Analysis of Morphological and Morphometric Changes in a Parenchymal Tissue after the Radiofrequency Ablation Procedure.

Background and Objectives: Prostate cancer is on the rise in the European Union, and radiofrequency ablation (RFA) is one of the minimally invasive treatment options used for its treatment. Therefore, the aim of this study was to investigate and analyze the effects of RFA on prostate tissues. Materials and Methods: A standard prostate RFA procedure was performed on 13 non-purebred dogs in three sessions: no cooling (NC), cooling with a 0.1% NaCl solution (C.01), and cooling using a 0.9% NaCl solution (C.09). Microtome-cut 2-3 µm sections of prostate samples were stained with hematoxylin and eosin and further examined. Results: A histopathologic evaluation identified four zones of exposure: direct, application, necrosis, and transitional, as the damage on tissues decreased going further from the ablation site. The areas and perimeters of these zones were calculated, and geometric shapes of ablative lesions were evaluated using the quotient formula. Areas and perimeters of prostate tissue lesions in the NC and C.09 sessions were of similar size, whereas those found in C.01 were statistically significantly smaller. Lesions observed in session C.01 were of the most regular geometric shape, while the most irregular ones were found in session C.09. The shapes of lesions closest to the ablation electrode were the most irregular, becoming more regular the further away from the electrode they were. Conclusions: Prostate RFA leads to tissue damage with distinct morphological zones. Notably, the prostate lesions were the smallest and the most regular in shape after RFA procedures using the 0.1% NaCl cooling solution. It can be argued that smaller ablation sites may result in smaller scars, thus allowing for faster tissue healing if the blood flow and innervation at the ablation site are not compromised.

Ursolic and Oleanolic Acids: Plant Metabolites with Neuroprotective Potential.

Ursolic and oleanolic acids are secondary plant metabolites that are known to be involved in the plant defence system against water loss and pathogens. Nowadays these triterpenoids are also regarded as potential pharmaceutical compounds and there is mounting experimental data that either purified compounds or triterpenoid-enriched plant extracts exert various beneficial effects, including anti-oxidative, anti-inflammatory and anticancer, on model systems of both human or animal origin. Some of those effects have been linked to the ability of ursolic and oleanolic acids to modulate intracellular antioxidant systems and also inflammation and cell death-related pathways. Therefore, our aim was to review current studies on the distribution of ursolic and oleanolic acids in plants, bioavailability and pharmacokinetic properties of these triterpenoids and their derivatives, and to discuss their neuroprotective effects in vitro and in vivo.

The Difference of Cholesterol, Platelet and Cortisol Levels in Patients Diagnosed with Chronic Heart Failure with Reduced Ejection Fraction Groups According to Neutrophil Count.

Background and Objectives: It is known that neutrophils are involved in the pro-inflammatory processes and thus, can have a great impact on the pathophysiology of heart failure (HF). Moreover, hypercholesterolemia heightens neutrophil production, thereby accelerating cardiovascular inflammation. However, there is a lack of information about the relation of low inflammation to the state of stress, hypercholesterolemia, and pro-thrombotic statement in patients with chronic HF. Therefore, we aimed to determine whether platelet, cholesterol and cortisol levels differ in a different inflammatory condition groups according to the neutrophil count in patients diagnosed with CHF with reduced ejection fraction (CHFrEF), and whether there is a correlation between those readings. Materials and Methods: The average of neutrophil count was 4.37 × 109 L; therefore, 180 patients were separated into two groups: one with relatively a higher inflammatory environment (neutrophil count ≥ 4.37 × 109 L (n = 97)) and one with a relatively lower inflammatory environment (neutrophil count < 4.38 × 109 L (n = 83)). We also determined the levels of lymphocytes, monocytes, platelet count (PLT), mean platelet volume (MPV), platelet aggregation, the levels of cortisol and cholesterol and the concentrations of C reactive protein (CRP) and fibrinogen. Results: We found that CRP, fibrinogen and cortisol concentrations were statistically significantly higher in the group with higher neutrophil counts. However, there were no differences among cholesterol concentration and other markers of platelet function between the groups. We also showed that PLT, leukocyte and monocyte counts were higher in the group with a higher neutrophil count, and the PLT correlated with other cell type count and CRP. In addition, the neutrophil count correlated with concentrations of fibrinogen, evening cortisol and CRP. Conclusions: Cortisol, fibrinogen and CRP levels, PLT and monocyte counts were higher in the CHFrEF patient group with higher neutrophil counts. The cholesterol levels and platelet function readings did not differ between the groups. The neutrophil count correlated with evening cortisol concentration.

Platelet Activity and Its Correlation with Inflammation and Cell Count Readings in Chronic Heart Failure Patients with Reduced Ejection Fraction.

Background and objectives: There has been an increasing interest in the role of inflammation in thrombosis complications in chronic heart failure (HF) patients. The incidence of thrombosis in HF has been shown to be the highest in patients classified as NYHA IV (New York Heart association). It is stated that inflammation is regulated by platelet-induced activation of blood leukocytes. We aimed to compare the platelet and cell count readings in chronic HF with reduced ejection fraction (HFrEF) patients according to NYHA functional class and to evaluate the correlation between those readings. Materials and methods: A total of 185 patients were examined. The results of heart echoscopy (TEE) testing; fibrinogen, N-terminal pro b-type natriuretic peptide (NT-proBNP), C reactive protein (CRP), and cortisol concentrations; complete blood counts; and a 6 min walking test were assessed and platelet aggregation was determined. Results: Mean platelet volume (MPV) increased with deterioration of a patient's state (p < 0.005). Lymphocyte count and percentage were the lowest in the NYHA IV group (p < 0.005). Neutrophil and monocyte percentage and count were the highest (p < 0.045) in the NYHA IV group. Adenosine diphosphate (ADP)- and ADR-induced platelet aggregation was higher in the NYHA III group compared to NYHA II and I groups (p < 0.023). NYHA functional class correlated with mean platelet volume (MPV) (r = 0.311, p = 0.0001), lymphocyte count (r = -0.186, p = 0.026), monocyte count (p = 0.172, p = 0.041), and percentage (r = 0.212, p = 0.011). CRP concentration correlated with NT-proBNP (r = 0.203, p = 0.005). MPV correlated with fibrinogen concentration (r = 0.244, p = 0.004). Conclusions: (1) MPV could be considered as an additional reading reflecting a patient's condition, however the use of MPV to identify patients at risk of hypercoagulable state should be evaluated in more extensive studies; (2) increased neutrophil and monocyte counts could indicate a higher inflammatory state in chronic HFrEF.

Anthocyanins: From plant pigments to health benefits at mitochondrial level.

Anthocyanins are water-soluble pigments providing certain color for various plant parts, especially in edible berries. Earlier these compounds were only known as natural food colorants, the stability of which depended on pH, light, storage temperature and chemical structure. However, due to the increase of the in vitro, in vivo experimental data, as well as of the epidemiological studies, today anthocyanins and their metabolites are also regarded as potential pharmaceutical compounds providing various beneficial health effects on either human or animal cardiovascular system, brain, liver, pancreas and kidney. Many of these effects are shown to be related to the free-radical scavenging and antioxidant properties of anthocyanins, or to their ability to modulate the intracellular antioxidant systems. However, it is generally overlooked that instead of acting exclusively as antioxidants certain anthocyanins affect the activity of mitochondria that are the main source of energy in cells. Therefore, the aim of the present review is to summarize the major knowledge about the chemistry and regulation of biosynthesis of anthocyanins in plants, to overview the facts on bioavailability, and to discuss the most recent experimental findings related to the beneficial health effects emphasizing mitochondria.

Anthocyanins in cardioprotection: A path through mitochondria.

Constantly growing experimental data from in vitro, in vivo and epidemiological studies show the great potential of anthocyanin-containing fruit and berry extracts or pure individual anthocyanins as cardioprotective food components or pharmacological compounds. In general it is regarded that the cardioprotective activity of anthocyanins is related to their antioxidant properties. However there are recent reports that certain anthocyanins may protect the heart against ischemia/reperfusion-induced injury by activating signal transduction pathways and sustaining mitochondrial functions instead of acting solely as antioxidants. In this review, we summarize the proposed mechanisms of direct or indirect actions of anthocyanins within cardiac cells with the special emphasis on recently discovered their pharmacological effects on mitochondria in cardioprotection: reduction of cytosolic cytochrome c preventing apoptosis and sustainment of electron transfer between NADH dehydrogenase and cytochrome c supporting oxidative phosphorylation in ischemia-damaged mitochondria.

Protecting the heart against ischemia/reperfusion-induced necrosis and apoptosis: the effect of anthocyanins.

BACKGROUND AND OBJECTIVE: It is well known that cardiomyocyte apoptosis contributes to ischemic heart damage. There is also increasing evidence that the polyphenolic compounds of natural origin, such as anthocyanins, may attenuate ischemia/reperfusion injury though the mechanisms of such protection are not clear. Following our previous studies showing the effect of certain anthocyanins on cytochrome c redox state, mitochondrial functions, and ischemia-induced caspase activation in the heart, here we investigated whether these anthocyanins can rescue cardiac cells from death by the mechanism involving the reduction of cytosolic cytochrome c. MATERIAL AND METHODS: Before global ischemia and reperfusion, isolated rat hearts were preloaded with cyanidin-3-O-glucoside (Cy3G) that has high cytochrome c-reducing capacity or pelargonidin-3-O-glucoside (Pg3G) that possesses low reducing activity. Cell death was evaluated assessing apoptosis by the TUNEL method or necrosis measuring the release of lactate dehydrogenase into perfusate. RESULTS: The perfusion of hearts with 20-µM Cy3G prevented ischemia/reperfusion-induced apoptosis of cardiomyocytes: the number of TUNEL-positive myocytes was decreased by 73% if compared with the untreated ischemic group. The same effect was observed measuring the activity of lactate dehydrogenase as the measure of necrosis: perfusion with 20-µM Cy3G reduced the level of LDH release into the perfusate to the control level. The perfusion of hearts with 20-µM Pg3G did not prevent ischemia/reperfusion-induced apoptosis as well as necrosis. CONCLUSIONS: Cy3G protected the rat heart from ischemia/reperfusion-induced apoptosis and necrosis; meanwhile, Pg3G did not exert any protective effect. The protective effect of Cy3G may be related due to its high capacity to reduce cytosolic cytochrome c.

LACTB is a filament-forming protein localized in mitochondria.

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.

Phenotypic and Functional Heterogeneity of Monocyte Subsets in Chronic Heart Failure Patients.

Chronic heart failure (CHF) results when the heart cannot consistently supply the body's tissues with oxygen and required nutrients. CHF can be categorized as heart failure (HF) with preserved ejection fraction (HFpEF) or HF with reduced ejection fraction (HFrEF). There are different causes and mechanisms underlying HF pathogenesis; however, inflammation can be regarded as one of the factors that promotes both HFrEF and HFpEF. Monocytes, a subgroup of leukocytes, are known to be cellular mediators in response to cardiovascular injury and are closely related to inflammatory reactions. These cells are a vital component of the immune system and are the source of macrophages, which participate in cardiac tissue repair after injury. However, these monocytes are not as homogenous as thought and can present different functions under different cardiovascular disease conditions. In addition, there is still an open question regarding whether the functions of monocytes and macrophages should be regarded as causes or consequences in CHF development. Therefore, the aim of this work was to summarize current studies on the functions of various monocyte subsets in CHF with a focus on the role of a certain monocyte subset in HFpEF and HFrEF patients, as well as the subsets' relationship to inflammatory markers.

Comparison of the Formulation, Stability and Biological Effects of Hydrophilic Extracts from Black Elder Flowers (Sambucus nigra L.).

Elderflower preparations have long been used to treat colds and flu, but their use is undeservedly reduced, and only dried flower teas, less often ethanolic extracts, can be purchased in pharmacies. In the case of homemade teas, the medicinal plant material is extracted with hot water for a relatively short time, thus only a small part of the active substances is extracted. The industrially produced ethanolic extract is rich in active substances, but its use is limited since ethanol in many countries is undesirable and unsuitable for children and geriatric patients. Therefore, the aim of this work was to produce extracts from elder flowers using water as extractant and a mixture of water + polyethylene glycol (PEG) 20%, to compare their chemical composition and stability, and to study the ability to neutralize reactive oxygen species (ROS) and to sustain the viability of C6 glial cells under oxidative stress conditions. The ethanolic extract was used as a standard. Thus, the extract with PEG contained more than two times higher amount of total phenolics (PC) than the aqueous one, and the stability at 6-8 °C was comparable to the stability of ethanolic extract. All three extracts showed an antioxidant effect in a concentration-dependent manner in vitro. However, only the PEG containing extract (at 20-40 µg/mL PC) was the most effective in reducing the intracellular level of ROS and sustaining the viability of glial cells. The results suggest that the co-solvent PEG increases the yield of phenolics in the extract, prolongs the stability, and enhances positive biological effects.

Uncoupling and antioxidant effects of ursolic acid in isolated rat heart mitochondria.

Ursolic acid (1), a pentacyclic triterpene acid, is one of the major components of certain traditional medicinal plants and possesses a wide range of biological effects, such as anti-inflammatory, antioxidative, and cytotoxic activities. Furthermore, 1, when present at 1.6-5 ng/mL concentrations in commercial herbal preparations used for patients with cardiac disorders, may also exert pro-cardiac activities. There are several indirect suggestions that the cardioprotective mechanism of ursolic acid could involve the mitochondria; however the mechanism of action is still not known. Therefore, the effects of 0.4-200 ng/mL ursolic acid (1) on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate, or palmitoyl-l-carnitine plus malate were investigated. It was found that 1 induced a statistically significant uncoupling of oxidative phosphorylation. A statistically significant decrease in H2O2 production in the mitochondria was observed after incubation with 5 ng/mL 1. This effect was comparable to the effectiveness of the classical uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Since mild mitochondrial uncoupling has been proposed as one of the mechanisms of cardioprotection, the present results indicate that ursolic acid (1) has potential use as a cardioprotective compound.

Direct effects of K(ATP) channel openers pinacidil and diazoxide on oxidative phosphorylation of mitochondria in situ.

K(ATP) channel openers protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, however, the protection mechanisms have not been fully clarified yet. Since the skinned fibers technique gives an opportunity to investigate an entire population of mitochondria in their native milieu, in this study we have investigated the effects of K(ATP) channel openers pinacidil and diazoxide on the respiration rate of rat heart mitochondria in situ, oxidizing physiological substrates pyruvate and malate (6+6 mM). Respiration rates were recorded by the means of Clark-type oxygen electrode in the physiological salt solution (37 degrees C). Our results showed that both pinacidil and diazoxide (60-1250 muM) in a concentration-dependent manner increased pyruvate-malate supported State 2 respiration rate of skinned cardiac fibers (59.1 +/- 5.1 nmol O/min/mg fiber dry weight, RCI 2.6 +/- 0.2, n=4) by 15-120%. Moreover, diazoxide did not affect, whereas pinacidil (60-1250 muM) decreased the State 3 respiration rate of skinned cardiac fibers (116.6 +/- 13.6 nmol O/min/mg fiber dry weight, RCI 2.3 +/- 0.2, n=4) by 4-27%. Thus, common effect for both K(ATP) channel openers is uncoupling of pyruvate and malate oxidizing mitochondria in skinned cardiac fibers, whereas pinacidil under same conditions also inhibits mitochondrial respiratory chain. Since mitochondria in situ resemble to the great extent mitochondria in vivo, our results suggest that uncoupling and/or respiratory chain inhibition could play a role in the cardioprotection by K(ATP) channel openers.

Fatty Acid Oxidation and Mitochondrial Morphology Changes as Key Modulators of the Affinity for ADP in Rat Heart Mitochondria.

Fatty acids are the main respiratory substrates important for cardiac function, and their oxidation is altered during various chronic disorders. We investigated the mechanism of fatty acid-oxidation-induced changes and their relations with mitochondrial morphology and ADP/ATP carrier conformation on the kinetics of the regulation of mitochondrial respiration in rat skinned cardiac fibers. Saturated and unsaturated, activated and not activated, long and medium chain, fatty acids similarly decreased the apparent KmADP. Addition of 5% dextran T-70 to mimic the oncotic pressure of the cellular cytoplasm markedly increased the low apparent KmADP value of mitochondria in cardiac fibers respiring on palmitoyl-l-carnitine or octanoyl-l-carnitine, but did not affect the high apparent KmADP of mitochondria respiring on pyruvate and malate. Electron microscopy revealed that palmitoyl-l-carnitine oxidation-induced changes in the mitochondrial ultrastructure (preventable by dextran) are similar to those induced by carboxyatractyloside. Our data suggest that a fatty acid oxidation-induced conformational change of the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier (M-state to C-state, condensed to orthodox mitochondria) may affect the oxidative phosphorylation affinity for ADP.

Anthocyanins: From the Field to the Antioxidants in the Body.

Anthocyanins are biologically active water-soluble plant pigments that are responsible for blue, purple, and red colors in various plant parts-especially in fruits and blooms. Anthocyanins have attracted attention as natural food colorants to be used in yogurts, juices, marmalades, and bakery products. Numerous studies have also indicated the beneficial health effects of anthocyanins and their metabolites on human or animal organisms, including free-radical scavenging and antioxidant activity. Thus, our aim was to review the current knowledge about anthocyanin occurrence in plants, their stability during processing, and also the bioavailability and protective effects related to the antioxidant activity of anthocyanins in human and animal brains, hearts, livers, and kidneys.

Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy.

BACKGROUND: Bacterial penicillin-binding proteins and beta-lactamases (PBP-betaLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-betaL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-betaL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-betaL proteins. RESULTS: Metazoan PBP-betaL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-betaL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-betaL homologs comprise four alloparalogus protein lineages that derive from alpha-proteobacteria. CONCLUSION: While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-betaL proteins were conserved and are found in metazoans including humans. Metazoan PBP-betaL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-betaL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding.

Long-term regulation of gene expression in muscle cells by systemically delivered siRNA.

Many muscular dystrophies, including lethal Duchenne muscular dystrophy, are incurable and require the sustained application of drugs that have only minor treatment effects and serious negative side effects. The mechanism of siRNA-mediated transcriptional gene regulation (TGR) appears to have a long-lasting effect and may be a viable solution to treat muscle disorders because single or at least rarely repeated therapies would be used. For the best results, siRNA should be delivered to all disease affected muscles, and systemic delivery of siRNA through blood vessels is probably the only applicable choice to achieve this goal. Unfortunately, there are many challenges to overcome such as siRNA degradation in blood, renal clearance, blood-muscle barrier, cell entry and endosomal escape. By exploiting and considering the unique features of muscles and the mechanism of TGR, we will discuss the possible ways to induce TGR in muscles by using non-viral systemic siRNA delivery methods.

5-Hydroxy-1,4-naphthalenedione exerts anticancer effects on glioma cells through interaction with the mitochondrial electron transport chain.

Survival of patients with glioblastoma remains within the range of several months despite advances in therapeutic options. We have already shown that 5-hydroxy-1,4-naphthalenedione (juglone) exerts antiproliferative, anti-invasive, and cytotoxic effects on glioma C6 cells. Here, we further revealed that juglone is relatively selective to glioma cells as compared to the normal glial culture, and investigated its mechanisms of action. The incubation of glioma C6 cells with juglone generated high levels of reactive oxygen species (ROS). The produced ROS accounted for the anticancer effect of juglone as antioxidant N-acetylcysteine reduced both cytotoxic and antiproliferative activities of juglone. Furthermore, high resolution respirometry revealed that juglone decreased oxygen consumption mainly by affecting pyruvate/malate- and glutamate/malate-induced mitochondrial respiration. The inhibition of respiratory complex I by amytal decreased juglone-triggered generation of ROS and diminished its anticancer activity. Thus, our results indicate that juglone generates ROS through interaction with respiratory complex I in glioma C6 cells, and, in turn, induces the anticancer effects.

Neuroprotective properties of anthocyanidin glycosides against H2O2-induced glial cell death are modulated by their different stability and antioxidant activity in vitro.

The neuroprotective effect of several anthocyanins in combination with their stability and antioxidant/pro-oxidant activity has been investigated against H2O2-induced oxidative stress in C6 glial cells. First it was found that delphinidin (Dp) 3-O-glucoside and 3-O-rutinoside were degraded within an hour, and at the same time stimulated the production of H2O2 in the micromolar concentration range. The stability of peonidin, pelargonidin (Pg), malvidin (Mv) and cyanidin (Cy) 3-O-glucosides and Cy 3-O-rutinoside was significantly higher than that of Dp 3-O-glycosides, with Pg3G showing the highest percent recovery over time. Based on these findings and chemical difference (according to the set of functional groups on the B-ring) of tested anthocyanins Cy3G, Mv3G and Pg3G were selected as candidates for the protection of glial cells against H2O2-induced oxidative stress. It was revealed that Cy3G (5-20µM) and Mv3G (10-20µM) but not Pg3G protected glial cells against H2O2-induced necrotic cell death. Moreover, these anthocyanins sustained the glutathione antioxidant defence system. Finally, to the extent of our knowledge we were the first to demonstrate the protective effect of Cy3G on the resting mitochondrial respiration rate in H2O2-affected glial cells. The results suggest that Cy3G, as the most prominent antioxidant among tested anthocyanins, could be a potential adjuvant for the prevention or reduction of necrotic glial cell death during the oxidative stress conditions met in neurodegenerative diseases. However, further elucidation of other possible mechanisms for anthocyanins to protect the nervous system is encouraged.

A user-friendly PC-based data acquisition and analysis system for respirometric investigations.

We have developed an easy-to-use computer-based system for recording, displaying, storing and analyzing signals generated by Clark-type oxygen electrodes. A user-friendly interface of Windows-based program BioMed significantly increases the productivity of investigations. It allows to process, control, present and archive the experimental data in real time. A 12-bit analog-to-digital-converter, analog and digital filters, a possibility to zoom the obtained respiratory curves and calculation of the respiration rates by a linear regression method increase the resolution of the estimated oxygen consumption rates. The new system enables to register even small changes, such as 3-5 ngatoms O/min, in respiration rates of biological objects -- enzymes, mitochondria and permeabilized muscle fibers. The system has been developed and is regularly used for the respirometric investigations at the Laboratory of Biochemistry, Institute for Biomedical Research, Kaunas University of Medicine.

Juglone Exerts Cytotoxic, Anti-proliferative and Anti-invasive Effects on Glioblastoma Multiforme in a Cell Culture Model.

In this study we evaluated the impact of juglone on rat glioma C6 cell culture viability, proliferation and invasiveness in vitro. Juglone induced C6 cell death with EC50 concentrations equal to 10.4 ± 1.6 µM after 24h incubation. At relatively low concentrations juglone significantly decreased cell proliferation, reduced spheroid invasiveness and suppressed "wound" healing. In addition, generation of intracellular reactive oxygen and nitrogen species (RS) was detected in cells treated with juglone. Noteworthy, juglone was relatively stable in cell culture medium and levels of H2O2 generated from juglone due to its probable reaction with medium components were not sufficient to affect the viability of glioma cells. Moreover, addition of catalase to the cell medium did not reduce the cytotoxicity of juglone. Therefore, we propose that cell death may be induced through the action of RS other than H2O2, However the direct effect of juglone on the cellular targets could not be excluded either. In conclusion, juglone exerted cytotoxic, anti-proliferative and anti-invasive effects on C6 rat glioma cells in vitro.