An insight into the complex roles of metallothioneins in malignant diseases with emphasis on (sub)isoforms/isoforms and epigenetics phenomena

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The Application of Curve Fitting on the Voltammograms of Various Isoforms of Metallothioneins–Metal Complexes

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

Zeptomole Electrochemical Detection of Metallothioneins

Thiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT.In this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals.This paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab-on-chip instrument

Drug Sequestration in Lysosomes as One of the Mechanisms of Chemoresistance of Cancer Cells and the Possibilities of Its Inhibition

Resistance to chemotherapeutics and targeted drugs is one of the main problems in successful cancer therapy. Various mechanisms have been identified to contribute to drug resistance. One of those mechanisms is lysosome-mediated drug resistance. Lysosomes have been shown to trap certain hydrophobic weak base chemotherapeutics, as well as some tyrosine kinase inhibitors, thereby being sequestered away from their intracellular target site. Lysosomal sequestration is in most cases followed by the release of their content from the cell by exocytosis. Lysosomal accumulation of anticancer drugs is caused mainly by ion-trapping, but active transport of certain drugs into lysosomes was also described. Lysosomal low pH, which is necessary for ion-trapping is achieved by the activity of the V-ATPase. This sequestration can be successfully inhibited by lysosomotropic agents and V-ATPase inhibitors in experimental conditions. Clinical trials have been performed only with lysosomotropic drug chloroquine and their results were less successful. The aim of this review is to give an overview of lysosomal sequestration and expression of acidifying enzymes as yet not well known mechanism of cancer cell chemoresistance and about possibilities how to overcome this form of resistance

Targeting hypoxic cancer stem cells (CSCs) with Doxycycline : implications for optimizing anti-angiogenic therapy

Here, we report new mechanistic insight into how chronic hypoxia increases ‘stemness’ in cancer cells. Using chemical inhibitors, we provide direct experimental evidence that ROS production and mitochondrial biogenesis are both required for the hypoxia-induced propagation of CSCs. More specifically, we show that hypoxic CSCs can be effectively targeted with i) simple mitochondrial anti-oxidants (Mito-TEMPO) and/or ii) inhibitors of mitochondrial biogenesis (Doxycycline). In this context, we discuss the idea that mitochondrial biogenesis itself may be a primary driver of “stemness” in hypoxic cancer cells, with metabolic links to fatty acid oxidation (FAO). As Doxycycline is an FDA-approved drug, we propose that it could be re-purposed to target hypoxic CSCs, either alone or in combination with chemotherapy, i.e., Paclitaxel. For example, we demonstrate that Doxycycline effectively targets the sub-population of hypoxia-induced CSCs that are Paclitaxel-resistant, overcoming hypoxia-induced drug-resistance. Finally, anti-angiogenic therapy often induces tumor hypoxia, allowing CSCs to survive and propagate, ultimately driving tumor progression. Therefore, we suggest that Doxycycline could be used in combination with anti-angiogenic agents, to actively prevent or minimize hypoxia-induced treatment failure. In direct support of this assertion, Paclitaxel is already known to behave as an angiogenesis inhibitor

An Adsorptive Transfer Technique Coupled with Brdicka Reaction to Reveal the Importance of Metallothionein in Chemotherapy with Platinum Based Cytostatics

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug

The comparison of cytotoxicity of the anticancer drugs doxorubicin and ellipticine to human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the cytotoxicity of ellipticine to human neuroblastoma derived cell lines IMR-32 and UKF-NB-4 was investigated. Treatment of neuroblastoma cells with ellipticine was compared with that of these cancer cells with doxorubicin. The toxicity of ellipticine was essentially the same as that of doxorubicin to UKF-NB-4 cells, but doxorubicin is much more effective to inhibit the growth of the IMR-32 cell line than ellipticine. Hypoxic conditions used for the cell cultivation resulted in a decrease in ellipticine and/or doxorubicin toxicity to IMR-32 and UKF-NB-4 neuroblastoma cells