Orsolya Láng

Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate.

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.

Vacuum ultraviolet photolysis of diclofenac and the effects of its treated aqueous solutions on the proliferation and migratory responses of Tetrahymena pyriformis

The effects of dissolved O2, phosphate buffer and the initial concentration of diclofenac on the vacuum ultraviolet photolysis of this contaminant molecule were studied. Besides kinetic measurements, the irradiated, multicomponent samples were characterized via the proliferation and migratory responses (in sublethal concentrations) of the bioindicator eukaryotic ciliate Tetrahymena pyriformis. The results suggest that hydroxyl radicals, hydrogen atoms and hydroperoxyl radicals may all contribute to the degradation of diclofenac. The aromatic by-products of diclofenac were presumed to include a hydroxylated derivative, 1-(8-chlorocarbazolyl)acetic acid and 1-(8-hydroxycarbazolyl)acetic acid. The biological activity of photoexposed samples reflected the chemical transformation of diclofenac and was also dependent on the level of dissolved O2. The increase in toxicity of samples taken after different irradiation times did not exceed a factor of two. Our results suggest that the combination of vacuum ultraviolet photolysis with toxicity and chemotactic measurements can be a valuable method for the investigation of the elimination of micropollutants.

Characterization of chemotactic ability of peptides containing N‐formyl‐methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C‐terminal f‐Met and N‐terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f‐MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f‐Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis‐activating processes.

Incidence and survival of central nervous system involvement in childhood malignancies: Hungarian experience

Direct extension and hematogenous metastasis of primary non-CNS malignant tumors to the CNS are rare complications in children. The authors analyzed the incidence and outcome of these complications in Hungary. During a 14-year period between 1989 and 2002, 406 patients younger than 18 years were studied at Semmelweis University, Second Department of Pediatrics, in Budapest. Among the 406 patients with non-CNS solid tumors, nine hematogenous metastases and five direct tumor extensions to the CNS occurred. Primary tumors included rhabdomyosarcoma, neuroblastoma, tumors of the Ewing sarcoma family, non-Hodgkin lymphoma, and malignant chordoma. Mean interval between the initial diagnosis and the diagnosis of CNS involvement was 11.4 months. Despite intensive treatment, the mean survival after detection of CNS involvement was 10.4 months. The frequency of CNS involvement in non-CNS tumors is low, with a very poor survival.

Daunomycin-polypeptide conjugates with antitumor activity

We have developed a group of water-soluble drug conjugates in which daunomycin (Dau) is coupled to cationic, amphoteric or anionic branched polypeptides and a new conjugate containing a cationic polypeptide carrier modified with a cell penetrating octaarginine. We investigated in vitro physiological activity of these conjugates in several aspects: in vitro cytotoxicity and cytostatic effect, adhesion and cellular uptake were examined on murine (J774 and L1210) and human (MonoMac6 and HL-60) leukemia cell lines and on murine bone marrow derived macrophages. We found that these processes are dependent on the properties of the carrier, on experimental conditions like concentration and incubation time. We found that attachment of polypeptide and cell penetrating peptide to the bioactive agent, depending on the cell line, could significantly improve the antitumor activity of the drug.

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2.

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis

The chemotactic properties of tuftsin (H‐TKPR‐OH), tuftsin derivatives (H‐KPR‐OH, H‐TKPKG‐NH2, Ac‐TKPKG‐NH2) and TKPKG‐based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N‐terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C‐terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10−9–10−6 M), while T30 had a wide‐ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin‐induced chemotaxis results from fairly short‐term signalling in Tetrahymena.

Impedimetric analysis of the effect of decellularized porcine heart scaffold on human fibrosarcoma, endothelial, and cardiomyocyte cell lines

Background Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. Material/Methods In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. Results Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. Conclusions Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts.

Effect of vasoactive peptides in Tetrahymena: chemotactic activities of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP)

Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny.

Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes

Selegiline (R-deprenyl), a monoamine oxidase-B (MAO-B) inhibitor, has complex pharmacological effect that contributes to treatment of neurodegenerative diseases such as Parkinson’s and presumably Alzheimer’s disease and might work as an inhibitor of tumor growth. In respect of tumorigenesis and metastasis formation, the controlled modifications of adhesion and migration have high therapeutic significance. In the present study, our purpose was to investigate cell physiological responses (adhesion, chemotaxis and proliferation) induced by selegiline, its metabolites and synthetic derivatives and to find some correlations between the molecular structure and the reported antitumor behavior of the derivatives. Our results demonstrated that both R- and S-deprenyls have the potency to elicit increased adhesion and a chemorepellent activity in monocyte model (Mono Mac 6 cell line derived from monoblastic leukemia); however, only the R-enantiomer proved to be cytotoxic. Among the metabolites R-amphetamine has retained the adhesion inducer and the chemorepellent effect of the parent drug on the most significant level. In contrast, a reversed chemotactic effect and an improved cytotoxic character were detected in the presence of fluoro group (p-fluoro-S-deprenyl). In summary, the adhesion inducer activity, chemorepellent and advantageous cytotoxic effects of selegiline and some derivatives indicate that these drug molecules might have inhibitory effects in metastasis formation in primary tumors.