Helena Gbelcová

Current approaches in SELEX: An update to aptamer selection technology.

Systematic evolution of ligands by exponential enrichment (SELEX) is a well-established and efficient technology for the generation of oligonucleotides with a high target affinity. These SELEX-derived single stranded DNA and RNA molecules, called aptamers, were selected against various targets, such as proteins, cells, microorganisms, chemical compounds etc. They have a great potential in the use as novel antibodies, in cancer theragnostics and in biomedical research. Vast interest in aptamers stimulated continuous development of SELEX, which underwent numerous modifications since its first application in 1990. Novel modifications made the selection process more efficient, cost-effective and significantly less time-consuming. This article brings a comprehensive and up-to-date review of recent advances in SELEX methods and pinpoints advantages, main obstacles and limitations. The post-SELEX strategies and examples of application are also briefly outlined in this review.

Differences in antitumor effects of various statins on human pancreatic cancer

Statins are widely used for the treatment of hypercholesterolemia. However, their inhibitory action on HMG‐CoA reductase also results in the depletion of intermediate biosynthetic products, which importantly contribute to cell proliferation. The aim of the present study was to compare the effects of the individual commercially available statins on experimental pancreatic cancer. The in vitro effects of individual statins (pravastatin, atorvastatin, simvastatin, lovastatin, cerivastatin, rosuvastatin and fluvastatin) on the viability of human pancreatic cancer were evaluated in CAPAN‐2, BxPc‐3 and MiaPaCa‐2 cell lines. The in vivo experiments were performed on nude mice xenotransplanted with CAPAN‐2 cells. The mice received oral treatments either with a placebo, or with the statins mentioned earlier in a daily dose corresponding to a hypocholesterolemic dose in humans. The effect of these statins on the intracellular Ras protein, trafficking in MiaPaCa‐2 transfected cells, was also investigated. Substantial differences in the tumor‐suppressive effects of all statins were detected in both in vitro and in vivo experiments. While simvastatin exerted the highest tumor‐suppressive effects in vitro, rosuvastatin (p = 0.002), cerivastatin (p = 0.002) and fluvastatin (p = 0.009) were the most potent compounds in an animal model. All statins (except pravastatin) inhibited intracellular Ras protein translocation. In summary, substantial tumor‐suppressive effects of various statins on the progression of experimental pancreatic adenocarcinoma were demonstrated, with marked differences among individual statins. These results support greatly the potential of statins for the chemoadjuvant treatment of pancreatic cancer.

Antiproliferative effects of carbon monoxide on pancreatic cancer

BACKGROUND Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. METHODS In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1h/day; n = 6 in each group). RESULTS Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p<0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30-50%, p<0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p<0.01), and doubled the survival rates (p<0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p=0.006). CONCLUSION These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer.

The complex understanding of Annexin A1 phosphorylation.

Annexin A1 (ANXA1) is the first characterized member of the annexins superfamily. It binds the cellular membrane phospholipids in Ca(2+) regulated manner. Annexin A1 has been found in several tissues and many physiological roles as hormones secretion, vesiculation, inflammatory response, apoptosis and differentiation have been shown. Its subcellular localization and binding with many partner proteins are altered accordingly with its physiological role. The Annexin A1 membrane localization is crucial for binding to receptors, suggesting a paracrine and juxtacrine extracellular action. Annexin A1 is subjected to several post-translational modifications. In particular the protein is phosphorylated on several residues both on the N-terminal functional domain and on the C-terminus core. Different kinases have been identified as responsible for the phosphorylation status of selective residues. The specific change in the phosphorylation status on the different sites alters ANXA1 localization, binding properties and functions. This review shows the physiological relevance of the ANXA1 phosphorylation leading to the conclusion that numerous and different roles of Annexin A1 could be associated with different phosphorylations to alter not only intracellular localization and bindings to its partners but also the extracellular receptor interactions.

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr(4)N[VO(O(2))(ox)(phen)], (Vphen), [phen=1,10-phenantroline, ox=oxalate(2-) and Pr(4)N=tetra(n-propyl)ammonium(1+)] with DNA. UV-Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60 min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλ(max)=6 nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30 min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100 μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1 μmol/L to 1.8 times for 100 μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC(50) values were 0.247 μg/mL (0.45 μmol/L) for 24h, 0.671 μg/mL (1.21 μmol/L) for 48 h and 0.627 μg/mL (1.13 μmol/L) for 72 h.

Variability in statin-induced changes in gene expression profiles of pancreatic cancer

Statins, besides being powerful cholesterol-lowering drugs, also exert potent anti-proliferative activities. However, their anti-cancer efficacy differs among the individual statins. Thus, the aim of this study was to identify the biological pathways affected by individual statins in an in vitro model of human pancreatic cancer. The study was performed on a human pancreatic cancer cell line MiaPaCa-2, exposed to all commercially available statins (12 μM, 24 h exposure). DNA microarray analysis was used to determine changes in the gene expression of treated cells. Intracellular concentrations of individual statins were measured by UPLC (ultra performance liquid chromatography)-HRMS (high resolution mass spectrometer). Large differences in the gene transcription profiles of pancreatic cancer cells exposed to various statins were observed; cerivastatin, pitavastatin, and simvastatin being the most efficient modulators of expression of genes involved namely in the mevalonate pathway, cell cycle regulation, DNA replication, apoptosis and cytoskeleton signaling. Marked differences in the intracellular concentrations of individual statins in pancreatic cancer cells were found (>11 times lower concentration of rosuvastatin compared to lovastatin), which may contribute to inter-individual variability in their anti-cancer effects. In conclusion, individual statins exert different gene expression modulating effects in treated pancreatic cancer cells. These effects may be partially caused by large differences in their bioavailability. We report large differences in gene transcription profiles of pancreatic cancer cells exposed to various statins. These data correlate to some extent with the intracellular concentrations of statins, and may explain the inter-individual variability in the anti-cancer effects of statins.

Fish oil emulsion supplementation might improve quality of life of diabetic patients due to its antioxidant and anti-inflammatory properties

Diabetes-related complications, including cardiovascular disease, retinopathy, nephropathy, and neuropathy, are a significant cause of increased morbidity and mortality among people with diabetes. Previous studies have confirmed that hyperglycemia has pro-oxidative and proinflammatory properties which cause diabetic complications. We hypothesized that supplementation of fish oil emulsion (FOE), rich in omega-3 polyunsaturated fatty acids, to diabetic patients might reduce hyperglycemia-induced pathological changes due to specific properties of FOE. Omega-3 polyunsaturated fatty acids have a wide range of biological effects. In this project, we have examined the potential protective effect of the FOE on hyperglycemia-induced oxidative stress and cytokine generation in monocytes/macrophages U937 system in vitro. The monocytes/macrophages U937 were cultivated under normal or hyperglycemic (35 mmol/L glucose) conditions with/without FOE for 72 hours. We have focused on specific markers of oxidative stress (antioxidant capacity; superoxide dismutase activity; oxidative damage to DNA, proteins, and lipids) and inflammation (tumor necrosis factor, interleukin-6, interleukin-8, monocytic chemotactic protein-1). Hyperglycemia caused reduction of antioxidant capacity, induction of DNA damage, and proinflammatory cytokine secretion. FOE significantly increased antioxidant capacity of cells as well as superoxide dismutase activity and significantly reduced tumor necrosis factor, interleukin-6, interleukin-8, and monocytic chemotactic protein-1 release. No effect was observed on oxidative damage to DNA, proteins, and lipids. Our results indicate that FOE can reduce hyperglycemia-induced pathological mechanisms by its antioxidant and anti-inflammatory properties.

PTEN sequence analysis in endometrial hyperplasia and endometrial carcinoma in Slovak women.

Phosphatase and tensin homolog (PTEN) is a protein that acts as a tumor suppressor by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate. Loss of PTEN function has been implicated in the pathogenesis of a number of different tumors, particularly endometrial carcinoma (ECa). ECa is the most common neoplasia of the female genital tract. Our study evaluates an association between the morphological appearance of endometrial hyperplasia and endometrial carcinoma and the degree of PTEN alterations. A total of 45 endometrial biopsies from Slovak women were included in present study. Formalin-fixed and paraffin-embedded tissue samples with simple hyperplasia (3), complex hyperplasia (5), atypical complex hyperplasia (7), endometrioid carcinomas G1 (20) and G3 (5), and serous carcinoma (5) were evaluated for the presence of mutations in coding regions of PTEN gene, the most frequently mutated tumor suppressor gene in endometrial carcinoma. 75% of the detected mutations were clustered in exons 5 and 8. Out of the 39 mutations detected in 24 cases, 20 were frameshifts and 19 were nonsense, missense, or silent mutations. Some specimens harboured more than one mutation. The results of current study on Slovak women were compared to a previous study performed on Polish population. The two sets of results were similar.

Metallomics for Alzheimer's disease treatment: Use of new generation of chelators combining metal-cation binding and transport properties

Alzheimers disease (AD) is a progressive neurodegenerative disorder affecting tens of million people. Currently marketed drugs have limited therapeutic efficacy and only slowing down the neurodegenerative process. Interestingly, it has been suggested that biometal cations in the amyloid beta (Aβ) aggregate deposits contribute to neurotoxicity and degenerative changes in AD. Thus, chelation therapy could represent novel mode of therapeutic intervention. Here we describe the features of chelators with therapeutically relevant mechanism of action. We have found that the tested compounds effectively reduce the toxicity of exogenous Aβ and suppress its endogenous production as well as decrease oxidative stress. Cholyl hydrazones were found to be the most active compounds. In summary, our data show that cation complexation, together with improving transport efficacy may represent basis for eventual treatment strategy in AD.

Autophagy in MCF-7 cancer cells induced by copper complexes

BACKGROUND Autophagy plays an important role in cancer cells. Targeting autophagy in cancer can provide new opportunities for drug development. METHODS In this study we tested four Schiff base Cu(II) complexes against human breast cancer cells (MCF-7) and human non-cancerous cells (HEK-293T). We have tested their cytotoxic effect by evaluating IC50 using MTT test. To detect morphological changes of the actin fibers we have used fluorescent microscopy. To determine the type of cell death we used electrophoretic analysis and western blot analysis (protein LC3). RESULTS IC50 values of the complexes increased with time of their influence, indicating acquired resistance of MCF-7 to the complexes. Healthy cells HEK-293T were not sensitive to the Cu(II) complexes. Compared with the control cells (cells without Cu(II) complexes) which were without morphological changes of actin fibers, Cu(II) complexes induced condensation and asymmetric conformational changes in actin filaments. To examine the type of cell death induced by the Cu(II) complexes we treated MCF-7 cells with Cu(II) complexes (1, 10, 50 and 100μmol/L) during a 72h incubation period. By electrophoresis we have not detected any DNA fragmentation. To determine whether Cu(II) complexes induced autophagy or necrotic cell death we used the western blot analysis. MCF-7 cells influenced with tested Cu(II) complexes produced LC3 protein after their 72h incubation indicating autophagy in MCF-7 cancer cells. CONCLUSIONS Tested Schiff base copper (II) complexes have antiproliferative activity against cancer cells but not against healthy cells. They have induced autophagy in the cancer cell line MCF-7.