Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.
- MeSH
- buněčné sféroidy patologie MeSH
- disulfiram farmakologie MeSH
- fluorouracil * farmakologie terapeutické užití MeSH
- kolorektální nádory * farmakoterapie patologie MeSH
- lidé MeSH
- měď farmakologie terapeutické užití MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.
- MeSH
- europium chemie MeSH
- hmotnostní spektrometrie MeSH
- imunoblotting MeSH
- kadmium chemie MeSH
- kovové nanočástice chemie MeSH
- kvantové tečky chemie MeSH
- lasery MeSH
- lidé MeSH
- limita detekce MeSH
- monoklonální protilátky chemie farmakologie MeSH
- nádorový supresorový protein p53 antagonisté a inhibitory MeSH
- stříbro chemie MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Chemotherapy plays an essential role in the management of cancer worldwide. However, it is a non-specific treatment limited by major drawbacks, thus identification and testing of new promising molecular structures representing potential drug candidates are urgently needed. In this work, ferrocene complexes as potential antitumor drugs that display cytotoxicity in low micromolar concentrations against ovarian cancer cells A2780 and SK-OV-3 were investigated to identify their mode of action. Their mechanism of cellular accumulation was studied using differential pulse voltammetry and inductively coupled plasma - mass spectrometry. Their mode of cell death induction was determined by changes in the mitochondrial membrane potential, production of reactive oxygen species and by Annexin V staining. Transferrin receptors were identified as key mediators of intracellular accumulation of ferrocenes and the extent of cellular uptake reflected the anticancer activity of individual compounds. Functional analysis revealed activation of intrinsic apoptosis as a dominant mechanism leading to regulated cell death induced in ovarian cancer cells by ferrocenes. Ferrocenes represent a group of promising sandwich organometallic complexes exerting cytotoxic activity. We suggest their application not only as standalone chemotherapeutics but also as modifying substituents of known drugs to improve their antitumor effects.
- MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- apoptóza účinky léků MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- metaloceny farmakologie terapeutické užití MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků farmakoterapie patologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- receptory transferinu metabolismus MeSH
- železnaté sloučeniny farmakologie terapeutické užití MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO3)2 NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO3)2 NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO3)2 NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB (NF-κB) and selected cytokines, such as tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (TGFβ1), interleukin 6(IL-6), IL-1α and IL-1β , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO3)2 NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.
- MeSH
- aktiny agonisté genetika imunologie MeSH
- alveolární makrofágy účinky léků imunologie patologie MeSH
- aplikace inhalační MeSH
- biologická dostupnost MeSH
- dusičnany farmakokinetika toxicita MeSH
- exprese genu MeSH
- inhalační expozice analýza MeSH
- interleukin-1alfa agonisté genetika imunologie MeSH
- interleukin-1beta agonisté genetika imunologie MeSH
- interleukin-6 agonisté genetika imunologie MeSH
- játra účinky léků imunologie patologie MeSH
- kovové nanočástice aplikace a dávkování toxicita MeSH
- látky znečišťující vzduch farmakokinetika toxicita MeSH
- myši inbrední ICR MeSH
- myši MeSH
- NF-kappa B agonisté genetika imunologie MeSH
- olovo farmakokinetika toxicita MeSH
- plíce účinky léků imunologie patologie MeSH
- poločas MeSH
- spektrofotometrie atomová MeSH
- tkáňová distribuce MeSH
- TNF-alfa agonisté genetika imunologie MeSH
- transformující růstový faktor beta1 agonisté genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
For the first time, the combination of molecularly imprinted polymer (MIP) technology with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is presented with focus on an optimization of the LA-ICP-MS parameters such as laser beam diameter, laser beam fluence, and scan speed using CdS quantum dots (QDs) as a template and dopamine as a functional monomer. A non-covalent imprinting approach was employed in this study due to the simplicity of preparation. Simple oxidative polymerization of the dopamine that creates the self-assembly monolayer seems to be an ideal choice. The QDs prepared by UV light irradiation synthesis were stabilized by using mercaptosuccinic acid. Formation of a complex of QD-antibody and QD-antibody-antigen was verified by using capillary electrophoresis with laser-induced fluorescence detection. QDs and antibody were connected together via an affinity peptide linker. LA-ICP-MS was employed as a proof-of-concept for detection method of two types of immunoassay: 1) antigen extracted from the sample by MIP and subsequently overlaid/immunoreacted by QD-labelled antibodies, 2) complex of antigen, antibody, and QD formed in the sample and subsequently extracted by MIP. The first approach provided higher sensitivity (MIP/NIP), however, the second demonstrated higher selectivity. A mixture of proteins with size in range 10-250 kDa was used as a model sample to demonstrate the capability of both approaches for detection of IgG in a complex sample.
- MeSH
- elektroforéza kapilární MeSH
- fluorescence MeSH
- hmotnostní spektrometrie * MeSH
- imunoanalýza metody MeSH
- imunoglobulin G analýza MeSH
- kvantové tečky chemie MeSH
- laserová terapie * MeSH
- molekulový imprinting * MeSH
- myši MeSH
- počítačové zpracování signálu MeSH
- polymery chemie MeSH
- proteiny analýza MeSH
- sloučeniny kadmia chemie MeSH
- sulfidy chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling.
- MeSH
- hmotnostní spektrometrie metody MeSH
- imunoanalýza metody MeSH
- indikátory a reagencie chemie MeSH
- kovové nanočástice chemie MeSH
- lasery * MeSH
- limita detekce MeSH
- ověření koncepční studie MeSH
- protilátky chemie MeSH
- specificita protilátek MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
We report a facile method for detection of metallothionein (MT), a promising clinically relevant biomarker, in spiked plasma samples. This method, for the first time, integrates molecularly imprinted polymers as purification/pretreatment step with matrix assisted laser desorption/ionization time-of-flight mass spectrometric detection and with laser ablation inductively coupled plasma mass spectrometry for analysis of MTs. The prepared MT-imprinted polydopamine layer showed high binding capacity and specific recognition properties toward the template. Optimal monomer (dopamine) concentration was found to be 16 mM of dopamine. This experimental setup allows to measure µM concentrations of MT that are present in blood as this can be used for clinical studies recognizing MT as marker of various diseases including tumour one. Presented approach not only provides fast sample throughput but also avoids the limitations of methods based on use of antibodies (e.g. high price, cross-reactivity, limited availability in some cases, etc.).
- MeSH
- hmotnostní spektrometrie MeSH
- indoly chemie MeSH
- lidé MeSH
- metalothionein krev MeSH
- molekulový imprinting * MeSH
- polymery chemie MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L-1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.
- MeSH
- chemické techniky analytické ekonomika metody MeSH
- Chlorella vulgaris chemie MeSH
- chromatografie na tenké vrstvě * MeSH
- hmotnostní spektrometrie * přístrojové vybavení MeSH
- lasery polovodičové MeSH
- Saccharomyces cerevisiae chemie MeSH
- selenocystein analýza MeSH
- selenomethionin analýza MeSH
- spektrální analýza MeSH
- volatilizace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu2+. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza MeSH
- komplexní sloučeniny metabolismus MeSH
- kumariny farmakologie MeSH
- lidé MeSH
- měď metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- subcelulární frakce metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
