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Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Background: Metallothioneins (MTs) family is a intracellular and cysteine-rich proteins with a high affinity to metals. MT-3 could play important neuromodulatory and neuroprotective roles. MT-3 has been also found up-regulated in a number of cancers. Neuroblastoma (Nbl) is a cancer is the most common extra-cranial solid tumour of childhood. The main aim was to provide novel insights into the molecular mechanisms of hMT-3 up-regulation and to elucidate the effects beneath the MT-3 up-regulation in Nbl cells., Methods: To increase the expression of MT-3 Nbl (UKF-NB-4) cells were transiently transfected with a plasmid containing MT-3 gene (pcDNA3.1-GFP-hMT-3-TOPO). Separations of tryptic digests were carried out on an Easy-nLC 1000 nano system. MS analysis was performed using a Q-Exactive MS. The mass spectrum *.raw file was searched against the human SwissProt 57.15 database using MASCOT search engine (version 2.3, Matrix Science)., Results: The efficiency of transfection analysed through a fluorescence of GFP tag expressed at the C-terminus of MT-3 showed more 70% transfection efficiency for both constructed plasmids (mock and MT-3). From the total of common proteins in dataset (hMT-3 vs. mock), 176, 20 and 1244 proteins were quantitatively identified with up-regulation, down-regulation, and no significant differences between hMT-3 and mock treatments. Noteworthy, the bioinformatical analyses revealed the exclusive expression (induced by MT-3) and up-regulation proteins of a number of proteins affecting biological pathways related to mitotic cell cycle, cellular responses to stress, positive regulation of proteolysis, negative regulation of cell cycle and programmed cell death., Conclusions: Our proteomic data shed some light on the proteins involved in inducing senescence and apoptosis in tested Nbl cells with up-regulated MT-3. Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. Cellular senescence and apoptosis are among those mechanisms. Further experiments will be performed to functionally verify these data to provide novel insights into the Nbl biology. These might be useful to develop novel therapeutic protocols utilizing MT-3 as prognostic biomarker or therapeutic target.

Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Michalkova H, Strmiska V, Kudr J, et al. Int J Nanomedicine. 2019;14:7609–7624The authors have advised due to an error at the time of figure assembly, Figure 1D on page 7614 and Figure 6A on page 7621 is incorrect. Read the original article &nbsp

The present study reports on a comprehensive investigation of mechanisms of in vitro cytotoxicity of high aspect ratio (HAR) bundles formed from anodic TiO2 nanotube (TNT) layers. Comparative cytotoxicity studies were performed using two types of HAR TNTs (diameter of similar to 110 nm), differing in initial thickness of the nanotubular layer (similar to 35 mu m for TNTs-1 vs. similar to 10 mu m for TNTs-2). Using two types of epithelial cell lines (MDA-MB-231, HEK-293), it was found that nanotoxicity is highly cell-type dependent and plausibly associates with higher membrane fluidity and decreased rigidity of cancer cells enabling penetration of TNTs to the cell membrane towards disruption of membrane integrity and reorganization of cytoskeletal network. Upon penetration, TNTs dysregulated redox homeostasis followed by DNA fragmentation and apoptotic/necrotic cell death. Both TNTs exhibited haemolytic activity and rapidly activated polarization of RAW 264.7 macrophages. Throughout the whole study, TNTs-2 possessing a lower aspect ratio manifested significantly higher cytotoxic effects. Taken together, this is the first report comprehensively investigating the mechanisms underlying the nanotoxicity of bundles formed from self-organised 1-D anodic TNT layers. Except for description of nanotoxicity of industrially-interesting nanomaterials, the delineation of the nanotoxicity paradigm in cancer cells could serve as solid basis for future efforts in rational engineering of TNTs towards selective anticancer nanomedicine., Tato studie představuje komplexní výzkum mechanizmu in vitro cytotoxicity svazků anodických vrstev TiO2 nanotrubic (TNT). Komparativní studie byly realizována s dvěma typy svazků se stejným nominálním průreme trubic (cca 110 nm), ale rozdílnou tloušťkou původních vrstev trubic: 35 mikrometrů pro TNTs-1 a 10 mikrometrů pro TNTs-2. S využitím dvou buněčných linií (MDA-MB-231, HEK-293) bylo zjištěno, že nanotoxicita je velmi závislá na použité bunečné linii a je pravděpodobně závislá od difuzních vlastností rakovinových buněčných membrán. Svazkům TNT je umožněno penetrovat tyto membrány směrem k porušení jejich celistvosti a reorganizaci cytoskeletálních sítí. Oba typy TNT svazků vykazují hemolytickou aktivitu a rapidně aktivují polarizaci RAW 264.7 makrofágů. V celé studii vykazovali svazky TNTs-2, které mají nižší poměr délky trubic vůči jejich průměru, o něco vyšší cytotoxický efekt. Toto je první report, který se uceleně zabývá mechanismem nanotoxicity svazků nanotrubic TiO2. Kromě popisu toxicity těchto aplikačně velmi zajímavých nanomateriálů, náčrt paradigmat toxicity rakovinových buněk by mohl sloužit jako pevná základna pro další optimalizaci využití TNT vrstev pro selektivní protirakovinovou nanomedicínu.

It has been shown that sarcosine supplementation stimulates proliferation and invassivenes of prostate cells. Nevertheless, the exact molecular mechanism responsible for this phenomenon is not known. In the present study we demonstrate that sarcosine increases expression of calmodulin (CaM), an important intracellular signaling molecule. Through this, sarcosine activates calmodulin-dependent protein kinases signaling. Pathway of activation CaM-dependent protein kinases can activate regulation of mitosis, proliferation, cell death, gene transcription and phosphorylation/ dephosphorlation of proteins. This is done through CaM binding of four Ca2+ ions. Interestingly, in this study, we identified decrese in free Ca2+ correlating with sarcosine-induced up-regulation of CaM. The influence of CaM to cell cycle changes was further verified using post transcritrional gene silencing using CaM-siRNA complex. Co-treatment of prostate cells with CaM-siRNA and sarcosine showed decrease in CaM-dependent kinases and cell invasiveness compared to sarcosine treatment only.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine- induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

Titanium dioxide nanoparticles (TiO2 NPs) are used in lots of human applications because of their extraordinary nano scaled properties. Particularly, due to their photoprotective properties, they are used in topical dermatologic preparation and also as white pigment. Due to these properties their use in human life is more and more frequent. Despite the fact that nano dimension brings various beneficial properties, it could bring also bad features. Therefore, in this study, we evaluated a cytotoxicity of two types of biphasic TiO2 NPs using distinct cells of epithelial origin. We found that TiO2 NPs can induce cytotoxic stress resulting in fragmentation of DNA.

The current study demonstrates design, preparation and characterization of biocompatible superparamagnetic iron oxide nanoparticles (SPIONs) coated with three different polymers polyvinylpyrrolidone (PVP) polyoxyethylene stearate (POES) and chitosan (Chit). Such modified nanoparticles were loaded with doxorubicin, as model anticancer drug. Resulting complex has an exceptional stability in physiological conditions. The highest release of complexed Dox was in endosomal environment in case SPIONs with POES. The cytotoxic effects of the complex were tested using breast cancer/healthy epithelial cell lines. Use of SPIONs increased the cytotoxicity of doxorubicin when compared to free doxorubicin and decreased the cytotoxicity in healthy cells. The results demonstrate that modification of SPIONs could have a potential in nanomedicine as versatile nanoplatform to enhance efficiency of anticancer therapy.

Hana Michalkova, 1 Vladislav Strmiska, 1 Jiri Kudr, 1, 2 Zuzana Skubalova, 1 Barbora Tesarova, 1 Pavel Svec, 1 Lukas Richtera, 1, 2 Ondrej Zitka, 1, 2 Vojtech Adam, 1, 2 Zbynek Heger 1, 2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno CZ-613 00, Czech Republic; 2Smart Nanodevices Research Group, Central European Institute of Technology, Brno University of Technology, Brno CZ-621 00, Czech RepublicCorrespondence: Zbynek HegerDepartment of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno CZ-613 00, Czech RepublicTel +420 54 513 3350Fax +420 54 521 2044Email heger@mendelu.czBackground: Iron oxide nanoparticles (IONs) have been increasingly utilized in a wide spectrum of biomedical applications. Surface coatings of IONs can bestow a number of exceptional properties, including enhanced stability of IONs, increased loading of drugs or their controlled release.Methods: Using two-step sonochemical protocol, IONs were surface-coated with polyoxyethylene stearate, polyvinylpyrrolidone or chitosan for a loading of two distinct topo II poisons (doxorubicin and ellipticine). The cytotoxic behavior was tested in vitro against breast cancer (MDA-MB-231) and healthy epithelial cells (HEK-293 and HBL-100). In addition, biocompatibility studies (hemotoxicity, protein corona formation, binding of third complement component) were performed.Results: Notably, despite surface-coated IONs exhibited only negligible cytotoxicity, upon tethering with topo II poisons, synergistic or additional enhancement of cytotoxicity was found in MDA-MB-231 cells. Pronounced anti-migratory activity, DNA fragmentation, decrease in expression of procaspase-3 and enhancement of p53 expression were further identified upon exposure to surface-coated IONs with tethered doxorubicin and ellipticine. Moreover, surface-coated IONs nanoformulations of topo II poisons exhibited exceptional stability in human plasma with no protein corona and complement 3 binding, an

The key to obtaining an optimum performance of an enzyme is often a question of devising a suitable enzyme and optimisation of conditions for its immobilization. In this study, laccases from the native isolates of white rot fungi Fomes fomentarius and/or Trametes versicolor, obtained from Czech forests, were used. From these, cross-linked enzyme aggregates (CLEA) were prepared and characterised when the experimental conditions were optimized. Based on the optimization steps, saturated ammonium sulphate solution (75 wt.%) was used as the precipitating agent, and different concentrations of glutaraldehyde as a cross-linking agent were investigated. CLEA aggregates formed under the optimal conditions showed higher catalytic efficiency and stabilities (thermal, pH, and storage, against denaturation) as well as high reusability compared to free laccase for both fungal strains. The best concentration of glutaraldehyde seemed to be 50 mM and higher efficiency of cross-linking was observed at a low temperature 4 degrees C. An insignificant increase in optimum pH for CLEA laccases with respect to free laccases for both fungi was observed. The results show that the optimum temperature for both free laccase and CLEA laccase was 35 degrees C for T. versicolor and 30 degrees C for F. fomentarius. The CLEAs retained 80% of their initial activity for Trametes and 74% for Fomes after 70 days of cultivation. Prepared cross-linked enzyme aggregates were also investigated for their decolourisation activity on malachite green, bromothymol blue, and methyl red dyes. Immobilised CLEA laccase from Trametes versicolor showed 95% decolourisation potential and CLEA from Fomes fomentarius demonstrated 90% decolourisation efficiency within 10 h for all dyes used. These results suggest that these CLEAs have promising potential in dye decolourisation.

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.

The hypothesis that dogs can detect malignant tumours through the identification of specific molecules is nearly 30 years old. To date, several reports have described the successful detection of distinct types of cancer. However, is still a lack of data regarding the specific molecules that can be recognized by a dog's olfactory apparatus. Hence, we performed a study with artificially prepared, well-characterized urinary specimens that were enriched with sarcosine, a widely reported urinary biomarker for prostate cancer (PCa). For the purposes of the study, a German shepherd dog was utilized for analyses of 60 positive and 120 negative samples. Our study provides the first evidence that a sniffer dog specially trained for the olfactory detection of PCa can recognize sarcosine in artificial urine with a performance [sensitivity of 90%, specificity of 95%, and precision of 90% for the highest amount of sarcosine (10 mu mol/ L)] that is comparable to the identification of PCa-diagnosed subjects (sensitivity of 93.5% and specificity of 91.6%). This study casts light on the unrevealed phenomenon of PCa olfactory detection and opens the door for further studies with canine olfactory detection and cancer diagnostics.

The current study demonstrates design preparation and characterization of biocompatible hybrid ruthenium core-shell nanoparticles (RuNPs) coated with polyvinylpyrrolidone (PVP) and polyoxyethylene stearate (POES). The resulting RuNPs were loaded with doxorubicin, as model anticancer drug. Resulting complex has an exceptional stability in physiological conditions. The cytotoxic effects of the complex were tested using cell lines representing breast and ovarian cancers and neuroblastoma. Although bare RuNPs had only negligible cytotoxicity, RuPDox caused an enhancement of doxorubicin cytotoxicity when compared to free doxorubicin. RuPDox promoted significantly increased stability of doxorubicin in human plasma and pronounced hemocompatibility assayed on human red blood cells. Results demonstrate that biocompatible RuNPs could have a great potential as versatile nanoplatform to enhance efficiency of anticancer therapy.

Chemotherapy often results in various side effects, which can negatively affect health. Neuroblastoma, one of the most common types of childhood cancer, is but one of the examples, where side effects of chemotherapeutic treatment lower the quality of patient's life. Modern way how to fight that is to enclose cytotoxic drug into some nanocarrier and its targeting to receptors overexpressed in membranes of cancer cells. Apoferritin (Apo), a natural protein cage, is very suitable as a nanocarrier, as it has no toxicity, immune system does not react to it, and drug can easily be loaded into its cavity. We enclosed ellipticine, clinical tested anti-cancer drug, into Apo cavity (creating ApoElli). The percentage of encapsulation was 61 % and size and transmission electron microscopy analysis showed the preserved Apo ~12 nm icosahedral structure after this encapsulation. Then we modified Apo outer surface with in silico-modelled peptides with hNET affinity and tested its toxicity and hemolytic activity. ApoElli modified with anti-hNET peptides was able to internalize into neuroblastoma cells and to deliver the drug. However, it proved to be safe for human RBC, unlike pure ellipticine, which caused observable hemolysis at the same concentration.

It has been shown that sarcosine suplementation stimulates the proliferation of prostate cells and also their invassiveness. In present study we show that enzymes conected with sarcosine conversion to glycine (sarcosine dehydrogenase, pipecolic acid oxidase) are stimulated due to sarcosine treatment. Further, sarcosine treatment increases S-adenosylmethioneine-to-S-adenosylhomocysteine ratio, which indicates a release and utilization of free methyl groups from sarcosine degradation pathway. We identified the highest induction of global methylation in non-malignant PNT1A cells, but global methylation profiles were altered also in malignant (22Rv1) and metastatic (LNCaP) cells. The influence on methylation changes was further verified using hypomethylating agent 5-azacytidine (5-aza). Co-treatment of prostate cells with 5-aza and sarcosine resulted in decrease in cells invassiveness when compared to treatment with sarcosine alone. This correlates with sarcosine-related hypermethylation of genes involved in cells growth and cell cycle.