Hana Buchtelova

Anticancerogenic effect of spices due to phenolic and flavonoid compounds – in vitro evaluation on prostate cells

This study shows the effects of spices, and their phenolic and flavonoid compounds, on prostate cell lines (PNT1A, 22RV1 and PC3). The results of an MTT assay on extracts from eight spices revealed the strongest inhibitory effects were from black pepper and caraway seed extracts. The strongest inhibitory effect on prostatic cells was observed after the application of extracts of spices in concentration of 12.5 mg·mL−1. An LC/MS analysis identified that the most abundant phenolic and flavonoid compounds in black pepper are 3,4-dihydroxybenzaldehyde and naringenin chalcone, while the most abundant phenolic and flavonoid compounds in caraway seeds are neochlorogenic acid and apigenin. Using an MTT assay for the phenolic and flavonoid compounds from spices, we identified the IC50 value of ~1 mmol·L−1 PNT1A. The scratch test demonstrated that the most potent inhibitory effect on PNT1A, 22RV1 and PC3 cells is from the naringenin chalcone contained in black pepper. From the spectrum of compounds assessed, the naringenin chalcone contained in black pepper was identified as the most potent inhibitor of the growth of prostate cells.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non‐malignant and non‐metastatic cells.

A two‐step protocol for isolation of influenza A (H7N7) virions and their RNA for PCR diagnostics based on modified paramagnetic particles

Annual epidemics of influenza cause death of hundreds of thousands people and they also have a significant economic impact. Hence, a need for fast and cheap influenza diagnostic method is arising. The conventional methods for an isolation of the viruses are time‐consuming and require expensive instrumentation as well as trained personnel. In this study, we modified the surface of nanomaghemite (γ‐Fe2O3) paramagnetic core with tetraethyl orthosilicate and (3‐aminopropyl)triethoxysilane and the resulting particles were utilized for the isolation of H7N7 influenza virions. Consequently, we designed γ‐Fe2O3 paramagnetic core modified with calcium tripolyphosphate which was employed for the isolation of viral nucleic acid after virions lysis. Both of these procedures can be performed rapidly in less than 10 min and, in combination with the RT‐PCR, the whole influenza detection can be shortened to few hours. Moreover, the whole protocol could be easily automated and/or miniaturized, and thus can serve as a basis for use in a lab‐on‐a‐chip device. We assume that magnetic isolation is an exceptional procedure which can significantly accelerate the diagnostic possibilities of a broad spectrum of diseases.

Comparative gene expression profiling of human metallothionein-3 up-regulation in neuroblastoma cells and its impact on susceptibility to cisplatin

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 (24IC50: 7.5 vs. 19.8 μg/ml), indicating its multipurpose biological significance.

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

BackgroundSuitable 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.ResultsMicrowave-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.ConclusionFor 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.

Novel vancomycin–peptide conjugate as potent antibacterial agent against vancomycin-resistant Staphylococcus aureus

Background Increase in vancomycin (Van)-resistant bacterial strains including vancomycin-resistant Staphylococcus aureus (VRSA) and lack of new effective antibiotics have become a formidable health problem. Materials and methods We designed a new conjugate composed of Van and a peptide Hecate (Hec; Van/Hec), and its potential antimicrobial activity was evaluated. Results Results from disk diffusion test, time-kill assay, determination of minimum inhibitory concentration (MIC), microscopy, and comet assay showed strong antimicrobial effects of Van/Hec against wild-type, methicillin-resistant Staphylococcus aureus (MRSA) and VRSA. Microscopy revealed that the exposure to Van/Hec results in disruption of bacterial cell integrity in all tested strains, which was not observed in case of Van or Hec alone. Conclusion Overall, we showed that the preparation of conjugates from antibiotics and biologically active peptides could help us to overcome the limitation of the use of antibiotic in the treatment of infections caused by multidrug-resistant bacteria.

Functional Analysis of Novicidin Peptide: Coordinated Delivery System for Zinc via Schiff Base Ligand

Novicidin (NVC), is a membrane-penetrating peptide, which forms a stable complex with Zn-Schiff base with interesting antitumor selectivity. We studied NVC derivatives to determine functional roles of key amino acids in toxicity, helicity, and binding of the Zn-Schiff base complex. Trimmed derivatives highlighted the role of peptide length and helicity in toxicity and membrane penetration. The removal of Lys from position 1 and 2 strongly increases the ability to disrupt the membranes. The trimming of the N-terminal residues significantly increases the stability of peptide helicity enhancing penetrating properties. Gly residue derivatives undermined a role of peptide bending in membrane penetration and toxicity. After the substitution of the central Gly derivatives with Ile or Lys, the peptides retained toxicity. These results illustrate the minor role of central helix bending in NVC toxicity. Binding-site-peptide derivatives identified His residue as the sole Zn-Schiff base binding site and eliminated the role of other aromatic residues.

Real-Time Visualization of Cell Membrane Damage Using Gadolinium-Schiff Base Complex-Doped Quantum Dots

Despite the importance of cell membranes for maintenance of integrity of cellular structures, there is still a lack of methods that allow simple real-time visualization of their damage. Herein, we describe gadolinium-Schiff base-doped quantum dots (GdQDs)-based probes for a fast facile spatial labeling of membrane injuries. We found that GdQDs preferentially interact through electron-rich and hydrophobic residues with a specific sequence motif of NHE-RF2 scaffold protein, exposed upon membrane damage. Such interaction results in a fast formation of intensively fluorescent droplets with a higher resolution and in a much shorter time compared to immunofluorescence using organic dye. GdQDs have high stability, brightness, and considerable cytocompatibility, which enable their use in long-term experiments in living cultures. To the best of our knowledge, this is the first report, demonstrating a method allowing real-time monitoring of membrane damage and recovery without any special requirements for instrumentation. Because of intensive brightness and simple signal pattern, GdQDs allow easy examination of interactions between cellular membranes and cell-penetrating peptides or cytostatic drugs. We anticipate that the simple and flexible method will also facilitate the studies dealing with host-pathogen interactions.

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.

Exceptional release kinetics and cytotoxic selectivity of oxidised MWCNTs double-functionalised with doxorubicin and prostate-homing peptide

Multiwall carbon nanotubes (MWCNTs) are among the frequently studied carbon materials, particularly because of their physical and chemical properties and high potential for application in materials chemistry, industry, and medicine. MWCNTs are very promising as transporters of bioactive molecules because of their π electrons and large surface area, which can be easily modified, mostly by the application of inorganic acids for the introduction of carboxylic moieties on the surface. In the present study, we designed an oxidised MWCNTs (oMWCNTs) transporter for the targeted delivery of doxorubicin (Dox). The modification of oMWCNTs with prostate-homing peptide (SMSIARL) promotes increased cytotoxicity for prostate cancer cells. Using advanced analytical techniques, we studied the loading efficiency, stability, and release kinetics of Dox from a oMWCNTs-Dox-Pep nanoconstruct. We show that pH strictly drives Dox release, and imitating the pH of intracellular acidic compartments, 60% of Dox is released from oMWCNTs-Dox-Pep, while in plasma conditions, only a 14% release of Dox was found during 24h. The nanoconstruct displayed no cytotoxicity in non-malignant prostate cells (PNT1A), while in metastatic prostate cancer cells (LNCaP), the cytotoxic effects were close to the cytotoxicity of free Dox. This indicates that peptide modification promotes interactions with malignant cells, resulting in efficient internalisation into the intracellular region. Overall, we show that oMWCNTs are exceptional platforms for simple and stable non-covalent modification with bioactive molecules.