Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Central European Institute of Technology Brno University of Technology Technicka 3058 10 616 00 Brno Czech Republic

Department of Chemistry and Faculty of AgriSciences Mendel University in Brno Zemedelska 1 613 00 Brno Czech Republic

Department of Oncology Charles University and University Hospital Motol 5 Uvalu 84 1 150 06 Prague 5 Czech Republic

Department of Paediatric Haematology and Oncology Charles University and University Hospital Motol 5 Uvalu 84 1 150 06 Prague 5 Czech Republic

Functional Proteomics Department of Cellular and Molecular Medicine and Proteomic Facility Centro de Investigaciones Biológicas Ramiro de Maeztu 9 28040 Madrid Spain

Instituto de Biomedicina Y Biotecnología de Cantabria Universidad de Cantabria 39011 Santander Spain

Zobrazit více v PubMed

Heger Z, et al. Metallothionein as a scavenger of free radicals—new cardioprotective therapeutic agent or initiator of tumor chemoresistance? Curr. Drug Targets. 2016;17:1438–1451. doi: 10.2174/1389450116666151001113304. PubMed DOI

Rodrigo MAM, et al. Fully automated two-step assay for detection of metallothionein through magnetic isolation using functionalized gamma-Fe2O3 particles. J. Chromatogr. B. 2016;1039:17–27. doi: 10.1016/j.jchromb.2016.10.018. PubMed DOI

Wojtczak B, et al. Metallothionein isoform expression in benign and malignant thyroid lesions. Anticancer Res. 2017;37:5179–5185. doi: 10.21873/anticanres.11940. PubMed DOI

Coyle P, et al. Metallothionein in human oesophagus, Barrett's epithelium and adenocarcinoma. Br. J. Cancer. 2002;87:533–536. doi: 10.1038/sj.bjc.6600473. PubMed DOI PMC

Sampaio FA, et al. A case-control study of Metallothionein-1 expression in breast cancer and breast fibroadenoma. Sci. Rep. 2019;9:1–5. doi: 10.1038/s41598-019-43565-0. PubMed DOI PMC

Faller P. Neuronal growth-inhibitory factor (metallothionein-3): reactivity and structure of metal-thiolate clusters*. Febs J. 2010;277:2921–2930. doi: 10.1111/j.1742-4658.2010.07717.x. PubMed DOI

Werynska B, et al. Expression of metallothionein-III in patients with non-small cell lung cancer. Anticancer Res. 2013;33:965–974. PubMed

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. doi: 10.1016/j.neuro.2007.12.006. PubMed DOI PMC

Sens MA, et al. Metallothionein isoform 3 as a potential biomarker for human bladder cancer. Environ. Health Perspect. 2000;108:413–418. doi: 10.1289/ehp.00108413. PubMed DOI PMC

Garrett SH, Sens MA, Todd JH, Somji S, Sens DA. Expression of MT-3 protein in the human kidney. Toxicol. Lett. 1999;105:207–214. doi: 10.1016/s0378-4274(99)00003-x. PubMed DOI

Hoey JG, Garrett SH, Sens MA, Todd JH, Sens DA. Expression of MT-3 mRNA in human kidney, proximal tubule cell cultures, and renal cell carcinoma. Toxicol. Lett. 1997;92:149–160. doi: 10.1016/s0378-4274(97)00049-0. PubMed DOI

Garrett SH, et al. Metallothionein isoform 3 expression in the human prostate and cancer-derived cell lines. Prostate. 1999;41:196–202. doi: 10.1002/(sici)1097-0045(19991101)41:3<196::aid-pros7>3.0.co;2-u. PubMed DOI

Gomulkiewicz A, et al. Expression of metallothionein 3 in ductal breast cancer. Int. J. Oncol. 2016;49:2487–2497. doi: 10.3892/ijo.2016.3759. PubMed DOI

Oldridge DA, et al. Genetic predisposition to neuroblastoma mediated by a LMO1 super-enhancer polymorphism. Nature. 2015;528:418–421. doi: 10.1038/nature15540. PubMed DOI PMC

de la Escosura-Muniz A, et al. In situ monitoring of PTHLH secretion in neuroblastoma cells cultured onto nanoporous membranes. Biosens. Bioelectron. 2018;107:62–68. doi: 10.1016/j.bios.2018.01.064. PubMed DOI

Georgakis MK, et al. Neuroblastoma among children in Southern and Eastern European cancer registries: Variations in incidence and temporal trends compared to US. Int. J. Cancer. 2018;142:1977–1985. doi: 10.1002/ijc.31222. PubMed DOI

Howman-Giles R, Shaw PJ, Uren RF, Chung DKV. Neuroblastoma and other neuroendocrine tumors. Semin. Nucl. Med. 2007;37:286–302. doi: 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. Oto-Rhino-Laryn. 2012;269:1967–1971. doi: 10.1007/s00405-011-1863-9. PubMed DOI

Nakamura Y, et al. Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma. Cancer Med. 2014;3:25–35. doi: 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. doi: 10.1002/ana.24659. PubMed DOI PMC

Lee SI, et al. Carfilzomib enhances cisplatin-induced apoptosis in SK-N-BE(2)-M17 human neuroblastoma cells. Sci. Rep. 2019;9:1–14. doi: 10.1038/s41598-019-41527-0. PubMed DOI PMC

Donzelli E, et al. Neurotoxicity of platinum compounds: comparison of the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. J. Neuro-Oncol. 2004;67:65–73. doi: 10.1023/B:NEON.0000021787.70029.ce. PubMed DOI

Groh T, et al. 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;47:343–352. doi: 10.3892/ijo.2015.2996. PubMed DOI

Rodrigo MAM, et al. Transcriptomic landscape of cisplatin-resistant neuroblastoma cells. Cells. 2019;8:1–19. doi: 10.3390/cells8030235. PubMed DOI PMC

Rodrigo MAM, et al. Comparative gene expression profiling of human metallothionein-3 up-regulation in neuroblastoma cells and its impact on susceptibility to cisplatin. Oncotarget. 2018;9:4427–4439. doi: 10.18632/oncotarget.23333. PubMed DOI PMC

Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer. 2009;9:274–U265. doi: 10.1038/nrc2622. PubMed DOI

Massague J, Obenauf AC. Metastatic colonization by circulating tumour cells. Nature. 2016;529:298–306. doi: 10.1038/nature17038. PubMed DOI PMC

Alizadeh AM, Shiri S, Farsinejad S. Metastasis review: from bench to bedside. Tumor Biol. 2014;35:8483–8523. doi: 10.1007/s13277-014-2421-z. PubMed DOI

Kmiecik AM, et al. Metallothionein-3 increases triple-negative breast cancer cell invasiveness via induction of metalloproteinase expression. PLoS ONE. 2015;10:1–13. doi: 10.1371/journal.pone.0124865. PubMed DOI PMC

Hishikawa Y, et al. Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus. Br. J. Cancer. 1999;81:712–720. doi: 10.1038/sj.bjc.6690753. PubMed DOI PMC

Kim HG, et al. Metallothionein-2A overexpression increases the expression of matrix metalloproteinase-9 and invasion of breast cancer cells. FEBS Lett. 2011;585:421–428. doi: 10.1016/j.febslet.2010.12.030. PubMed DOI

Irvine GW, Pinter TBJ, Stillman MJ. Defining the metal binding pathways of human metallothionein 1a: Balancing zinc availability and cadmium seclusion. Metallomics. 2016;8:71–81. doi: 10.1039/c5mt00225g. PubMed DOI

Chen SH, Russell WK, Russell DH. Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein. Anal. Chem. 2013;85:3229–3237. doi: 10.1021/ac303522h. PubMed DOI

Adams SV, et al. Genetic variation in metallothionein and metal-regulatory transcription factor 1 in relation to urinary cadmium, copper, and zinc. Toxicol. Appl. Pharmacol. 2015;289:381–388. doi: 10.1016/j.taap.2015.10.024. PubMed DOI PMC

Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009;4:44–57. doi: 10.1038/nprot.2008.211. PubMed DOI

Kanehisa M, Sato Y. KEGG Mapper for inferring cellular functions from protein sequences. Protein Sci. 2020 doi: 10.1002/pro.3711. PubMed DOI PMC

Almond JB, Cohen GM. The proteasome: A novel target for cancer chemotherapy. Leukemia. 2002;16:433–443. doi: 10.1038/sj.leu.2402417. PubMed DOI

Yogev O, et al. In vivo modeling of chemoresistant neuroblastoma provides new insights into chemorefractory disease and metastasis. Cancer Res. 2019;79:5382–5393. doi: 10.1158/0008-5472.can-18-2759. PubMed DOI

Cimpean AM, et al. VEGF-A/HGF induce Prox-1 expression in the chick embryo chorioallantoic membrane lymphatic vasculature. Clin. Exper. Med. 2010;10:169–172. doi: 10.1007/s10238-009-0085-6. PubMed DOI

Kalirai H, Shahidipour H, Coupland SE, Luyten GPM. Use of the chick embryo model in uveal melanoma. Ocular Oncol. Pathol. 2015;1:133–140. doi: 10.1159/000370151. PubMed DOI PMC

Klingenberg M, Becker J, Eberth S, Kube D, Wilting J. The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma. BMC Cancer. 2014;14:1–12. doi: 10.1186/1471-2407-14-339. PubMed DOI PMC

Lokman NA, Elder ASF, Ricciardelli C, Oehler MK. Chick Chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis. Int. J. Mol. Sci. 2012;13:9959–9970. doi: 10.3390/ijms13089959. PubMed DOI PMC

Swadi R, et al. Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery. BMC Cancer. 2018;18:1–11. doi: 10.1186/s12885-017-3978-x. PubMed DOI PMC

Ribatti D, Tamma R. The chick embryo chorioallantoic membrane as an in vivo experimental model to study human neuroblastoma. J. Cell. Physiol. 2019;234:152–157. doi: 10.1002/jcp.26773. PubMed DOI

Valentiner U, Haane C, Nehmann N, Schumacher U. Effects of bortezomib on human neuroblastoma cells in vitro and in a metastatic xenograft model. Anticancer Res. 2009;29:1219–1225. PubMed

Rodrigo MAM, et al. Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin. J. Proteome Res. 2019;18:1255–1263. doi: 10.1021/acs.jproteome.8b00867. PubMed DOI

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. doi: 10.1002/pros.10097. PubMed DOI

Lujambio A, Banito A. Functional screening to identify senescence regulators in cancer. Curr. Opin. Genet. Dev. 2019;54:17–24. doi: 10.1016/j.gde.2019.02.001. PubMed DOI

You JQ, et al. Cellular senescence and anti-cancer therapy. Curr. Drug Targets. 2019;20:705–715. doi: 10.2174/1389450120666181217100833. PubMed DOI

Rishi AK, et al. Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437. J. Biol. Chem. 2003;278:33422–33435. doi: 10.1074/jbc.M303173200. PubMed DOI

Tang WT, Dong KR, Li K, Dong R, Zheng S. MEG3, HCN3 and linc01105 influence the proliferation and apoptosis of neuroblastoma cells via the HIF-1 alpha and p53 pathways. Sci. Rep. 2016;6:1–9. doi: 10.1038/srep36268. PubMed DOI PMC

Urfali-Mamatoglu C, Kazan HH, Gunduz U. Dual function of programmed cell death 10 (PDCD10) in drug resistance. Biomed. Pharmacother. 2018;101:129–136. doi: 10.1016/j.biopha.2018.02.020. PubMed DOI

Lambertz N, et al. Downregulation of programmed cell death 10 is associated with tumor cell proliferation, hyperangiogenesis and peritumoral edema in human glioblastoma. BMC Cancer. 2015;15:1–11. doi: 10.1186/s12885-015-1709-8. PubMed DOI PMC

Ma X, et al. PDCD10 interacts with Ste20-related kinase MST4 to promote cell growth and transformation via modulation of the ERK pathway. Mol. Biol. Cell. 2007;18:1965–1978. doi: 10.1091/mbc.E06-07-0608. PubMed DOI PMC

Hou XF, et al. ECRG2 enhances the anti-cancer effects of cisplatin in cisplatin-resistant esophageal cancer cells via upregulation of p53 and downregulation of PNCA. World J. Gastroenterol. 2017;23:1796–1803. doi: 10.3748/wjg.v23.i10.1796. PubMed DOI PMC

Kawahara B, et al. Carbon monoxide sensitizes cisplatin-resistant ovarian cancer cell lines toward cisplatin via attenuation of levels of glutathione and nuclear metallothionein. J. Inorg. Biochem. 2019;191:29–39. doi: 10.1016/j.jinorgbio.2018.11.003. PubMed DOI

Maleckaite R, et al. DNA methylation of metallothionein genes is associated with the clinical features of renal cell carcinoma. Oncol. Rep. 2019;41:3535–3544. doi: 10.3892/or.2019.7109. PubMed DOI

Skowron MA, et al. Multifaceted mechanisms of cisplatin resistance in long-term treated urothelial carcinoma cell lines. Int. J. Mol. Sci. 2018;19:1–17. doi: 10.3390/ijms19020590. PubMed DOI PMC

Wong DL, Stillman MJ. Capturing platinum in cisplatin: Kinetic reactions with recombinant human apo-metallothionein 1a. Metallomics. 2018;10:713–721. doi: 10.1039/c8mt00029h. PubMed DOI

Shedden K, Xie XT, Chandaroy P, Chang YT, Rosania GR. Expulsion of small molecules in vesicles shed by cancer cells: Association with gene expression and chemosensitivity profiles. Cancer Res. 2003;63:4331–4337. PubMed

Wang SS, Hu CH, Wu F, He SS. Rab25 GTPase: Functional roles in cancer. Oncotarget. 2017;8:64591–64599. doi: 10.18632/oncotarget.19571. PubMed DOI PMC

Guerra F, Bucci C. Role of the RAB7 protein in tumor progression and cisplatin chemoresistance. Cancers. 2019;11:1–19. doi: 10.3390/cancers11081096. PubMed DOI PMC

Nogami S, et al. Taxilin; a novel syntaxin-binding protein that is involved in Ca2+-dependent exocytosis in neuroendocrine cells. Genes Cells. 2003;8:17–28. doi: 10.1046/j.1365-2443.2003.00612.x. PubMed DOI

Ohtomo N, et al. Expression of alpha-taxilin in hepatocellular carcinoma correlates with growth activity and malignant potential of the tumor. Int. J. Oncol. 2010;37:1417–1423. doi: 10.3892/ijo_00000793. PubMed DOI

Machado E, et al. Regulated lysosomal exocytosis mediates cancer progression. Sci. Adv. 2015;1:1–16. doi: 10.1126/sciadv.1500603. PubMed DOI PMC

Rudzinska M, et al. The role of cysteine cathepsins in cancer progression and drug resistance. Int. J. Mol. Sci. 2019;20:1–21. doi: 10.3390/ijms20143602. PubMed DOI PMC

Don S, et al. Neuronal-associated microtubule proteins class III beta-tubulin and MAP2c in neuroblastoma: Role in resistance to microtubule-targeted drugs. Mol. Cancer Ther. 2004;3:1137–1146. doi: 10.4161/cbt.3.11.1216. PubMed DOI

Verma H, Bahia MS, Choudhary S, Singh PK, Silakari O. Drug metabolizing enzymes-associated chemo resistance and strategies to overcome it. Drug Metab. Rev. 2019;51:196–223. doi: 10.1080/03602532.2019.1632886. PubMed DOI

Akhdar, H., Legendre, C., Aninat, C. & Morel, F. Anticancer Drug Metabolism: Chemotherapy Resistance and New Therapeutic Approaches. (2012).

Fuqua SAW, et al. Heat-shock proteins and drug-resistance. Breast Cancer Res. Treat. 1994;32:67–71. doi: 10.1007/bf00666207. PubMed DOI

Zhou JW, Tang JJ, Sun W, Wang H. PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma. Mol. Med. 2019;25:1–12. doi: 10.1186/s10020-019-0079-0. PubMed DOI PMC

Chatterjee S, Burns TF. Targeting heat shock proteins in cancer: A promising therapeutic approach. Int. J. Mol. Sci. 2017;18:1–39. doi: 10.3390/ijms18091978. PubMed DOI PMC

Gesualdi NM, et al. Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis. Stress. 2007;10:342–350. doi: 10.1080/10253890701314863. PubMed DOI

Yamashita M, et al. Dnajb8, a member of the heat shock protein 40 family has a role in the tumor initiation and resistance to docetaxel but is dispensable for stress response. PLoS ONE. 2016;11:1–12. doi: 10.1371/journal.pone.0146501. PubMed DOI PMC

Weinberg F, Ramnath N, Nagrath D. Reactive oxygen species in the tumor microenvironment: An overview. Cancers. 2019;11:1–20. doi: 10.3390/cancers11081191. PubMed DOI PMC

Sosa V, et al. Oxidative stress and cancer: An overview. Ageing Res. Rev. 2013;12:376–390. doi: 10.1016/j.arr.2012.10.004. PubMed DOI

Yu WG, et al. Cisplatin generates oxidative stress which is accompanied by rapid shifts in central carbon metabolism. Sci. Rep. 2018;8:1–12. doi: 10.1038/s41598-018-22640-y. PubMed DOI PMC

Traverso N, et al. Role of glutathione in cancer progression and chemoresistance. Oxidat. Med. Cell. Longev. 2013;1–10:2013. doi: 10.1155/2013/972913. PubMed DOI PMC

Haas B, et al. Thioredoxin confers intrinsic resistance to cytostatic drugs in human glioma cells. Int. J. Mol. Sci. 2018;19:1–13. doi: 10.3390/ijms19102874. PubMed DOI PMC

Nakamura H, et al. Expression of thioredoxin and glutaredoxin, redox-regulating proteins, in pancreatic cancer. Cancer Detect. Prev. 2000;24:53–60. PubMed

Gumulec J, et al. Cisplatin-resistant prostate cancer model: Differences in antioxidant system, apoptosis and cell cycle. Int. J. Oncol. 2014;44:923–933. doi: 10.3892/ijo.2013.2223. PubMed DOI

Kwee JK. A paradoxical chemoresistance and tumor suppressive role of antioxidant in solid cancer cells: A strange case of Dr Jekyll and Mr. Hyde. Biomed Res. Int. 2014 doi: 10.1155/2014/209845. PubMed DOI PMC

Kim JW, Sahm H, You J, Wang M. Knock-down of superoxide dismutase 1 sensitizes cisplatin-resistant human ovarian cancer cells. Anticancer Res. 2010;30:2577–2581. PubMed

Hur GC, et al. Manganese superoxide dismutase expression correlates with chemosensitivity in human gastric cancer cell lines. Clin. Cancer Res. 2003;9:5768–5775. PubMed

Urano Y, et al. 6-Hydroxydopamine induces secretion of PARK7/DJ-1 via autophagy-based unconventional secretory pathway. Autophagy. 2018;14:1943–1958. doi: 10.1080/15548627.2018.1493043. PubMed DOI PMC

Bedrnicek J, et al. Characterization of drug-resistant neuroblastoma cell lines by comparative genomic hybridization. Neoplasma. 2005;52:415–419. PubMed

Herrero A, et al. Small molecule inhibition of ERK dimerization prevents tumorigenesis by RAS-ERK pathway oncogenes. Cancer Cell. 2015;28:170–182. doi: 10.1016/j.ccell.2015.07.001. PubMed DOI

Deryugina EI, Quigley JP. Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis. Histochem. Cell Biol. 2008;130:1119–1130. doi: 10.1007/s00418-008-0536-2. PubMed DOI PMC

Guran R, et al. MALDI MSI of MeLiM melanoma: Searching for differences in protein profiles. PLoS ONE. 2017;12:1–15. doi: 10.1371/journal.pone.0189305. PubMed DOI PMC

Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells

Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism

Single-cell transcriptomics of neuroblastoma identifies chemoresistance-associated genes and pathways

Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells