Tanya Topouzova-Hristova

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-L-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-L-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.

Mitochondria are involved in stress response of A549 alveolar cells to halothane toxicity.

During inhalation anaesthesia lung epithelial cells are directly exposed to halogenated hydrocarbons such as halothane. Information about the effects of volatile anaesthetics on lung cells is rather limited although their noxious effect on the A549 alveolar cells has been shown recently. The present study indicated that halothane decreases cell viability, impairs DNA integrity and provokes stress-induced apoptosis in A549 cells when applied at clinically relevant concentrations. Data obtained clearly demonstrated intensive expression of anti-apoptotic Bcl-2 protein during treatment with all tested concentrations. In post-treatment periods the increased DNA injury was accompanied by reduction of Bcl-2 expression. We concluded that the in vitro effect of halothane on lung cells involved alteration in the expression of proteins of the mitochondrial apoptotic pathway.

Methanol and Chloroform Extracts from Lamium Album L. Affect Cell Properties of A549 Cancer Lung Cell Line

ABSTRACT Lamium album L. (white dead nettle) is a plant widely used in official and folk medicine. It possess astringent, anti-inflammotary, antibiotic, antispasmotic, antioxidant and bacteriostatic properties and is used in bladder, kidney and menstrual problems. The spectrum of its therapeutic activity is related to a variety of biologically active substances. In present study we investigated potential effects on A549 cancer lung cell line of several concentrations and combinations of methanol and chloroform extracts from in vivo and in vitro propagated Lamium album L., obtained by Soxhlet extraction. MTT test for assessment of cell viability, test for cell attachment and FACS-analyses after 24 and 48 hours of cultivation were performed. After 24h and 48h of incubation, all extracts showed reduction in cell viability, accept methanol (below 2.5 mg/ml) and chloroform (0.5 mg/ml) extracts from in vivo plants. Strongest cytotoxic effect was observed when methanol extract (5.0 mg/ml) from in vivo cultivated plant was applied. All studied extracts decreased adhesion properties of the cells. The strongest effect showed methanol extract from in vivo plants with concentration 4.5mg/ml. FACS—analyses revealed retention of the cell cycle in G2 period for cells, cultivated in all extracts, while in the population of cells, cultivated in the combination of extracts and in the chloroform extract from in vitro propagated plants of L. album L., small amount of apoptotic cells was observed. Extracts from in vivo cultivated plants were more active then those, from in vitro propagated. We consider that Lamium album L. extracts possess potential anti-cancer effects that need to be investigated in more details.

Influence of Volatile Anaesthetics on Lung Cells and Lung Surfactant

ABSTRACT Volatile anaesthetics as halothane, enflurane, isoflurane, sevoflurane and desfluane have significant role in surgery. Anaesthesia could affect breath holding and might cause a dose-dependent depression in respiration. Lung epithelial cells are the first barrier against inhaled agents. Among them the pneumocytes type II are the main producers of pulmonary surfactant. Damage of lung surfactant (LS) is one of the possible reasons for the respiratory complications. In contrast to studies, concerning the influence of general anaesthetics on lung surfactant functionality, there is still poor information about the influence, based on the alveolar cell cultures. In this review we focus our attention on the data indicating the harmful effect of anaesthetics and mainly of halothane on the properties of lung cells, biosynthesis, secretion and functionality of LS components, using an appropriate cell culture model system and we discuss as far as possible the mechanism underlying the halothane toxicity.

Preparation and Biological Activity of New Collagen Composites, Part I: Collagen/Zinc Titanate Nanocomposites.

The aim of this investigation was to develop new antimicrobial collagen/zinc titanate (ZnTiO3) biomaterials using a sol–gel cryogenic draying technology in keeping the native collagen activity. Broad-spectrum antimicrobial activity was demonstrated against Firmicutes (Staphylococcus epidermidis, Bacillus cereus, and Candida lusitaniae) and Gracilicutes (Escherichia coli, Salmonella enterica, and Pseudomonas putida) microorganisms. The antimicrobial activity as well as the cytotoxicity were specific for the different test microorganisms (Gram-positive and Gram-negative bacteria and fungi) and model eukaryotic cells (osteosarcoma, fibroblast, and keratinocyte cells), respectively, and both were depending on the ZnTiO3 concentration. Three mechanisms of the antimicrobial action were supposed, including (i) mechanical demolition of the cell wall and membrane by the crystal nanoparticles of the ZnTiO3 entrapped in the collagen matrix, (ii) chelation of its metal ions, and (iii) formation of free oxygen radicals due to the interaction between the microbial cells and antimicrobial agent. It was concluded that the optimal balance between antimicrobial activity and cytotoxicity could be achieved by a variation of the ZnTiO3 concentration. The antifungal and broad-spectrum antibacterial activity of the studied collagen/ZnTiO3 nanocomposites, combined with a low cytotoxicity, makes them a promising anti-infection biomaterial.

Antitumor Lipids—Structure, Functions, and Medical Applications

Cell proliferation and metastasis are considered hallmarks of tumor progression. Therefore, efforts have been made to develop novel anticancer drugs that inhibit both the proliferation and the motility of tumor cells. Synthetic antitumor lipids (ATLs), which are chemically divided into two main classes, comprise (i) alkylphospholipids (APLs) and (ii) alkylphosphocholines (APCs). They represent a new entity of drugs with distinct antiproliferative properties in tumor cells. These compounds do not interfere with the DNA or mitotic spindle apparatus of the cell, instead, they incorporate into cell membranes, where they accumulate and interfere with lipid metabolism and lipid-dependent signaling pathways. Recently, it has been shown that the most commonly studied APLs inhibit proliferation by inducing apoptosis in malignant cells while leaving normal cells unaffected and are potent sensitizers of conventional chemo- and radiotherapy, as well as of electrical field therapy. APLs resist catabolic degradation to a large extent, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. They are internalized in the cell membrane via raft domains and cause downstream reactions as inhibition of cell growth and migration, cell cycle arrest, actin stress fibers collapse, and apoptosis. This review summarizes the in vitro, in vivo, and clinical trials of most common ATLs and their mode of action at molecular and biochemical levels.

Effects of Ca2+ ions on bestrophin-1 surface films

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Effects of vipoxin and its components on HepG2 cells.

Snake venom Phospholipases A2 (svPLA2) are among the main toxic venom components with a great impact on different tissues and organs based on their catalytic specificity and a variety of pharmacological effects, whose mechanism is still under debate. The main toxic component, isolated from the venom of Vipera ammodytes meridionalis, is the heterodimeric postsynaptic ionic complex vipoxin, composed of a basic and toxic PLA2 enzyme subunit (GIIA secreted PLA2) and an acidic, enzymatically inactive and nontoxic subunit - vipoxin acidic component (VAC). This study demonstrates for the first time that vipoxin and its individual subunits affect integrity and viability of HepG2 cells displaying differences in their pharmacological activities. Under the experimental conditions, the individual PLA2 subunit induces cytotoxicity, cytoskeletal rearrangements and triggers early apoptosis in a concentration-dependent manner related to its enzymatic activity. Vipoxin and VAC do not affect cell viability but manifest high degree of genotoxicity, whereas DNA damage induced by PLA2 subunit could be defined as moderate and not associated with its catalytic activity. Our results suggest that the interactions between vipoxin subunits play an important role in HepG2 cell response and most likely affect the observed distinction between cyto- and genotoxicity.

Halothane-induced alterations in cellular structure and proliferation of A549 cells

Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.

Polymeric vehicles for transport and delivery of DNA via cationic micelle template method

This work describes the preparation of polymeric non-viral system for transport and delivery of nucleic acids. An amphiphilic poly(2-(dimethylamino)ethyl methacrylate)-block-poly(ε-caprolactone)-block-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA20-b-PCL70-b-PDMAEMA20) triblock copolymer was synthesized and used for formation of cationic micelles and subsequent complexation with DNA. Next, a protective polymer coating on the complex surface and removal of copolymer from the particle interior were conducted. In this way, polymer nanocapsules containing DNA molecules were obtained. The synthesized polymer, cationic micelles, complexes, and polymer capsules were investigated by proton nuclear magnetic resonance, gel permeation chromatography, dynamic and electrophoretic light scattering, and transmission electron microscopy. In vitro cytotoxicity assessment of the different systems revealed very good tolerance to human cells.