The Interaction of Selective A1 and A2A Adenosine Receptor Antagonists with Magnesium and Zinc Ions in Mice: Behavioural, Biochemical and Molecular Studies.

The purpose of the study was to investigate whether the co-administration of Mg2+ and Zn2+ with selective A1 and A2A receptor antagonists might be an interesting antidepressant strategy. Forced swim, tail suspension, and spontaneous locomotor motility tests in mice were performed. Further, biochemical and molecular studies were conducted. The obtained results indicate the interaction of DPCPX and istradefylline with Mg2+ and Zn2+ manifested in an antidepressant-like effect. The reduction of the BDNF serum level after co-administration of DPCPX and istradefylline with Mg2+ and Zn2+ was noted. Additionally, Mg2+ or Zn2+, both alone and in combination with DPCPX or istradefylline, causes changes in Adora1 expression, DPCPX or istradefylline co-administered with Zn2+ increases Slc6a15 expression as compared to a single-drug treatment, co-administration of tested agents does not have a more favourable effect on Comt expression. Moreover, the changes obtained in Ogg1, MsrA, Nrf2 expression show that DPCPX-Mg2+, DPCPX-Zn2+, istradefylline-Mg2+ and istradefylline-Zn2+ co-treatment may have greater antioxidant capacity benefits than administration of DPCPX and istradefylline alone. It seems plausible that a combination of selective A1 as well as an A2A receptor antagonist and magnesium or zinc may be a new antidepressant therapeutic strategy.

Cytotoxic activity of methanolic fractions of different Marrubium spp. against melanoma cells is independent of antioxidant activity and total phenolic content.

The Marrubium genus (horehound) has proved to be an abundant source of biologically active compounds, but there is little knowledge about its potential anticancer activity. Moreover, some Marrubium species have not been the subject of study in this regard. In this study, we performed comparative analysis of phenolic acid (PhA) content and total phenolic content in fractions obtained from methanolic extracts of Marrubium vulgare L. (common horehound), Marrubium cylleneum Boiss. & Heldr. and Marrubium friwaldskyanum Boiss herbs. We examined the cytotoxicity of these fractions against a human melanoma cancer cell line (A375) and normal human skin fibroblasts (BJ) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test, cell cycle analysis and real-time monitoring of cell viability. We detected caffeic, p-coumaric, ferulic and gentisic acids among the PhAs. Although the extracts obtained demonstrated low total phenolic content and did not show significant antioxidative properties, the nonhydrolyzed PhA fraction exhibited cytotoxic activity against a human melanoma cancer cell line, without affecting normal fibroblasts. Both acidic and alkaline hydrolysis abolished this activity, indicating that the esterified forms of phenolic compounds caused the observed cytotoxic effects. Further investigation of these compounds may facilitate the development of novel drugs for cancer treatment.

Ethanol extracts of Allium sp. regulate cyclooxygenase-2 and E-cadherin expression in gastric cancer MKN74 cell line and enhance doxorubicin toxicity.

BACKGROUND: Gastric cancer (GC) remains one of the leading causes of cancer-related death. Its aetiology is multifactorial, but the major risk factor is a high in salt diet. During gastric carcinogenesis, cadherin-1 (CDH1) down-expression and cyclooxygenase 2 (COX2) overexpression may be observed. The intensity of these alterations contributes to the GC invasion, its metastases and poor prognosis. As the diet plays a significant role in the aetiology of GC, it is reasonable to include the nutritional chemoprevention agents. One of the plant genus demonstrating chemoprotective properties is Allium genus, which includes garlic. The relationship between CDH1 and COX2 in GC cells treated with Allium species extract has never been evaluated. METHODS: In this study, the MKN28 and MKN74 GC cell lines were treated with ethanol extracts of Allium angulosum L., Allium lusitanicum Lam., Allium sativum L. (from Malaysia and Poland), Allium tibeticum Rendle and Allium ursinum L. The cytotoxicity of the extracts and their influence on COX2 and CDH1 mRNA and protein expression were evaluated as well as their influence on doxorubicin's (DOX) efficacy - a drug that has been used in GC treatment. RESULTS: Among the tested species, ethanol extracts of A. sativum L. (Poland and Malaysia), A. tibeticum Rendle and A. ursinum L. influenced the levels of CDH1 and COX2, but only in the MKN74 cell line. Thus, it is possible that tumours with increased COX2 expression will be more susceptible to garlic treatment. Observed phenomenon was independent of Allium extract's toxicity. In comparison to DOX, tested extracts were more toxic. Moreover, A. sativum revealed synergistic effect with the drug. CONCLUSION: In conclusion, the results indicate the potential application of Allium genus to GC chemoprevention and treatment support through CDH restoration and COX2 downregulation. This issue needs further investigations as it might be used in clinics.

Edaravone presents antidepressant-like activity in corticosterone model of depression in mice with possible role of Fkbp5, Comt, Adora1 and Slc6a15 genes.

Chronic exposure to environmental-like stress leads to dysregulation of hypothalamic-pituitary-adrenal (HPA) axis and to appearance of oxidative stress, which is implicated in the development of depression-like behaviour. Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) exhibits a neuroprotective effect attributed to the potent free radical scavenging. This study was designed to assess antidepressant-like activity of edaravone based on behavioural tests in the animal model of depression. Furthermore, to elucidate its mechanisms, the expression of Fkbp5, Comt, Adora and Slc6a15 genes involved in turnover of neurotransmitters was analysed. In order to evaluate the antioxidant features of edaravone, DNA's oxidative damage was determined. The mice were injected subcutaneously (sc) with 40 mg/kg corticosterone, chronically for 21 days. Paroxetine (10 mg/kg) (a selective serotonin reuptake inhibitor) and edaravone (10 mg/kg) were administered separately (ip) 30 min prior to the corticosterone injection. After 21-days of treatment with respective drugs, the mice were decapitated and the prefrontal cortex was rapidly dissected and used for determination of DNA's oxidative damage and the real-time PCR analysis. Edaravone ameliorated behavioural impairments in sucrose preference test (SPT) and forced swim test (FST). A possible role in Fkbp5, Comt, Adora1 and Slc6a15 genes' expression in mediating this effect is postulated. Both edaravone and paroxetine have no effect on corticosterone-induced DNA's oxidative damage.

Apigenin and hesperidin augment the toxic effect of doxorubicin against HepG2 cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies, with an increasing incidence. Despite the fact that systematic chemotherapy with a doxorubicin provides only marginal improvements in survival of the HCC patients, the doxorubicin is being used in transarterial therapies or combined with the target drug - sorafenib. The aim of the study was to evaluate the effect of natural flavonoids on the cytotoxicity of the doxorubicin against human hepatocellular carcinoma cell line HepG2. METHODS: The effect of apigenin and its glycosides - cosmosiin, rhoifolin; baicalein and its glycosides - baicalin as well as hesperetin and its glycosides - hesperidin on glycolytic genes expression of HepG2 cell line, morphology and cells' viability at the presence of doxorubicin have been tested. In an attempt to elucidate the mechanism of observed results, the fluorogenic probe for reactive oxygen species (ROS), the DNA oxidative damage, the lipid peroxidation and the double strand breaks were evaluated. To assess impact on the glycolysis pathway, the mRNA expression for a hexokinase 2 (HK2) and a lactate dehydrogenase A (LDHA) enzymes were measured. The results were analysed statistically with the one-way analysis of variance (ANOVA) and post hoc multiple comparisons. RESULTS: The apigenin and the hesperidin revealed the strongest effect on the toxicity of doxorubicin. Both flavonoids simultaneously changed the expression of the glycolytic pathway genes - HK2 and LDHA, which play a key role in the Warburg effect. Although separate treatment with doxorubicin, apigenin and hesperidin led to a significant oxidative DNA damage and double strand breaks, simultaneous administration of doxorubicin and apigenin or hesperidin abolished these damage with the simultaneous increase in the doxorubicin toxicity. CONCLUSION: The obtained results indicate the existence of a very effective cytotoxic mechanism in the HepG2 cells of the combined effect of doxorubicin and apigenin (or hesperidin), not related to the oxidative stress. To explain this synergy mechanism, further research is needed, The observed intensification of the cytotoxic effect of doxorubicin by this flavonoids may be a promising direction of the research on the therapy of hepatocellular carcinoma, especially in a chemoembolization.

Inhibition of glycolysis disrupts cellular antioxidant defense and sensitizes HepG2 cells to doxorubicin treatment.

Increased glucose consumption is a known hallmark of cancer cells. Increased glycolysis provides ATP, reducing agents and substrates for macromolecular synthesis in intensely dividing cells. Therefore, inhibition of glycolysis is one strategy in anticancer therapy as well as in improved efficacy of conventional anticancer chemotherapeutic agents. One such agent is doxorubicin (DOX), but the mechanism of sensitization of tumor cells to DOX by inhibition of glycolysis has not been fully elucidated. As oxidative stress is an important phenomenon accompanying DOX action and antioxidant defense is closely related to energy metabolism, the aim of the study was the evaluation of oxidative stress markers and antioxidant abilities of cancer cells treated with DOX while glycolysis is inhibited. HepG2 cells were treated with DOX and one of three glycolysis inhibitors: 2-deoxyglucose, dichloroacetate or 3-promopyruvate. To evaluate the possible interaction mechanisms, we assessed mRNA expression of selected genes related to energy metabolism and antioxidant defense; oxidative stress markers; and reduced glutathione (GSH) and NADPH levels. Additionally, glutamine consumption was measured. It was demonstrated that the chemotherapeutic agent and glycolysis inhibitors induced oxidative stress and associated damage in HepG2 cells. However, simultaneous treatment with both agents resulted in even greater lipid peroxidation and a significant reduction in GSH and NADPH levels. Moreover, in the presence of the drug and an inhibitor, HepG2 cells had a reduced ability to take up glutamine. These results indicated that cells treated with DOX while glycolysis was inhibited had significantly reduced ability to produce NADPH and antioxidant defenses.

Altered expression of genes involved in brain energy metabolism as adaptive responses in rats exposed to chronic variable stress; changes in cortical level of glucogenic and neuroactive amino acids.

Brain metabolism is closely associated with neuronal activity and enables the accurate synthesis and function of neurotransmitters. Although previous studies have demonstrated that chronic stress is associated with the overproduction of reactive oxygen species (ROS), which leads to oxidative stress and the disruption of glucose metabolism, the molecular mechanisms and cerebral gluconeogenesis in depression have not yet been completely elucidated. In order to examine this subject, the present study evaluated changes in the expression of selected genes involved in the glycolytic pathway and the levels of glucogenic and neuroactive amino acids in the brain of rats exposed to chronic variable stress. Male Wistar rats (50­55 days old, weighing 200­250 g) were divided into two groups: control and stressed, and the rats in the stressed group were exposed to stress conditions for 40 days. Depressive­like states were observed and recorded by measuring the body weight and forced swim test (FST). The mRNA levels of Slc2a3 (coding GLUT3) and Tfam (activator of mitochondrial transcription and a participant in mitochondrial genome replication) were markedly increased, while a decrease in the expression of Ldhb and GAPDH was also observed. These modifications were associated with the redirection of glucose metabolism to appropriate defensive pathways under chronic stress conditions, and an increased ability to maintain mitochondrial function as potential adaptive responses. A marked reduction of glucogenic and neuroactive amino acids levels indicate the support of energy metabolism by stimulation of the gluconeogenesis pathway. The findings of the present study provide a novel insight into the molecular and biochemical events that impact the development of depression under chronic stress conditions, and they may identify novel targets for therapeutic intervention.

Withdrawal of caffeine after its chronic administration modifies the antidepressant-like activity of atypical antidepressants in mice. Changes in cortical expression of Comt, Slc6a15 and Adora1 genes.

RATIONALE: Depressed patients often present increased consumption of caffeine. OBJECTIVES: We aimed to investigate the effects of chronic treatment with caffeine (5 mg/kg, twice daily for 14 days) on the activity of single, ineffective doses of agomelatine (20 mg/kg) or mianserin (10 mg/kg) given on day 15 alone or simultaneously with caffeine. METHODS: We used the forced swim test (FST), tail suspension test (TST), and locomotor activity test in mice and quantitative real-time PCR analysis of the selected genes in the cerebral cortex (Cx). RESULTS: There were no changes in the immobility time between mice that received saline and caffeine for 14 days. Administration of agomelatine or mianserin on day 15 did not produce an antidepressant-like effect, but such effect was observed after administration of agomelatine or mianserin simultaneously with caffeine on day 15, in both mice that received saline and caffeine for 14 days. In mice treated with caffeine for 14 days, joint administration of agomelatine or mianserin and caffeine on day 15 decreased solute carrier family 6, member 15 (Slc6a15), messenger RNA (mRNA) level in the Cx, compared to the group which received only the respective antidepressant on this day. Moreover, in mice treated with caffeine for 14 days, joint administration of mianserin and caffeine on day 15 decreased adenosine A1 receptor (Adora1) and catechol-O-methyltransferase (Comt) mRNA level in the Cx, compared to the group which received mianserin without caffeine on this day. CONCLUSIONS: Withdrawal of caffeine after its chronic intake can modify the activity of antidepressants. Adora1, Slc6a15, and Comt may be involved in the antidepressant-like effect observed after joint administration of caffeine and mianserin or agomelatine, following chronic treatment with caffeine.

Chronic treatment with caffeine and its withdrawal modify the antidepressant-like activity of selective serotonin reuptake inhibitors in the forced swim and tail suspension tests in mice. Effects on Comt, Slc6a15 and Adora1 gene expression.

Recent preclinical and clinical data suggest that low dose of caffeine enhances the effects of common antidepressants. Here we investigated the effects of chronic administration of caffeine (5mg/kg, twice daily for 14days) and its withdrawal on day 15th on the activity of per se ineffective doses of fluoxetine (5mg/kg) and escitalopram (2mg/kg) given on day 15th. We found decreased immobility time in the forced swim and tail suspension tests in mice in which caffeine was administered simultaneously with antidepressants on day 15th following a 14-day caffeine treatment and no alterations in the spontaneous locomotor activity. A decrease in the level of escitalopram and an increase in the level of caffeine in serum were observed after concomitant administration of these compounds, while the joint administration of caffeine and fluoxetine was not associated with changes in their levels in serum or brain. Caffeine withdrawal caused a decrease in Adora1 mRNA level in the cerebral cortex (Cx). Administration of escitalopram or fluoxetine followed by caffeine withdrawal caused an increase in this gene expression, whereas administration of escitalopram, but not fluoxetine, on day 15th together with caffeine caused a decrease in Adora1 mRNA level in the Cx. Furthermore, antidepressant-like activity observed after joint administration of the tested drugs with caffeine was associated with decreased Slc6a15 mRNA level in the Cx. The results show that withdrawal of caffeine after its chronic intake may change activity of antidepressants with concomitant alterations within monoamine, adenosine and glutamate systems.

Evaluation of the phytochemical composition and protective activities of methanolic extracts of Centaurea borysthenica and Centaurea daghestanica (Lipsky) Wagenitz on cardiomyocytes treated with doxorubicin.

Centaurea L. is a genus of the family Asteraceae that comprises over 600 taxa. Representatives of the Centaurea genus were used as natural medications for many diseases. Methanolic-aqueous extracts from aerial parts of two Centaurea species: C. borysthenica Gruner and C. daghestanica (Lipsky) Wagenitz were studied for their polyphenolic composition and potential protective effect on cardiomyocytes treated with doxorubicin. Effectiveness of doxorubicin in cancer therapy is limited by a dose-dependent cardiotoxicity. Oxidative stress is a widely recognized mechanism of this phenomenon. One of the most important strategies has been an application of drug together with antioxidant agents. A cardioprotective effect of selected extracts of Centaurea species was suspected in this study. Cell viability, oxidative stress, and mitochondrial membrane potential analyses showed protective activity of the methanolic extract of C. borysthenica and C. daghestanica on rat cardiomyocytes treated with doxorubicin. Although C. borysthenica is more effective as a cardiomyocyte protective agent, in higher concentrations it weakened the drug activity. C. daghestanica extract did not change the doxorubicin efficacy in the evaluated experiment. Interestingly, both tested extracts were cytotoxic for myeloma cells. The detected antioxidant activity of the studied extracts can be used in the prevention of doxorubicin-induced cardiotoxicity.

The effect of a number of H9C2 rat cardiomyocytes passage on repeatability of cytotoxicity study results.

The embryonic cardiomyocyte cell line H9C2 is commonly used in numerous in vitro studies, including cardiotoxicity analyses of new drugs. So far no results were published for studies on cell parameters variability during the cell line ageing process. For this reason the aim of the study was to evaluate the effect of a number of H9C2 rat embryonic cardiomyocytes passages on repeatability of study results for selected cytotoxicity parameters, with doxorubicin as a model toxic agent. The cultures were passaged twenty-five times. Cells from passage 1, 5, 10, 15, 20 and 25 were treated with doxorubicin for 24 h. Then drug cytotoxicity was evaluated with the MTT test and additionally the nuclear factor erythroid 2-related factor (Nrf2) gene expression was examined. The analysis of oxidative stress intensity and cell morphology was also assessed. The microscopic appearance of cells indicates that untreated cardiomyocytes morphology changes as well as sensitivity to toxic effects increases with the number of passages. Also an increase in oxidative stress in cells occurs with further passaging of cardiomyocytes. Statistical significance of differences in conducted tests results depended on doxorubicin concentration but in many cases the H9C2 line was found to be a reliable in vitro model only for the first five passages. For this reason it is important to take into consideration that further culturing of cardiomyocytes may not ensure repeatability of study results due to the culture ageing.

The genus Milnesium Doyère, 1840 (Tardigrada) in South America with descriptions of two new species from Argentina and discussion of the feeding behaviour in the family Milnesiidae.

BACKGROUND: The diversity and distribution of the tardigrades in South America are rather poor and selective, as is information about their feeding behaviour and diet. To date, only ca. 210 tardigrade taxa have been reported from the region of South America. In the present paper, we provide an update of the distribution of the genus Milnesium inSouth America and discuss some aspects of the feeding behaviour in the family Milnesiidae. RESULTS: In seven moss samples collected in the Argentinean province of Río Negro, 31 specimens, 4 exuviae and 32 eggs belonging to the genus Milnesium were found. Among them, four species were identified: Milnesium argentinum sp. nov., Milnesium beatae sp. nov., Milnesium brachyungue and Milnesium granulatum. By its dorsal sculpture,M.argentinum sp.nov. is most similar to M.beatae sp.nov., Milnesiumbeasleyi andMilnesiumberladnicorum, butit differs from M.beasleyi andM.berladnicorum mainlyby having a different claw configuration and from M. beatae and M. beasleyi by having stylet supports inserted in a more caudal position and by some other morphometriccharacters. In the width of its buccal tube and the claw configuration [3-3]-[3-3], M.beatae sp.nov. is most similar to Milnesiumbohleberi,M.brachyungue andMilnesiumeurystomum,but it differs from them mainly by having a sculptured dorsal cuticle and by some other morphometric characters. CONCLUSIONS: The study discusses distribution and taxonomic problems of the Milnesium species known from South America. As of now, nine Milnesium taxa are known from this region (including two new species reported in this paper). Additionally, the study broadens our knowledge of tardigrades' feeding behaviour, provides some details about their diet and suggests that the type of prey chosen by some species belonging to the family Milnesiidae may be associated with the width of their buccal tube.

Adenosine ecto-deaminase (ecto-ADA) from porcine cerebral cortex synaptic membrane.

We have purified and investigated the role of adenosine ecto-deaminase (ecto-ADA) in porcine brain synaptic membranes and found a low activity of ecto-ADA in synaptic preparations from the cerebral cortex, hippocampus, striatum and medulla oblongata in the presence of purine transport inhibitors (NBTI, dipyridamole and papaverine). The purification procedure with affinity chromatography on epoxy-Toyopearl gel/purine riboside column as a crucial step of purification allowed a 214-fold purification of synaptic ecto-ADA with a yield of 30%. Gel filtration chromatography revealed a molecular mass estimated at 42.4+/-3.9 kDa. The enzyme had a broad optimum pH and was not affected by mono- and divalent cations. Ecto-ADA revealed a low affinity to adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) (K(M)=286.30+/-40.38 microM and 287.14+/-46.50 microM, respectively). We compared the affinity of ecto-ADA to the substrates with the physiological and pathological concentrations of the extracellular Ado in brains that do not exceed a low micromolar range even during ischemia and hypoxia, and with the affinity of adenosine receptors to Ado not exceeding a low nanomolar (A(1) and A(2A) receptors) or low micromolar (A(2B) and A(3)) range. Taken together, our data suggest that the role of synaptic ecto-ADA in the regulation of the ecto-Ado level in the brain and in the termination of adenosine receptor signaling is questionable. The porcine brain synapses must have other mechanisms for the ecto-Ado removal from the synaptic cleft and synaptic ecto-ADA may also play an extra-enzymatic role in cell adhesion and non-enzymatic regulation of adenosine receptor activity.