Biological analyses of the effects of TiO2 and PEG-b-PLA nanoparticles on three-dimensional spheroid-based tumor.

The aim of our study was to monitor the antiproliferative/ cytotoxic and genotoxic effects of both, poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) and titanium dioxide (TiO2) nanoparticles on the tumor (HT-29, MCF-7, U118MG) and healthy (HEK-293T) cell lines during 2D cultivation and during cultivation in the spheroid form (3D cultivation). Cells or spheroids were cultivated with nanoparticles (0.01, 0.1, 1, 10, 50, and 100 ?g/ml) for 72 hours. The cytotoxic effect was determined by the MTT test and the genotoxic effect by the comet assay. We found that 2D cultivation of tumor cell lines with PEG-b-PLA and TiO2 nanoparticles had an anti-proliferative effect on human colon cancer cell line HT-29, human breast cancer cell line MCF-7, human glioma cell line U-118MG during 72h cultivation, but not on control/healthy HEK-293T cells. At the concentrations used, the tested nanoparticles caused no cytotoxic effect on tumor cell lines. Nanoparticles PEG-b-PLA induced significant damage to DNA in HT-29 and MCF-7 cells, while TiO2 nanoparticles in MCF-7 and U-118MG cells. Only PEG-b-PLA nanoparticles caused cytotoxic (IC50 = 7 mikrog/ml) and genotoxic effects on the healthy cell line HEK-293T after 72h cultivation. The cells which were cultivated in spheroid forms were more sensitive to both types of nanoparticles. After 72h cultivation, we observed the cytotoxic effect on both, the tumor and healthy cell lines.

Autonomic Nervous System Function in Newly Diagnosed Multiple Sclerosis: Association With Lipid Levels and Insulin Resistance.

Autonomic nervous system (ANS) disorders are common in multiple sclerosis (MS). Previous studies showed differences in insulin resistance (IR) and lipoprotein levels in MS subjects compared to controls. Lipolysis caused by increased sympathetic activity could be one of the possible linking mechanisms leading to dyslipidemia in MS. Our study aimed to evaluate ANS activity in the context of glucose and lipid metabolism in people with MS. We prospectively measured short-term heart rate variability (HRV), fasting lipoprotein concentrations, and calculated IR indices based on plasma glucose and insulin levels during oral glucose tolerance test (oGTT) in 32 patients with MS and 29 healthy controls matched for age, sex and body mass index in our study. There was no significant difference in HRV parameters and lipoprotein levels between MS and controls. A significant positive correlation was found between low/high-frequency power ratio (LF/HF) and triglycerides (r=0.413, p=0.021) in MS subjects but not in controls. A significantly lower whole-body insulin sensitivity index (ISIMat) was found in patients with MS compared to the control group (7.3±3.7 vs. 9.8±5.6, p=0.041). No significant correlations were found between LF/HF and IR parameters. In MS subjects, the positive correlation of LF/HF with triglycerides could reflect the effects of sympathetic activity on lipolysis. Positive correlations of sympathetic activity with increased lipoprotein levels could rather reflect processes associated with immune system activation/inflammation, than processes involved in glucose homeostasis maintenance.

Effect of natural polyphenols on thromboxane levels in children with Crohn's disease.

OBJECTIVES: The aim of this study was to investigate the relationship between thromboxane levels and oxidative stress in children with Crohn´s disease (CD), and examine the effect of natural polyphenolic compounds on thromboxane levels. METHODS: This study involved 14 children suffering from CD and 15 healthy controls. Patients were receiving the polyphenolic extract Pycnogenol for 10 weeks. Plasma levels of the static and dynamic forms of thromboxane B2 as well as their metabolite 11-dehydro thromboxane B2 in urine were determined. RESULTS: In comparison to controls, CD patients had significantly higher levels of the static and dynamic forms of thromboxane B2. Pycnogenol decreased the level of the dynamic form of thromboxane B2 after 10 weeks of administration. CONCLUSIONS: Paediatric Crohn's disease is associated with higher thromboxane levels. Our results indicate that Pycnogenol administration reduces thromboxane levels, which may positively influence some clinical symptoms of CD such as thromboembolic episodes (Tab. 3, Ref. 49).

Schiff base Cu(II) complexes as inhibitors of proteasome in human cancer cells.

BACKGROUND: It has been demonstrated that proteasome inhibitors might be potential anticancer drugs. The copper complexes can be used as specific proteasome inhibitors in tumor cells able to induce apoptosis by the ubiquitin-proteasome pathway. The goal of our study was to test the cytotoxic and proteasome inhibitory effects of five Schiff base Cu(II) complexes - [Cu2(sal-D,L-glu)2(isoquinoline)2] . 2C2H5OH (1), [Cu(sal-5-met-L-glu)(H2O)].H2O (2), [Cu(ethanol)2(imidazole)4][Cu2(sal-D,L-glu)2(imidazole)2] (3), [Cu(sal-D,L-glu)(2-methylimidazole)] (4) on human lung carcinoma cells A549, cervix carcinoma cells HeLa and glioblastoma cells U-118MG. MATERIAL AND METHODS: For the cytotoxic analysis we used MTT test and for monitoring the proteasome inhibition western blot analysis. RESULTS: We have observed different cytotoxic effects of tested complexes on human cancer cells depending on the ligand present in their structure. Cu(II) complexes 4 and 5 were the most effective against A549 cells; all complexes were cytotoxic against HeLa cells and the complex 4 was the most effective against U-118MG. Moreover, we have detected the inhibition of the proteasome activity in human cancer cells A549 by Cu(II) complexes 1, 2 and 4 at IC50 concentration. CONCLUSION: Results of our study suggest that isoquinoline- and imidazole-based copper complexes could be used as inhibitors of the proteasome system in cancer cells A549 (Tab. 1, Fig. 1, Ref. 26).

Role of oxidative stress, adiponectin and endoglin in the pathophysiology of erectile dysfunction in diabetic and non-diabetic men.

Erectile dysfunction (ED) and diabetes mellitus (DM) share common pathophysiological risk factors including endothelial dysfunction which together with hyperglycemia contribute to the increased oxidative/glycooxidative stress. A reduced NO concentration is insufficient for relaxation processes in the penis. Chronic inflammation and endoglin are involved in the regulation of endothelial function. Adiponectin from the adipose tissue has anti-inflammatory effects. Our study aimed to investigate the relation between erectile function in patients with and without DM and the oxidative stress, hormone adiponectin, and endothelial dysfunction marker endoglin. Men (n=32) with ED evaluated by the International Index of Erectile function (IIEF-5) questionnaire (17 without DM (NDM); 15 with type 2 diabetes mellitus (DM)) and 31 controls were included. Advanced glycation end products (AGEs), 8-isoprostanes (8-isoP), protein carbonyls, antioxidant capacity, adiponectin and endoglin were determined in the blood. DM patients compared to NDM patients and controls, had increased levels of glucose, C-reactive protein, triacylglycerols, 8-isoP, AGEs, endoglin and BMI. IIEF-5 score, NO and adiponectin levels were decreased. We are the first to find out that endoglin shows a negative correlation with erectile function in NDM, but not in DM patients. Endoglin can be considered as endothelial dysfunction marker in nondiabetic men suffering from ED.

Oxidative stress and Down syndrome. Do antioxidants play a role in therapy?

Oxidative stress is a phenomenon associated with imbalance between production of free radicals and reactive metabolites (e.g. superoxide and hydrogen peroxide) and the antioxidant defences. Oxidative stress in individuals with Down syndrome (DS) has been associated with trisomy of the 21st chromosome resulting in DS phenotype as well as with various morphological abnormalities, immune disorders, intellectual disability, premature aging and other biochemical abnormalities. Trisomy 21 in patients with DS results in increased activity of an important antioxidant enzyme Cu/Zn superoxide dismutase (SOD) which gene is located on the 21st chromosome along with other proteins such as transcription factor Ets-2, stress inducing factors (DSCR1) and precursor of beta-amyloid protein responsible for the formation of amyloid plaques in Alzheimer disease. Mentioned proteins are involved in the management of mitochondrial function, thereby promoting mitochondrial theory of aging also in people with DS. In defence against toxic effects of free radicals and their metabolites organism has built antioxidant defence systems. Their lack and reduced function increases oxidative stress resulting in disruption of the structure of important biomolecules, such as proteins, lipids and nucleic acids. This leads to their dysfunctions affecting pathophysiology of organs and the whole organism. This paper examines the impact of antioxidant interventions as well as positive effect of physical exercise on cognitive and learning disabilities of individuals with DS. Potential therapeutic targets on the molecular level (oxidative stress markers, gene for DYRK1A, neutrophic factor BDNF) after intervention of natural polyphenols are also discussed.

Effect of natural polyphenols (Pycnogenol) on oxidative stress markers in children suffering from Crohn's disease--a pilot study.

Crohn's disease (CD) is a nonspecific, chronic inflammatory disease of the gastrointestinal tract. It is supposed that in etiopathogenesis oxidative stress (OS) plays a role. However, its precise role in the active and non-active states of disease is not known yet. We conducted a pilot study focusing on the relationship between OS of CD in remission and the possibility to influence clinical parameters and markers of OS by polyphenolic extract, Pycnogenol® (Pyc). Compared to 15 healthy controls 15 pediatric CD patients (all were in remission according to their disease activity index - PCDAI) had reduced the activity of Cu/Zn-superoxide dismutase (SOD) and increased the oxidative damage to proteins. We found negative correlations between markers of inflammation (calprotectin, CRP) as well as between PCDAI and total antioxidant capacity (TAC). Activities of antioxidant enzymes, SOD, and glutathione peroxidase (GPX) negatively correlated with calprotectin and PCDAI. Pyc (2 mg/kg) positively influenced the parameters of OS in CD patients after 10 weeks of administration.

Iron accumulation in human spleen in autoimmune thrombocytopenia and hereditary spherocytosis.

BACKGROUND: Although the iron is an essential element for the physiological functions of cells, tissues and organs, it is also an important inductor of reactive oxygen species (ROS). MATERIAL AND METHODS: Three groups of human spleen with autoimmune thrombocytopenia (AITP), hereditary spherocytosis (HS) and reference samples stained by haematoxylin and eosin, Perls' reaction for nonheme Fe(III) iron and Alcian blue for glycoconjugates detection were studied. RESULTS: Positive Perls' reaction in both AITP and HS groups was seen. Higher positivity in the HS than in AITP group was observed. HS group showed a higher amount of acidic glycoconjugates deposits than AITP group. Iron overload in HS and AITP leads to overproduction of ROS. CONCLUSION: We suggest that acidic glycoconjugates deposits are involved in antioxidant defence by elimination and restriction of iron as a ROS inducer (Fig. 4, Ref. 19).

Complex steroid-peptide-receptor cascade controls insect ecdysis.

Insect ecdysis sequence is composed of pre-ecdysis, ecdysis and post-ecdysis behaviors controlled by a complex cascade of peptide hormones from endocrine Inka cells and neuropeptides in the central nervous system (CNS). Inka cells produce pre-ecdysis and ecdysis triggering hormones (ETH) which activate the ecdysis sequence through receptor-mediated actions on specific neurons in the CNS. Multiple experimental approaches have been used to determine mechanisms of ETH expression and release from Inka cells and its action on the CNS of moths and flies. During the preparatory phase 1-2 days prior to ecdysis, high ecdysteroid levels induce expression of ETH receptors in the CNS and increased ETH production in Inka cells, which coincides with expression of nuclear ecdysone receptor (EcR) and transcription factor cryptocephal (CRC). However, high ecdysteroid levels prevent ETH release from Inka cells. Acquisition of Inka cell competence to release ETH requires decline of ecdysteroid levels and beta-FTZ-F1 expression few hours prior to ecdysis. The behavioral phase is initiated by ETH secretion into the hemolymph, which is controlled by two brain neuropeptides-corazonin and eclosion hormone (EH). Corazonin acts on its receptor in Inka cells to elicit low level ETH secretion and initiation of pre-ecdysis, while EH induces cGMP-mediated ETH depletion and consequent activation of ecdysis. The activation of both behaviors is accomplished by ETH action on central neurons expressing ETH receptors A and B (ETHR-A and B). These neurons produce numerous excitatory or inhibitory neuropeptides which initiate or terminate different phases of the ecdysis sequence. Our data indicate that insect ecdysis is a very complex process characterized by two principal steps: (1) ecdysteroid-induced expression of receptors and transcription factors in the CNS and Inka cells. (2) Release and interaction of Inka cell peptide hormones and multiple central neuropeptides to control consecutive phases of the ecdysis sequence.

Activity of paraoxonase 1 and lipid profile in healthy children.

Paraoxonase 1 (PON1) seems to have a relevant role in detoxifying processes and in atherosclerosis. The aim of this study was to determine PON1 activity, the total antioxidant capacity, as well as entire lipid profile in children for screening of possible risk of atherosclerosis development. Serum PON1 arylesterase/paraoxonase activities were determined spectrophotometrically. The total antioxidant capacity of the serum was measured by TEAC method. Parameters of lipid profile were analyzed by routine laboratory methods. It has been shown that PON1 arylesterase/ paraoxonase activities were very similar to values found in adults. In children, no significant correlation between PON1 arylesterase activity and HDL was observed. PON1 paraoxonase activity correlated only with atherogenic index. PON1 arylesterase activity was significantly higher in girls than in boys. The antioxidant capacity was inversely related to the body mass index. In this study, PON1 activity was determined in healthy children aged 11 to 12 years and we found a similarity in PON1 activities of children and adults. Moreover, the results of our study support the hypothesis that higher body weight of children may contribute to a greater risk for development of atherosclerosis in which oxidative stress plays a role.

The combined effect of pycnogenol with ascorbic acid and trolox on the oxidation of lipids and proteins.

Pycnogenol (PYC), a procyanidin-rich extract of French maritime pine bark (Pinus pinaster) has strong antioxidant potential and promotes cellular health. The aim of this study was to investigate a possible cooperation of natural antioxidant PYC with synthetic antioxidants ascorbic acid and trolox in the model system of lipid peroxidation determined as conjugated dienes formation in liposomes and on the oxidation of proteins (in BSA and plasma proteins) determined as protein carbonyls. The present study shows that PYC and trolox significantly increased inhibition of lipid peroxidation initiated by copper acetate and tert-butylhydroperoxide in concentration and time dependence compared with untreated unilamellar liposomes. PYC and trolox added simultaneously to the oxidized liposomes exerted an additive preventive effect. PYC s inhibitory effect on formation of carbonyl compounds in BSA and plasma proteins, oxidized by two oxidative systems--H2O2/FeSO4 and HOCl, were studied in co-operation with other synthetic antioxidants--ascorbic acid and trolox. We found the synergistic or additive effect of PYC with mentioned antioxidants.

Conservation of ecdysis-triggering hormone signalling in insects.

Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, we found Inka cells in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviours in pharate larvae of Bombyx, indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae. Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioural sequence.

Effects of aminoguanidine Schiff's base on biomarkers of the oxidative stress, 4-hydroxy-2-nonenal and conjugated dienes, in the model diabetes mellitus.

Diabetes mellitus evoked by streptozotocine in rats is associated with the oxidative stress. We examined the effect of Schiff's base 2,5-dihydroxybenzaldehyde with a well-known antidiabetic drug aminoguanidine, 2,5-dihydroxybenzilideneaminoguanidine (BAG) on the production of markers of oxidative stress such as 4-hydroxy-2-nonenal (4HNE) and conjugated dienes in diabetic rats. BAG administration did not affect glucose level in diabetic rats but significantly decreased the production of 4HNE and conjugated dienes. On the other hand, BAG caused the elevation of conjugated dienes and an insignificant increase of 4HNE levels in the control animals.

Dual ecdysteroid action on the epitracheal glands and central nervous system preceding ecdysis of Manduca sexta.

Initiation of the ecdysis behavioural sequence in insects requires activation of the central nervous system (CNS) by pre-ecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which are released from the Inka cells of the epitracheal glands. Here, we show that the developmental events preceding larval and pupal ecdysis of Manduca sexta involve a dual action of ecdysteroids on the epitracheal glands and CNS. The low steroid levels in freshly ecdysed and feeding larvae are associated with small-sized epitracheal glands, reduced peptide production in Inka cells and insensitivity of the CNS to ETH. The elevated ecdysteroid levels before each ecdysis lead to a dramatic enlargement of Inka cells and increased production of peptide hormones and their precursors. As blood ecdysteroids reach peak levels, the CNS becomes responsive to Inka cell peptides. These effects of natural ecdysteroid pulses can be experimentally induced by injection of 20-hydroxyecdysone or the ecdysteroid agonist tebufenozide (RH-5992) into ecdysed larvae, thus stimulating peptide production in Inka cells and inducing CNS sensitivity to ETH. A direct steroid action on the CNS is demonstrated by subsequent treatment of isolated nerve cords from ecdysed larvae with 20-hydroxyecdysone and ETH, which results in pre-ecdysis or ecdysis bursts. Our data show that ecdysteroid-induced transcriptional activity in both the epitracheal glands and the CNS are necessary events for the initiation of the ecdysis behavioural sequence.

Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence.

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.

The superoxide dismutase-like activity of some copper(II) complexes derived from tridentate Schiff bases.

Oxygen free radicals are the final or intermediate products of many metabolic reactions. Of greatest significance to the organism are superoxide anion radical (O2-.), hydrogen peroxide (H2O2), hydroxyl radical (.OH), singlet oxygen (1O2) etc. A proper ratio between both production and breakdown of oxy-radicals is essential for the maintenance of a dynamic equilibrium of vital processes. The superoxide dismutases protect cells against toxic influence of the superoxide. In addition, some square-pyramidally pentacoordinated copper(II) complexes, derived from tridentate Schiff bases of the N-salicylideneaminoalcanoate type, show remarkable SOD-like activity. A selected set of complexes of this type have been tested: potassium [aqua-(N-salicylideneglutamato) cuprate] (L- and D,L-form), potassium [(isothiocyanato)-(N-salicylideneglycinato) cuprate], potassium [(isothiocyanato)-(N-salicylidene-D,L-alaninato) cuprate], potassium [(isothiocyanato)-(N-salicylidene-beta-alaninato) cuprate] and potassium [(isocyanato)-(N-salicylideneglycinato) cuprate]. Our results suggest that the copper complexes are not only antioxidants, but may also possess anti-inflammatory, cytostatic and radioprotective properties.