BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
The efficiency of cisplatin (CDDP) is significantly hindered by the development of resistance during the treatment course. To gain a detailed understanding of the molecular mechanisms underlying the development of cisplatin resistance, we comparatively analyzed established a CDDP-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its susceptible parental cells (UKF-NB-4). We verified increased chemoresistance of UKF-NB-4CDDP cells by analyzing the viability, induction of apoptosis and clonal efficiency. To shed more light on this phenomenon, we employed custom cDNA microarray (containing 2234 probes) to perform parallel transcriptomic profiling of RNA and identified that 139 genes were significantly up-regulated due to CDDP chemoresistance. The analyses of molecular pathways indicated that the top up-regulation scoring functions were response to stress, abiotic stimulus, regulation of metabolic process, apoptotic processes, regulation of cell proliferation, DNA repair or regulation of catalytic activity, which was also evidenced by analysis of molecular functions revealing up-regulation of genes encoding several proteins with a wide-spectrum of enzymatic activities. Functional analysis using lysosomotropic agents chloroquine and bafilomycin A1 validated their potential to re-sensitize UKF-NB-4CDDP cells to CDDP. Taken together, the identification of alterations in specific genes and pathways that contribute to CDDP chemoresistance may potentially lead to a renewed interest in the development of novel rational therapeutics and prognostic biomarkers for the management of CDDP-resistant neuroblastoma.
- Publikační typ
- časopisecké články MeSH
Východiska: Rezistence nádorových buněk k cytostatikům je způsobena řadou mechanizmů, které se často kombinují. Patří k nim redukovaný vstup cytostatika do buňky nebo jeho zvýšený eflux, zvýšená oprava DNA, defekty apoptotických drah, zvýšené odbourávání cytostatika a také zvýšená hladina intracelulárních thiolů glutathionu a metalothioneinů (MT). Bylo popsáno, že vysoká koncentrace thiolových skupin v cytoplazmě váže platinové alkylační deriváty a chemorezistence je způsobena přenosem platiny z cytostatika na MT, které je inaktivují. Protože jsme v našich předchozích studiích prokázali zvýšení hladin MT v rezistentních neuroblastomových (NB) liniích, ale ne v senzitivních liniích po inkubaci s platinovými cytostatiky, zabývali jsme se významem MT-3 pro buňky NB. Metoda: Buňky NB linie SiMa transfekované vektorem obsahujícím lidský MT-3 a GFP nebo pouze GFP (kontrola). Expresní microarray Human Cancer 3711 ElectraSense medium density 4 × 2k array slides s 1 609 DNA probami (Custom Array, Bothell, WA, USA), exprese MT-3 a nejvíce exprimovaných genů validována real-time polymerázová řetězová reakce (RT-PCR). Citlivost k CDDP (cisplatina) – MTT test, vyšetření klonogenicity, průkaz štěpení kaspázy 3 Western blottem a volné kyslíkové radikály fluorescenční mikroskopií po barvení CellROX Deep Red Reagent. Hladiny mRNA MT-3 ve 23 vzorcích vysoce rizikových NB buňkách normální lidské kůry nadledviny a bovinní nadledviny byly vyšetřeny RT-PCR. Výsledky: Expresní microarray prokázala downregulaci 3 a over-expresi 19 genů u MT-3 transfekovaných NB buněk. Pomocí genové ontologie bylo zjištěno, že over-exprimované geny řídí onkogeny indukovanou senescenci (CDKN2B a ANAPC5), a zvýšeně exprimované byly i geny glutathion S-transferázy M3, kaspázy 4 a DNAJB6 (chaperon neuronálních proteinů). Prokázali jsme sníženou senzitivitu MT-3 transfekovaných buněk k CDDP (24 h IC50 7,48 ± 0,97 a 19,81 ± 1,2 µg/ml), vyšší počet kolonií po inkubaci s CDDP, snížené štěpení kaspázy 3 po inkubaci s CDDP a nižší volné kyslíkové radikály po jejich indukci CDDP. Buňky vysoce rizikových NB exprimovaly MT-3 významně více než nenádorové buňky nadledviny, ale nepodařilo se prokázat jednoznačný vztah k průběhu onemocnění. Závěr: Prokázali jsme vztah mezi MT-3 a geny onkogeny indukované senescence a některými dalšími geny významnými pro osud buňky (glutathion S-transferáza M3, kaspáza 4 a DNAJB6) a významný podíl MT-3 na rezistenci k CDDP. Vysoké hladiny MT-3 u vysoce rizikového NB by mohly být jednou z příčin častých recidiv u tohoto nádoru.
Background: Resistance of cancer cells to cytostatics is caused by a number of mechanisms that are often combined. These include reduced cell entry or increased efflux, increased DNA repair, defects of, apoptotic pathways, increased cytostatic degradation as well as elevated levels of intracellular thiols of glutathione and metallothioneins (MT). It has been reported that high concentrations of thiol groups in the cytoplasm bind platinum alkylation derivatives and chemorezistence is due to the transfer of platinum from the cytostatic to MT, which inactivates them. Because we have shown an increase in MT levels in resistant neuroblastoma (NB) lines, but not in sensitive lines after incubation with platinum cytostatics, we have considered MT-3 for NB cells in our previous studies. Method: SiMa NB cell lines transfected with vector containing human MT-3 and GFP or GFP only (control). Expression Microarray Human Cancer 3711 ElectraSense medium density 4 × 2k array slides with 1,609 DNA probes (Custom Array, Bothell, WA, USA), MT-3 expression and most expressed genes validated by real-time polymerase chain reaction. Sensitivity to CDDP (cisplatin) – MTT assay, clonogenicity test, Western blott caspase cleavage and free oxygen radicals fluorescence microscopy after CellROX Deep Red Reagent staining. Levels of MT-3 mRNA in 23 samples of high-risk NB, normal human cortex and bovine adrenal glands were investigated by reverse transcription polymerase chain reaction. Results: Expression microarray showed downregulation 3 and overexpression of 19 genes in MT-3 transfected NB cells. Using gene ontology, over-expressed genes have been shown to drive senescence-induced oncogenes (CDKN2B and ANAPC5), and the genes of glutathione S-transferase M3, caspase 4 and DNAJB6 (chaperone neuronal proteins) were also expressed. We have demonstrated a reduced sensitivity of MT-3 transfected cells to CDDP (24h IC50 of 7.48 ? 0.97 and 19.81 ? 1.2 μg/ml), a higher number of colonies after incubation with CDDP, reduced caspase 3 after incubation with CDDP and lower free oxygen radicals after induction of CDDP. High-grade NB cells expressed MT-3 significantly more than non-tumoral adrenal cells but failed to show a clear relationship to disease course. Conclusion: We have demonstrated the relationship between MT-3 and senescence-induced oncogene genes and some other genes relevant to cell fate (glutathione S-transferase M3, caspase 4 and DNAJB6) and a significant proportion of MT-3 on CDDP resistance. High levels of MT-3 in high-risk NB could be one of the causes of frequent relapses in this tumor.
- MeSH
- chemorezistence * MeSH
- cisplatina terapeutické užití MeSH
- lidé MeSH
- metalothionein 3 MeSH
- mikro RNA MeSH
- nádorové buněčné linie MeSH
- neuroblastom * genetika metabolismus patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny nervové tkáně * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
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.
- MeSH
- antracykliny aplikace a dávkování škodlivé účinky MeSH
- chemorezistence MeSH
- kardiomyopatie etiologie prevence a kontrola MeSH
- kardiotonika farmakologie MeSH
- lidé MeSH
- metalothionein metabolismus farmakologie MeSH
- nádory farmakoterapie patologie MeSH
- oxidační stres účinky léků MeSH
- protinádorová antibiotika aplikace a dávkování škodlivé účinky MeSH
- scavengery volných radikálů farmakologie MeSH
- srdeční selhání chemicky indukované prevence a kontrola MeSH
- volné radikály metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
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 interaction of a plethora nanoparticles with major biota such as plants and animals/humans has been the subject of various multidisciplinary studies with special emphasis on toxicity aspects. However, reports are meager on the transport phenomena of nanoparticles in the plant-animal/human system. Since plants and animals/humans are closely linked via food chain, discussion is imperative on the main processes and mechanisms underlying the transport phenomena of nanoparticles in the plant-animal/human system, which is the main objective of this paper. Based on the literature appraised herein, it is recommended to perform an exhaustive exploration of so far least explored aspects such as reproducibility, predictability, and compliance risks of nanoparticles, and insights into underlying mechanisms in context with their transport phenomenon in the plant-animal/human system. The outcomes of the suggested studies can provide important clues for fetching significant benefits of rapidly expanding nanotechnology to the plant-animal/human health-improvements and protection as well.
- MeSH
- biologický transport MeSH
- lidé MeSH
- nanočástice metabolismus MeSH
- potravní řetězec MeSH
- rostliny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
In recent years, MALDI imaging mass spectrometry, also known as MALDI imaging or MALDI MSI, has been getting wider recognition thanks to an increasing number of studies devoted to various tumor markers, comparing 2D mass maps of healthy and diseased tissue sections. By determining the spatial distribution of markers in the tissue we step forward to clarify the physiological processes in tumors and to better understand them. Moreover, by combining 2D mass maps with microscopic photos of the sections from histology, we get a more comprehensive picture of the distribution of the examined analytes. However, one of the disadvantages of MALDI imaging is a complicated ionization of analytes in a complex matrix of tissues and thus low detection limits. In this paper we report on the current state of MALDI imaging in oncology research. Development option of this method in the near future is outlined according to authors’ opinion.
- MeSH
- biopsie MeSH
- histocytologické preparační techniky MeSH
- lidé MeSH
- nádorové biomarkery * analýza MeSH
- nádory * patofyziologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody přístrojové vybavení využití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen occurring not only in hospitals but also in foodstuff. Currently, discussions on the issue of the increasing resistance, and timely and rapid diagnostic of resistance strains have become more frequent and sought. Therefore, the aim of this study was to design an effective platform for DNA isolation from different species of microorganisms as well as the amplification of mecA gene that encodes the resistance to β-lactam antibiotic formation and is contained in MRSA. For this purpose, we fabricated 3D-printed chip that was suitable for bacterial cultivation, DNA isolation, PCR, and detection of amplified gene using gold nanoparticle (AuNP) probes as an indicator of MRSA. Confirmation of the MRSA presence in the samples was based on a specific interaction between mecA gene with the AuNP probes and a colorimetric detection, which utilized the noncross-linking aggregation phenomenon of DNA-functionalized AuNPs. To test the whole system, we analyzed several real refractive indexes, in which two of them were positively scanned to find the presence of mecA gene. The aggregation of AuNP probes were reflected by 75% decrease of absorbance (λ = 530 nm) and change in AuNPs size from 3 ± 0.05 to 4 ± 0.05 nm (n = 5). We provide the one-step identification of mecA gene using the unique platform that employs the rapid, low-cost, and easy-to-use colorimetric method for MRSA detection in various samples.
- MeSH
- absces mikrobiologie MeSH
- bakteriální proteiny genetika MeSH
- design vybavení MeSH
- DNA bakterií analýza genetika MeSH
- kovové nanočástice chemie MeSH
- lidé středního věku MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus genetika izolace a purifikace MeSH
- molekulární typizace MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů přístrojové vybavení metody MeSH
- stafylokokové infekce mikrobiologie MeSH
- techniky amplifikace nukleových kyselin MeSH
- zlato chemie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- hmotnostní spektrometrie MeSH
- ionty metabolismus farmakologie MeSH
- kovy metabolismus farmakologie MeSH
- metabolom MeSH
- metabolomika metody MeSH
- metalothionein chemie metabolismus MeSH
- mikrobiální viabilita účinky léků MeSH
- RNA ribozomální genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Staphylococcus aureus účinky léků genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
