Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistance.

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.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells.

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% ß-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Improving cytocompatibility of CdTe quantum dots by Schiff-base-coordinated lanthanides surface doping.

BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.

Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone Nanoparticles and Their Effect on Pathogenic Microorganisms.

Herein we describe a novel alternative synthesis route of polyvinylpyrrolidone nanoparticles using salting-out method at a temperature close to polyvinylpyrrolidone decomposition. At elevated temperatures, the stability of polyvinylpyrrolidone decreases and the opening of pyrrolidone ring fractions occurs. This leads to cross-linking process, where separate units of polyvinylpyrrolidone interact among themselves and rearrange to form nanoparticles. The formation/stability of these nanoparticles was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, mass spectrometry, infrared spectroscopy, and spectrophotometry. The obtained nanoparticles possess exceptional biocompatibility. No toxicity and genotoxicity was found in normal human prostate epithelium cells (PNT1A) together with their high hemocompatibility. The antimicrobial effects of polyvinylpyrrolidone nanoparticles were tested on bacterial strains isolated from the wounds of patients suffering from hard-to-heal infections. Molecular analysis (qPCR) confirmed that the treatment can induce the regulation of stress-related survival genes. Our results strongly suggest that the polyvinylpyrrolidone nanoparticles have great potential to be developed into a novel antibacterial compound.

Label-free bead-based metallothionein electrochemical immunosensor.

A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 µL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 µg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

3D-printed biosensor with poly(dimethylsiloxane) reservoir for magnetic separation and quantum dots-based immunolabeling of metallothionein.

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 µL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 µL), with low acquisition costs and high portability.

Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser-induced fluorescence detection.

Quantum dots (QDs) are one of the most promising nanomaterials, due to their size-dependent characteristics as well as easily controllable size during the synthesis process. They are promising label material and their interaction with biomolecules is of great interest for science. In this study, CdTe QDs were synthesized under optimal conditions for 2 nm size. Characterization and verification of QDs synthesis procedure were done by fluorimetric method and with CE. Afterwards, QDs interaction with chicken genomic DNA and 500 bpDNA fragment was observed employing CE-LIF and gel electrophoresis. Performed interaction relies on possible matching between size of QDs and major groove of the DNA, which is approximately 2.1 nm.

Remote-controlled robotic platform ORPHEUS as a new tool for detection of bacteria in the environment.

Remote-controlled robotic systems are being used for analysis of various types of analytes in hostile environment including those called extraterrestrial. The aim of our study was to develop a remote-controlled robotic platform (ORPHEUS-HOPE) for bacterial detection. For the platform ORPHEUS-HOPE a 3D printed flow chip was designed and created with a culture chamber with volume 600 µL. The flow rate was optimized to 500 µL/min. The chip was tested primarily for detection of 1-naphthol by differential pulse voltammetry with detection limit (S/N = 3) as 20 nM. Further, the way how to capture bacteria was optimized. To capture bacterial cells (Staphylococcus aureus), maghemite nanoparticles (1 mg/mL) were prepared and modified with collagen, glucose, graphene, gold, hyaluronic acid, and graphene with gold or graphene with glucose (20 mg/mL). The most up to 50% of the bacteria were captured by graphene nanoparticles modified with glucose. The detection limit of the whole assay, which included capturing of bacteria and their detection under remote control operation, was estimated as 30 bacteria per µL.

Preconcentration based on paramagnetic microparticles for the separation of sarcosine using hydrophilic interaction liquid chromatography coupled with coulometric detection.

Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphate buffer pH 9). The optimal mixing ratio was A + B/C 1:1 with a flow rate of 0.80 mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reached a good resolution for separation also with a good LOD (100 nM). In addition, we successfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.

"Golgi-customized Trojan horse" nanodiamonds impair GLUT1 plasma membrane localization and inhibit tumor glycolysis.

Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation strategy remains a formidable task. The Golgi morphology is crucial in maintaining the function of transport proteins (such as GLUT1) driving glycolysis. Thus, in this study, we present a "Golgi-customized Trojan horse" based on tellurium loaded with apigenin (4',5,7-trihydroxyflavone) and human serum albumin, which was able to induce GLUT1 plasma membrane localization disturbance via Golgi dispersal leading to the inhibition of tumor glycolysis. Diamond-shaped delivery system can efficiently penetrate into cells as a gift like Trojan horse, which decomposes into tellurite induced by intrinsically high H2O2 and GSH levels. Consequently, tellurite acts as released warriors causing up to 3.8-fold increase in Golgi apparatus area due to the down-regulation of GOLPH3. Further, this affects GLUT1 membrane localization and glucose transport disturbance. Simultaneously, apigenin hinders ongoing glycolysis and causes significant decrease in ATP level. Collectively, our "Golgi-customized Trojan horse" demonstrates a potent antitumor activity because of its capability to deprive energy resources of cancer cells. This study not only expands the applications of tellurium-based nanomaterials in the biomedicine but also provides insights into glycolysis restriction for anticancer therapy.

Microfluidic tool coupled with electrochemical assay for detection of lactoferrin isolated by antibody-modified paramagnetic beads.

Lactoferrin (LF) is approximately 80 kDa iron-binding protein, which is important part of saliva and other body fluids. Due to its ability to bind metal ions, it has many biologically important functions. In this study, a method for the isolation of LF from a biological sample using robotically prepared antibody-modified paramagnetic particles was developed using robotic pipetting station. The method consisted of the following optimised steps. Protein G was bound on the paramagnetic particles, on which goat antibody (10 µg) was linked. LF was subsequently added to microtitration plate, which had affinity to goat antibody and the interaction lasted for 30 min. We found that the highest signals were obtained using the combination of goat antibody 1:3000, murine antibody 1:5000 and conjugate 1:1500. Horseradish peroxidase reducing 3,3,5,5-tetramethylbenzidine (TMB) was linked to the merged complex. The resulted product of this reaction was subsequently analysed spectrometrically with detection limit (3 S/N) as 5 ng/mL. In addition, we also determined TMB by stopped flow injection analysis with electrochemical detection. The limit of detection (3 S/N) was estimated as 0.1 µg/mL. To compare spectrometric and electrochemical approach for detection of TMB, calibration range of bead-LF-antibodies complex was prepared and was determined using a least-squares correlation with coefficient R² higher than 0.95, indicating a very good agreement of the results obtained.

Electrophoretic study of peptide-mediated quantum dot-human immunoglobulin bioconjugation.

The bioconjugation of quantum dots (QDs) is a key process in their application for bioanalysis as well as imaging. The coupling of QDs with biologically active molecules such as peptides, nucleic acids, and/or antibodies enables their fluorescent labeling, and therefore, selective and sensitive tracking during the bioanalytical process, however, the efficiency of the labeling and preservation of the biological activity of the bioconjugate have to be considered. In this study, a new approach of the bioconjugation of CdTe-QDs and human immunoglobulin employing a small peptide is described. The heptapeptide (HWRGWVC) was synthesized and characterized by mass spectrometry, liquid chromatography, and capillary electrophoresis. Moreover, the peptide was used as a capping agent for QDs synthesis. The CdTe-QDs were synthesized by microwave synthesis (600 W, 20 min) using 3.2 mM CdCl2 and 0.8 mM Na2TeO3. The bioconjugation of QDs capped by this peptide with immunoglobulin was investigated by capillary electrophoresis and magnetic immunoextraction coupled with electrochemical detection by differential pulse voltammetry. Furthermore, the applicability of prepared bioconjugates for fluorescent immunodetection was verified using immobilized goat antihuman IgG antibody.

Use of brightness wavelet transformation for automated analysis of serum metallothioneins- and zinc-containing proteins by Western blots to subclassify childhood solid tumours.

In this study, we determined serum levels of metallothioneins (MTs) and zinc in children with solid tumours (neuroblastoma, Hodgkin lymphoma, medulloblastoma, osteosarcoma, Ewing sarcoma and nephroblastoma) by differential pulse voltammetry Brdicka reaction and ELISA. Zn(II) level in patients sera was 40% compared to controls, contrariwise, MT level was 4.2 × higher in patients. No significant differences among single diagnoses were found both for Zn(II) and MT. When determined Zn(II)/MT ratio, in controls its value was 24.6, but it was 2.6 in patients. After Western-blotting with anti-MT and anti-Zn chicken antibodies, variable intensities of the bands within the samples were observed. The brightness curve obtained for each sample both for MT- and Zn blots was further analysed to produce a list of band positions together with some complementary information related to the intensity of the observed bands by the optimised algorithm. We constructed from those profiles decision trees that enable to distinguish different groups of tumours. The blood samples were heat-treated, in which we supposed mainly MT, but samples contained other thermostable Zn-containing proteins that were helpful for identification of embryonal tumours with 88% accuracy and for identification of sarcomas with 78% accuracy. In MT blots the accuracies were 53 and 45%, respectively. Simultaneous analysis of MT and Zn blots did not increased accuracy of identification neither in embryonal tumours (80%) nor in sarcomas. Those results are promising not only from diagnostic point of view but particularly in the area of studying of individual MT isoforms and their aggregates in malignant tumours.

Rapid superparamagnetic-beads-based automated immunoseparation of Zn-proteins from Staphylococcus aureus with nanogram yield.

Pathogenic bacteria have become a serious socio-economic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn-proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn-proteins by superparamagnetic beads modified with chicken anti-Zn(II) antibody. S. aureus culture was treated with ZnCl(2). Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle-surface interactions were tested and associated protein profiles were identified using SDS-PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS-PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.

Molecular biology of beta-estradiol-estrogen receptor complex binding to estrogen response element and the effect on cell proliferation.

Group of estrogen pollutants, where the highest estrogen activity is reported at estradiol, is characterized by the fact that even at very low concentrations have potential to cause xenoestrogenic effects. During exposure of excessive amounts of estradiols may be produced undesirable effects resulting in the feminization of males of water organisms. The presence of estradiols in drinking water implies also a risk for the human population in the form of cancers of endocrine systems, abnormalities in reproduction or dysfunctions of neuronal and immune system. Currently, the research is focused mainly to uncover the relationship between the estrogen receptors binding affinity with an estrogen response element and estradiol. In this review we summarized facts about molecular biological principles of ß estradiol-estrogen receptor complex binding with estrogen response element and its successive effect on cancer genes expression.

Beads-based electrochemical assay for the detection of influenza hemagglutinin labeled with CdTe quantum dots.

In this study we describe a beads-based assay for rapid, sensitive and specific isolation and detection of influenza vaccine hemagglutinin (HA). Amplification of the hemagglutinin signal resulted from binding of an electrochemical label as quantum dots (QDs). For detection of the metal and protein part of the resulting HA-CdTe complex, two differential pulse voltammetric methods were used. The procedure includes automated robotic isolation and electrochemical analysis of the isolated product. The isolation procedure was based on the binding of paramagnetic particles (MPs) with glycan (Gly), where glycan was used as the specific receptor for linkage of the QD-labeled hemagglutinin.

Remodeling of the liver fibrosis microenvironment based on nilotinib-loaded multicatalytic nanozymes with boosted antifibrogenic activity.

Liver fibrosis is a reversible pathological process caused by chronic liver damage and a major risk factor for hepatocellular carcinoma (HCC). Hepatic stellate cell (HSC) activation is considered the main target for liver fibrosis therapy. However, the efficiency of this strategy is limited due to the complex microenvironment of liver fibrosis, including excessive extracellular matrix (ECM) deposition and hypoxia-induced imbalanced ECM metabolism. Herein, nilotinib (NIL)-loaded hyaluronic acid (HA)-coated Ag@Pt nanotriangular nanozymes (APNH NTs) were developed to inhibit HSCs activation and remodel the microenvironment of liver fibrosis. APNH NTs efficiently eliminated intrahepatic reactive oxygen species (ROS) due to their inherent superoxide dismutase (SOD) and catalase (CAT) activities, thereby downregulating the expression of NADPH oxidase-4 (NOX-4) and inhibiting HSCs activation. Simultaneously, the oxygen produced by the APNH NTs further alleviated the hypoxic microenvironment. Importantly, the released NIL promoted collagen depletion by suppressing the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), thus synergistically remodeling the microenvironment of liver fibrosis. Notably, an in vivo study in CCl4-induced mice revealed that APNH NTs exhibited significant antifibrogenic effects without obvious long-term toxicity. Taken together, the data from this work suggest that treatment with the synthesized APNH NTs provides an enlightening strategy for remodeling the microenvironment of liver fibrosis with boosted antifibrogenic activity.

Metallothioneins and zinc in cancer diagnosis and therapy.

Metallothioneins (MTs) are involved in protection against oxidative stress (OS) and toxic metals and they participate in zinc metabolism and its homeostasis. Disturbing of zinc homeostasis can lead to formation of reactive oxygen species, which can result in OS causing alterations in immunity, aging, and civilization diseases, but also in cancer development. It is not surprising that altered zinc metabolism and expression of MTs are of great interest in the case of studying of oncogenesis and cancer prognosis. The role of MTs and zinc in cancer development is tightly connected, and the structure and function of MTs are strongly dependent on Zn²âº redox state and its binding to proteins. Antiapoptic effects of MTs and their interactions with proteins nuclear factor kappa B, protein kinase C, esophageal cancer-related gene, and p53 as well as the role of MTs in their proliferation, immunomodulation, enzyme activation, and interaction with nitric oxide are reviewed. Utilization of MTs in cancer diagnosis and therapy is summarized and their importance for chemoresistance is also mentioned.

Immunoextraction of zinc proteins from human plasma using chicken yolk antibodies immobilized onto paramagnetic particles and their electrophoretic analysis.

Zinc(II) as the only transition metal lacking redox activity is an essential part of approximately 10% proteins as a cofactor of these proteins. Considering the fact that there are numerous zinc(II) containing proteins, proteomics and metallomics studies aimed on them require accurate methods for preparation of real biological samples prior to their subsequent analysis using 2DE and MS. For this purpose, we suggested a new method based on chicken anti-zinc antibodies and magnetizable particles. Antibodies were covalently immobilized to the surface of paramagnetic beads activated with tosyl group. Binding of the antibody to the beads was confirmed by secondary anti-chicken antibody conjugated with horseradish peroxidase. The immunoextraction conditions, such as concentration of the beads (6-18 µg/mL of the sample), time of immunoextraction (6-34 min), pH and composition of the elution buffer, and time of extraction (48-300 s) were optimized. Subsequently, zinc proteins were extracted from human plasma and total concentration of zinc was monitored by electrochemical detection in the extracts. Under optimal conditions it was possible to monitor the proteins and zinc removal from the sample by chip CE, SDS-PAGE, and indirectly using electrochemistry.