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Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.

Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.

Background Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. Objectives The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. Material and methods Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. Results In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. Conclusions The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.

Cardiotoxicity is a serious complication of anticancer therapy by anthracycline antibiotics. Except for intercalation into DNA/RNA structure, inhibition of DNA-topoisomerase and histone eviction from chromatin, the main mechanism of their action is iron-mediated formation of various forms of free radicals, which leads to irreversible damage to cancer cells. The most serious adverse effect of anthracyclines is, thus, cardiomyopathy leading to congestive heart failure, which is caused by the same mechanisms. Here, we briefly summarize the basic types of free radicals formed by anthracyclines and the main processes how to scavenge them. From these, the main attention is paid to metallothioneins. These low-molecular cysteine-rich proteins are introduced and their functions and properties are reviewed. Further, their role in detoxification of metals and drugs is discussed. Based on these beneficial roles, their use as a new therapeutic agent against oxidative stress and for cardioprotection is critically evaluated with respect to their ability to increase chemoresistance against some types of commonly used cytostatics.

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 (24IC(50): 7.5 vs. 19.8 mu g/ml), indicating its multipurpose biological significance.

In this study, we focused on the effect of heavy metal ions in resistant strains of gram-positive bacteria Staphylococcus aureus using biochemical methods and mass spectrometry. Five nitrate solutions of heavy metals (Ag(+), Cu(2+), Cd(2+), Zn(2+) and Pb(2+)) were used to create S. aureus resistant strains. Biochemical changes of resistant strains in comparison with the non-resistant control strain of S. aureus were observed by microbiological (measuring - growth curves and inhibition zones) and spectrophotometric methods (antioxidant activity and alaninaminotransferase, aspartateaminotransferase, alkaline phosphatase, γ-glutamyltransferase activities). Mass spectrometry was employed for the qualitative analysis of the samples (changes in S. aureus protein composition) and for the identification of the strains database MALDI Biotyper was employed. Alterations, in terms of biochemical properties and protein composition, were observed in resistant strains compared to non-resistant control strain. Our results describe the possible option for the analysis of S. aureus resistant strains and may thus serve as a support for monitoring of changes in genetic information caused by the forming of resistance to heavy metals.

Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S. aureus were observed. At a concentration of 300 μM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3 μg mg(-1) protein) but was increased by 29% after addition of SeNPs (63 μg mg(-1) protein) compared with the S. aureus control (49 μg mg(-1) protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.

Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.

Quantum dots (QDs) are fluorescence nanoparticles (NPs) with unique optic properties which allow their use as probes in chemical, biological, immunological, and molecular imaging. QDs linked with target ligands such as peptides or small molecules can be used as tumor biomarkers. These particles are a promising tool for selective, fast, andsensitive tagging and imaging in medicine. In thisstudy, an attempt was made to use QDs as a marker forhuman metallothionein (MT) isoforms 1 and 2. Fourkinds of CdTe QDs of different sizes bioconjugatedwith MT were analyzed using the chip-CE technique.Based on the results, it can be concluded that MT iswilling to interact with QDs, and the chip-CE technique enables the observation of their complexes.It was also observed that changes ranging roughly6–7 kDa, a value corresponding to the MT monomer,depend on the hydrodynamic diameters of QDs; also,the MT sample without cadmium interacted strongerwith QDs than MT saturated with cadmium. Resultsshow that MT is willing to interact with smaller QDs(blue CdTe) rather than larger ones QDs (red CdTe).To our knowledge, chip-CE has not previously beenapplied in the study of CdTe QDs interaction with MT. Kvantové tečky (QDS) jsou fluorescenční nanočástice (NPS) s unikátními optickými vlastnostmi, které umožňují jejich použití jako sondy s chemickým, biologickým, imunologickým, a molekulárním zobrazování. QDS spojeny s cílovými ligandy, jako jsou peptidy nebo malé molekuly mohou být použity jako nádorové biomarkery. Tyto částice jsou slibným nástrojem pro selektivní, rychlé, a citlivé označování a zobrazování v medicíně. V téhle studii byl učiněn pokus o použití QDS jako marker pro lidský metalothionein (MT) izoformy 1 a 2. Čtyři druhy CdTe QDS různých velikostí bioconjugatů s MT byly analyzovány použitím techniky chip-CE. Na základě výsledků, lze dojít k závěru, že je MT schopna interakce s QDS, a technika chip-CE umožňuje pozorování jejich komplexů. Bylo také pozorováno, že změny v rozmezí zhruba KDa 6 až 7, je hodnota, která odpovídá MT monomeru,a ataké závisí na hydrodynamických průměru QDS Vzorek MT bez kadmia styku silnější s QDS než MT nasycené kadmium. Výsledky ukazují, že MT je ochoten interakci s menšími QDS (modrá CdTe) spíše než ty větší QDS (červená CdTe).

Metallothioneins (MTs), low molecular mass cysteine-rich proteins, which are able to bind up to 20 monovalent and up to 7 divalent heavy metal ions are widely studied due to their functions in detoxification of metals, scavenging free radicals and cells protection against the oxidative stress. It was found that the loss of the protective effects of MT leads to an escalation of pathogenic processes and carcinogenesis. The most extensive area is MTs expression for oncological applications, where the information about gene patterns is helpful for the identification biological function, resistance to drugs and creating the correct chemotherapy. In other medical applications the effect of oxidative stress to cell lines exposed to heavy metals and hydrogen peroxide is studied as well as influence of drugs and cytokines on MTs expression and MTs expression in the adipose tissue. The precise detection of low metallothionein concentrations and its isoforms is necessary to understand the connection between quantity and isoforms of MTs to size, localization and type of cancer. This information is necessary for well-timed therapy and increase the chance to survival. Microarray chips appear as good possibility for finding all information about expression of MTs genes and isoforms not only in cancer, but also in other diseases, especially diabetes, obesity, cardiovascular diseases, ageing, osteoporosis, psychiatric disorders and as the effects of toxic drugs and pollutants, which is discussed in this review.

The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of this study was to determine the behavior of MT in dependence on various externally modelled environments, using electrochemistry. In our study, the mass distribution of MTs was characterized using MALDI-TOF. After that, adsorptive transfer stripping technique with differential pulse voltammetry was selected for optimization of electrochemical detection of MTs with regard to accumulation time and pH effects. Our results show that utilization of 0.5 M NaCl, pH 6.4, as the supporting electrolyte provides a highly complicated fingerprint, showing a number of non-resolved voltammograms. Hence, we further resolved the voltammograms exhibiting the broad and overlapping signals using curve fitting. The separated signals were assigned to the electrochemical responses of several MT complexes with zinc(II), cadmium(II), and copper(II), respectively. Our results show that electrochemistry could serve as a great tool for metalloproteomic applications to determine the ratio of metal ion bonds within the target protein structure, however, it provides highly complicated signals, which require further resolution using a proper statistical method, such as curve fitting., The work has been supported by the Agency for the Czech Republic Health Research (AZV) project no. 15-28334A. The presented research was financed by the Czech Ministry of Education in frame of the National Sustainability Program, the grant LO1401 INWITE.

Sarcosine is currently one of the most discussed markers of prostate cancer. It is involved in amino acid metabolism and methylation processes that occur during the progression of prostate cancer. In this study, we monitored the effect of the addition of sarcosine (0; 10; 250; 500; 1,000 and 1,500 µM) in a time-dependent manner (0-72 h) on the PC-3 prostate cancer cell line. For the assessment of cell viability, the commonly used MTT test was employed. Furthermore, ion-exchange liquid chromatography was used for the determination of sarcosine content in the PC-3 cells. We also determined metallothionein (MT) levels by chip capillary electrophoresis and Brdicka reaction in the cells treated with sarcosine. Sarcosine levels in the cells increased in a concentration-dependent manner levels increased from only 270 nM with the lowest applied concentration of sarcosine (10 µM) to 106 µM with the highest applied concentration of sarcosine (1,500 µM). There was a marginal change observed in the MT concentration. Finally, the antioxidant activity of the PC-3 cells was determined using five different spectrophotometric methods [2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP), free radicals, N,N-dimethyl-p-phenylenediamine (DMPD) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)]. A significant negative correlation was observed between DPPH and FRAP (r=-0.68 at p

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatin's therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose+atorvastatin (30 mg/kg/day) Sprague-Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid β-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (x 0.74) and liver triglyceride (x 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (x 0.45) and its target genes, and increased the hepatic activity of the fatty acid β-oxidation system (x 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (x 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.