Purpose: The chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential. Methods: The Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed. Results: Ex ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM. Conclusions: Taken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- chemorezistence genetika MeSH
- ionty metabolismus MeSH
- karcinogeneze genetika patologie MeSH
- kovy metabolismus MeSH
- lidé MeSH
- metalothionein genetika metabolismus MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádory diagnóza farmakoterapie genetika patologie MeSH
- prognóza MeSH
- progrese nemoci MeSH
- protein - isoformy genetika metabolismus MeSH
- protinádorové látky farmakologie terapeutické užití MeSH
- regulace genové exprese u nádorů MeSH
- staging nádorů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analyzed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analyzed using nano liquid chromatography with tandem mass spectrometry. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ATP-binding cassette superfamily proteins (ATP = adenosine triphosphate), solute carrier-mediated trans-membrane transporters, proteasome complex subunits, and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of the key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.
- MeSH
- apoptóza účinky léků MeSH
- chemorezistence genetika MeSH
- cisplatina škodlivé účinky farmakologie MeSH
- lidé MeSH
- lyzozomy genetika MeSH
- nádorové buněčné linie MeSH
- neuroblastom farmakoterapie genetika patologie MeSH
- proliferace buněk účinky léků MeSH
- proteasomový endopeptidasový komplex genetika MeSH
- proteomika * MeSH
- regulace genové exprese u nádorů genetika MeSH
- transkriptom genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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 inN-Mycamplifying 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 (CDKN2BandANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC50: 7.5vs. 19.8 μg/ml), indicating its multipurpose biological significance.
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- chlorid sodný chemie MeSH
- elektrochemie MeSH
- elektrolyty MeSH
- komplexní sloučeniny chemie metabolismus MeSH
- kovy chemie metabolismus MeSH
- metalothionein chemie metabolismus MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- gelová chromatografie metody MeSH
- játra chemie MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- laboratorní automatizace MeSH
- magnetické nanočástice chemie MeSH
- metalothionein analýza izolace a purifikace MeSH
- potkani Wistar MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. As a mass of protection, many of them, develop mechanisms of full resistance or at least exhibit partially resisting toward these effects. In this way, based on the chemical similarity of the involved metallic species, they are able, to replace them with viable metals necessary for the effective functioning of the cell. These heavy metals may be bound to the functional groups of proteins and modify their structure and through this also affect their physiological function 1, 2. Higher plants, algae, certain yeasts and animals are able to respond to heavy metals by synthesizing phytochelatins (PCs) and related cysteine-rich polypeptides. Phytochelatin synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs 3, 4. PCs, cysteine-rich peptides, are produced from glutamine, cysteine and glycine. Unlike commonmetal-binding structures, MT and GSH, PCs are not gene-encoded, but enzymatically synthesized peptides 5. PCs have been identified in a wide variety of plant species, microorganisms and some invertebrates 6-10. They are structurally related to glutathione (GSH) and were presumed to be the products of a biosynthetic pathway. Numerous physiological, biochemical and genetic studies have confirmed GSH as the substrate for PCs biosynthesis 11, 12. The general structure of PCs is (c-Glu-Cys)n-Gly, with increasing repetitions of the dipeptide Glu-Cys linked through a c-carboxylamide bond (Fig 1), where n varies from 2 to 11, but typically reaching not further than five 13. Except glycine, also other amino acid residues can be found on C-terminal end of (γ-Glu-Cys)n peptides. Examples of which, like Ser, Glu, Gln and Ala are often found at this position in some plant species, and they are assumed to be functionally analogous and synthesised via essentially similar biochemical pathways 14, 15. In in vitro studies of PC synthase expressed in E. coli or in S. cerevisiae, the enzyme was activated to varying extents by Cd, Cu, Ag, Hg, Zn and Pb ions 16-18. PC synthase genes were also isolated in A.thaliana 16 and T.aestivum 18. Genes homologous to those from A.thaliana and T.aestivum were also found in S.pombe and C.elegans, suggesting the existence of PC synthase genes in more species 19.
- MeSH
- chemické techniky analytické metody MeSH
- fytochelatiny * biosyntéza chemie metabolismus MeSH
- rostliny metabolismus MeSH
- těžké kovy * metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Metallothioneins (MTs) were discovered in 1957 and identified as low-molecular weight sulfhydryl-rich proteins. MTs belong to a superfamily of intracellular metal-binding proteins, present in virtually all living organisms, with features common to the archetypal. MT was first isolated from horse kidney and characterized by Margoshes and Vallee [1]. In this work, we wish to briefly summarize the current knowledge regarding the MT forms. All vertebrates examined contain two or more distinct MT isoforms designated MT-1 through MT-4. The three-dimensional structures of MTs from mammalian that have been determined so far show a monomeric protein composed of two globular domains, each encompassing a metal–thiolate cluster. The metallothionein isoform A (MTA) is a 64-residue metalloprotein, which contains essentially the same number of metal-chelating Cys–Cys and Cys–Xxx–Cys motifs (where Xxx stands for any amino acid, other than Cys) and metal ions [2, 3]. These cysteine-rich proteins are localized in cytoplasm and some organelles, predominantly in mitochondria, where their presence is sensitively and strictly regulated by the oxidative state induced by mitochondrial respiration. Depending on the cell state, but especially presence of oxidative stress, MTs are rapidly translocated to the nucleus through nuclear pore complexes. MT localized in the nuclei is oxidized there and it is transported to cytosol; this system is balanced [3].
- MeSH
- kovy MeSH
- lidé MeSH
- metalothionein * analýza fyziologie chemie MeSH
- nádorové biomarkery * analýza metabolismus MeSH
- nádory diagnóza metabolismus MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
