Metallothioneins (MTs) family is a intracellular and cysteine-rich proteins with a high affinity to metals. MT-3 could play important neuromodulatory and neuroprotective roles. MT-3 has been also found up-regulated in a number of cancers. Neuroblastoma (Nbl) is a cancer is the most common extra-cranial solid tumour of childhood. The main aim was to provide novel insights into the molecular mechanisms of hMT-3 up-regulation and to elucidate the effects beneath the MT-3 up-regulation in Nbl cells.
To increase the expression of MT-3 Nbl (UKF-NB-4) cells were transiently transfected with a plasmid containing MT-3 gene (pcDNA3.1-GFP-hMT-3-TOPO). Separations of tryptic digests were carried out on an Easy-nLC 1000 nano system. MS analysis was performed using a Q-Exactive MS. The mass spectrum *.raw file was searched against the human SwissProt 57.15 database using MASCOT search engine (version 2.3, Matrix Science).
The efficiency of transfection analysed through a fluorescence of GFP tag expressed at the C-terminus of MT-3 showed more 70% transfection efficiency for both constructed plasmids (mock and MT-3). From the total of common proteins in dataset (hMT-3 vs. mock), 176, 20 and 1244 proteins were quantitatively identified with up-regulation, down-regulation, and no significant differences between hMT-3 and mock treatments. Noteworthy, the bioinformatical analyses revealed the exclusive expression (induced by MT-3) and up-regulation proteins of a number of proteins affecting biological pathways related to mitotic cell cycle, cellular responses to stress, positive regulation of proteolysis, negative regulation of cell cycle and programmed cell death.
Our proteomic data shed some light on the proteins involved in inducing senescence and apoptosis in tested Nbl cells with up-regulated MT-3. Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. Cellular senescence and apoptosis are among those mechanisms. Further experiments will be performed to functionally verify these data to provide novel insights into the Nbl biology. These might be useful to develop novel therapeutic protocols utilizing MT-3 as prognostic biomarker or therapeutic target.
Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, CZ.
European Research Council (ERC) under the European Uniońs Horizon 2020 Research and Innovation Programme (grant agreement No. 759585).
All authors have declared no conflicts of interest.