Strategies. Christiansen H, Christiansen NM, editors. Karger. 2015 https://doi.org/10.1159/isbn.978-3-318-05497-2 DOI

He QY, Zhu R. Serological protein profiling by ProteinChip-SELDI-TOF technology for biomarker identification of neuroblastoma. Ann Neurol. 2005;58:35. PubMed

Yang HJ, Wang M, Wang L, Cheng BF, Lin XY, Feng ZW. NF-kappa B Regulates Caspase-4 Expression and Sensitizes Neuroblastoma Cells to Fas-Induced Apoptosis. PLoS One. 2015;10:1–16. https://doi.org/10.1371/journal.pone.0117953 PubMed DOI PMC

Chuang JZ, Zhou H, Zhu M, Li SH, Li XJ, Sung CH. Characterization of a brain-enriched chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity independently. J Biol Chem. 2002;277:19831–38. https://doi.org/10.1074/jbc.M109613200 PubMed DOI

Gumulec J, Raudenska M, Adam V, Kizek R, et al. Immunohistochemical Cancer Biomarker: A Meta-Analysis. PLoS One. 2014;9:e85346. https://doi.org/10.1371/journal.pone.0085346 PubMed DOI PMC

Babula P, Masarik M, Adam V, Eckschlager T, Stiborova M, Trnkova L, Skutkova H, Provaznik I, Hubalek J, Kizek R. Mammalian metallothioneins: properties and functions. Metallomics. 2012;4:739–50. https://doi.org/10.1039/c2mt20081c PubMed DOI

Dutsch-Wicherek M, Sikora J, Tomaszewska R. The possible biological role of metallothionein in apoptosis. Front Biosci. 2008;13:4029–38. https://doi.org/10.2741/2991 PubMed DOI

Krizkova S, Ryvolova M, Hrabeta J, Adam V, Stiborova M, Eckschlager T, Kizek R. Metallothioneins and zinc in cancer diagnosis and therapy. Drug Metab Rev. 2012;44:287–301. https://doi.org/10.3109/03602532.2012.725414 PubMed DOI

Werynska B, Pula B, Kobierzycki C, Dziegiel P, Podhorska-Okolow M. Metallothioneins in the lung cancer. Folia Histochem Cytobiol. 2015;53:1–10. https://doi.org/10.5603/FHC.a2015.0009 PubMed DOI

Dziegiel P. Expression of metallothioneins in tumor cells. Pol J Pathol. 2004;55:3–12. PubMed

Dutta R, Sens DA, Somji S, Sens MA, Garrett SH. Metallothionein isoform 3 expression inhibits cell growth and increases drug resistance of PC-3 prostate cancer cells. Prostate. 2002;52:89–97. https://doi.org/10.1002/pros.10097 PubMed DOI

Howells C, West AK, Chung RS. Neuronal growth-inhibitory factor (metallothionein-3): evaluation of the biological function of growth-inhibitory factor in the injured and neurodegenerative brain. FEBS J. 2010;277:2931–39. https://doi.org/10.1111/j.1742-4658.2010.07718.x PubMed DOI

Loaiza N, Demaria M. Cellular senescence and tumor promotion: is aging the key? Biochim Biophys Acta. 2016;1865:155–67. https://doi.org/10.1016/j.bbcan.2016.01.007 PubMed DOI

Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M. Tumour biology: Senescence in premalignant tumours. Nature. 2005;436:642. https://doi.org/10.1038/436642a PubMed DOI

Dimri GP. What has senescence got to do with cancer? Cancer Cell. 2005;7:505–12. https://doi.org/10.1016/j.ccr.2005.05.025 PubMed DOI PMC

Bassi CL, Martelli L, Cipolotti R, Scrideli CA, Defávery R, Tone LG. Lack of evidence for mutations or deletions in the CDKN2A/p16 and CDKN2B/p15 genes of Brazilian neuroblastoma patients. Braz J Med Biol Res. 2004;37:1683–87. https://doi.org/10.1590/S0100-879×2004001100014 PubMed DOI

Park KH, Choi SE, Eom M, Kang Y. Downregulation of the anaphase-promoting complex (APC)7 in invasive ductal carcinomas of the breast and its clinicopathologic relationships. Breast Cancer Res. 2005;7:R238–47. https://doi.org/10.1186/bcr978 PubMed DOI PMC

Singh S. Cytoprotective and regulatory functions of glutathione S-transferases in cancer cell proliferation and cell death. Cancer Chemother Pharmacol. 2015;75:1–15. https://doi.org/10.1007/s00280-014-2566-x PubMed DOI

Hall AG, McGuckin AG, Pearson AD, Cattan AR, Malcolm AJ, Reid MM. Glutathione S-transferase in bone marrow metastases of disseminated neuroblastoma. J Clin Pathol. 1994;47:468–69. https://doi.org/10.1136/jcp.47.5.468 PubMed DOI PMC

Tew KD, Manevich Y, Grek C, Xiong Y, Uys J, Townsend DM. The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer. Free Radic Biol Med. 2011;51:299–313. https://doi.org/10.1016/j.freeradbiomed.2011.04.013 PubMed DOI PMC

He QY, Zhu R, Ren Y, Tam PK, Chiu JF. Serological protein profiling of neuroblastoma by ProteinChip SELDI-TOF technology. J Cell Biochem. 2005;95:165–72. https://doi.org/10.1002/jcb.20417 PubMed DOI

McIlwain DR, Berger T, Mak TW. Caspase Functions in. Cell Death and Disease Cold Spring Harbor Perspect Biol. 2015;7:1–28. PubMed PMC

Pedersen MO, Larsen A, Stoltenberg M, Penkowa M. The role of metallothionein in oncogenesis and cancer prognosis. Prog Histochem Cytochem. 2009;44:29–64. https://doi.org/10.1016/j.proghi.2008.10.001 PubMed DOI

Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev. 2012;64:706–21. https://doi.org/10.1124/pr.111.005637 PubMed DOI PMC

Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo M, Kroemer G. Molecular mechanisms of cisplatin resistance. Oncogene. 2012;31:1869–83. https://doi.org/10.1038/onc.2011.384 PubMed DOI

Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, Harel-Bellan A, Castedo M, Kroemer G. Systems biology of cisplatin resistance: past, present and future. Cell Death Dis. 2014;5:e1257. https://doi.org/10.1038/cddis.2013.428 PubMed DOI PMC

Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 2003;22:7265–79. https://doi.org/10.1038/sj.onc.1206933 PubMed DOI

Florea AM, Büsselberg D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers (Basel) 2011;3:1351–71. https://doi.org/10.3390/cancers3011351 PubMed DOI PMC

Kartalou M, Essigmann JM. Mechanisms of resistance to cisplatin. Mutat Res. 2001;478:23–43. https://doi.org/10.1016/S0027-5107(01)00141-5 PubMed DOI

Iolascon A, Borriello A, Giordani L, Cucciolla V, Moretti A, Monno F, Criniti V, Marzullo A, Criscuolo M, Ragione FD. Caspase 3 and 8 deficiency in human neuroblastoma. Cancer Genet Cytogenet. 2003;146:41–47. https://doi.org/10.1016/S0165-4608(03)00125-0 PubMed DOI

Heger Z, Michalek P, Guran R, Havelkova B, Kominkova M, Cernei N, Richtera L, Beklova M, Adam V, Kizek R. Exposure to 17β-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida. PLoS One. 2015;10:e0145426. https://doi.org/10.1371/journal.pone.0145426 PubMed DOI PMC

Roth KM, Peyvan K, Schwarzkopf KR, Ghindilis A. Electrochemical detection of short DNA oligomer hybridization using the CombiMatrix ElectraSense Microarray reader. Electroanalysis. 2006;18:1982–88. https://doi.org/10.1002/elan.200603603 DOI

Heger Z, Merlos Rodrigo MA, Michalek P, Polanska H, Masarik M, Vit V, Plevova M, Pacik D, Eckschlager T, Stiborova M, Adam V. Sarcosine Up-Regulates Expression of Genes Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer. PLoS One. 2016;11:e0165830. https://doi.org/10.1371/journal.pone.0165830 PubMed DOI PMC

Heger Z, Polanska H, Merlos Rodrigo MA, Guran R, Kulich P, Kopel P, Masarik M, Eckschlager T, Stiborova M, Kizek R, Adam V. Prostate tumor attenuation in the nu/nu murine model due to anti-sarcosine antibodies in folate-targeted liposomes. Sci Rep. 2016;6:33379. https://doi.org/10.1038/srep33379 PubMed DOI PMC

Metallothionein-3 promotes cisplatin chemoresistance remodelling in neuroblastoma

Transcriptomic Landscape of Cisplatin-Resistant Neuroblastoma Cells