Pharmacological Akt and JNK Kinase Inhibitors 10-DEBC and SP600125 Potentiate Anti-Glioblastoma Effect of Menadione and Ascorbic Acid Combination in Human U251 Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, characterized by a highly invasive nature and therapy resistance. Combination of menadione and ascorbic acid (AA+MD) exerts strong ROS-mediated anti-GBM activity in vitro. The objective of this study was to improve AA+MD anti-GBM potential by modulating the activity of Akt and c-Jun N-terminal kinase (JNK), molecules with an important role in GBM development. The effects of Akt and JNK modulation on AA+MD toxicity in U251 human glioblastoma cells were assessed by cell viability assays, flow cytometry, RNA interference and plasmid overexpression, and immunoblot analysis. The AA+MD induced severe oxidative stress, an early increase in Akt phosphorylation followed by its strong inhibition, persistent JNK activation, and U251 cell death. Small molecule Akt kinase inhibitor 10-DEBC enhanced, while pharmacological and genetic Akt activation decreased, AA+MD-induced toxicity. The U251 cell death potentiation by 10-DEBC correlated with an increase in the combination-induced autophagic flux and was abolished by genetic autophagy silencing. Additionally, pharmacological JNK inhibitor SP600125 augmented combination toxicity toward U251 cells, an effect linked with increased ROS accumulation. These results indicate that small Akt and JNK kinase inhibitors significantly enhance AA+MD anti-GBM effects by autophagy potentiation and amplifying deleterious ROS levels.

The Exploitation of Lysosomes in Cancer Therapy with Graphene-Based Nanomaterials.

Graphene-based nanomaterials (GNMs), including graphene, graphene oxide, reduced graphene oxide, and graphene quantum dots, may have direct anticancer activity or be used as nanocarriers for antitumor drugs. GNMs usually enter tumor cells by endocytosis and can accumulate in lysosomes. This accumulation prevents drugs bound to GNMs from reaching their targets, suppressing their anticancer effects. A number of chemical modifications are made to GNMs to facilitate the separation of anticancer drugs from GNMs at low lysosomal pH and to enable the lysosomal escape of drugs. Lysosomal escape may be associated with oxidative stress, permeabilization of the unstable membrane of cancer cell lysosomes, release of lysosomal enzymes into the cytoplasm, and cell death. GNMs can prevent or stimulate tumor cell death by inducing protective autophagy or suppressing autolysosomal degradation, respectively. Furthermore, because GNMs prevent bound fluorescent agents from emitting light, their separation in lysosomes may enable tumor cell identification and therapy monitoring. In this review, we explain how the characteristics of the lysosomal microenvironment and the unique features of tumor cell lysosomes can be exploited for GNM-based cancer therapy.

Dual anticancer role of metformin: an old drug regulating AMPK dependent/independent pathways in metabolic, oncogenic/tumorsuppresing and immunity context.

Metformin has been known to treat type 2 diabetes for decades and is widely prescribed antidiabetic drug. Recently, its anticancer potential has also been discovered. Moreover, metformin has low cost thus it has attained profound research interest. Comprehensing the complexity of the molecular regulatory networks in cancer provides a mode for advancement of research in cancer development and treatment. Metformin targets many pathways that play an important role in cancer cell survival outcome. Here, we described anticancer activity of metformin on the AMPK dependent/independent mechanisms regulating metabolism, oncogene/tumor suppressor signaling pathways together with the issue of clinical studies. We also provided brief overwiev about recently described metformin's role in cancer immunity. Insight in these complex molecular networks, will simplify application of metformin in clinical trials and contribute to improvement of anti-cancer therapy.

Comparative analysis of cell death mechanisms induced by lysosomal autophagy inhibitors.

We performed a comparative analysis of molecular cytotoxic mechanisms of lysosomal autophagy inhibitors bafilomycin A1, chloroquine, and ammonium chloride in B16 mouse melanoma cells. All agents caused oxidative stress, mitochondrial depolarization, and caspase-dependent apoptotic death, which was not affected by genetic inactivation of autophagy. Cathepsin inhibition reduced only the cytotoxicity of chloroquine, indicating its ability to cause lysosomal membrane permeabilization. Bafilomycin reduced the mRNA levels of anti-apoptotic Bcl-2, while chloroquine and ammonium chloride increased the mRNA expression of pro-apoptotic Pten and Puma, as well as anti-apoptotic Bcl-xL. Ammonium chloride additionally increased the mRNA expression of pro-apoptotic Bim and p53. All three agents decreased the activity of mechanistic target of rapamycin (mTOR) and increased the activation of p38 mitogen-activated protein kinase (MAPK). Chloroquine and ammonium chloride additionally stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), respectively, while only bafilomycin increased the phosphorylation of the energy sensor AMP-activated protein kinase (AMPK). mTOR activator leucine did not affect the cytotoxicity of lysosomal inhibitors. p38 MAPK inhibitor SB203580 reduced the cytotoxicity of bafilomycin but increased that of chloroquine and ammonium chloride. The pharmacological inhibition of ERK1/2, JNK, and AMPK potentiated the cytotoxicity of chloroquine, ammonium chloride, and bafilomycin, respectively. The observed mechanistic differences were associated with antagonistic interactions of lysosomal inhibitors in B16 cell killing. In conclusion, all investigated lysosomal inhibitors cause autophagy-independent mitochondrial dysfunction and apoptotic death, but differ in the ability to affect lysosomal permeabilization, balance between pro- and anti-apoptotic molecules of Bcl-2 family, and MAPK/AMPK signaling.

Autophagy-independent increase of ATG5 expression in T cells of multiple sclerosis patients.

Autophagy, a process of controlled self-digestion which regulates cell homeostasis, is involved in innate and adaptive immunity. We investigated the expression of autophagy genes and autophagic activity in distinct lymphocyte populations in treatment-naive MS patients. The mRNA and protein levels of autophagy-related (ATG)5, required for autophagosome formation, were increased in CD4+ and CD4- T cells, but not B cells of MS patients compared to control subjects. The expression of other investigated autophagy genes, as well as the autophagic activity, did not significantly differ between the two groups. ATG5 mRNA levels in CD4+ T cells from MS patients were positively correlated with those of the proinflammatory cytokine tumor necrosis factor. These data suggest that autophagy-independent increase in ATG5 expression might be associated with the proinflammatory capacity of T cells in multiple sclerosis.

Graphene quantum dots suppress proinflammatory T cell responses via autophagy-dependent induction of tolerogenic dendritic cells.

Graphene quantum dots (GQD) are atom-thick nanodimensional carbon sheets with excellent physico-chemical and biological properties, making them attractive for application in theranostics. However, their immunoregulatory properties are insufficiently investigated, especially in human primary immune cells. We found that non-toxic doses of GQD inhibit the production of proinflammatory and T helper (Th)1 cytokines, and augment the production of anti-inflammatory and Th2 cytokines by human peripheral blood mononuclear cells. While unable to affect T cells directly, GQD impaired the differentiation and functions of monocyte-derived dendritic cells (DC), lowering their capacity to stimulate T cell proliferation, development of Th1 and Th17 cells, and T-cell mediated cytotoxicity. Additionally, GQD-treated DC potentiated Th2 polarization, and induced suppressive CD4+CD25highFoxp3+ regulatory T cells. After internalization in a dynamin-independent, cholesterol-dependent manner, GQD lowered the production of reactive oxygen species and nuclear translocation of NF-κB in DC. The activity of mammalian target of rapamycin (mTOR) was reduced by GQD, which correlated with the increase in transcription of autophagy genes and autophagic flux in DC. Genetic suppression of autophagy impaired the pro-tolerogenic effects of GQD on DC. Our results suggest that GQD-triggered autophagy promotes tolerogenic functions in monocyte-derived DC, which could be beneficial in inflammatory T-cell mediated pathologies, but also harmful in GQD-based anti-cancer therapy.

Downregulation of autophagy gene expression in endometria from women with polycystic ovary syndrome.

Autophagy, a process of controlled cellular self-digestion, could be involved in cyclic remodeling of the human endometrium. We investigated endometrial mRNA expression of 23 autophagy-related (ATG) genes and transcription factors in healthy controls (n = 12) and anovulatory polycystic ovary syndrome (PCOS) patients (n = 24), as well as in their subgroup (n = 12) before and after metformin treatment. The mRNA levels of transcription factor forkhead box protein O1 (FOXO1) and several molecules involved in autophagosome formation (ATG13, RB1-inducible coiled-coil 1), autophagosome nucleation (ATG14, beclin 1, SH3-domain GRB2-like endophilin B1), autophagosome elongation (ATG3, ATG5, γ-aminobutyric acid receptor-associated protein - GABARAP), and delivery of ubiquitinated proteins to autophagosomes (sequestosome 1), were significantly reduced in anovulatory PCOS compared to healthy endometrium. Free androgen index, but not free estrogen index, insulin levels, or body mass index, negatively correlated with the endometrial expression of ATG3, ATG14, and GABARAP in PCOS patients. Treatment of PCOS patients with metformin (2 g/day for 3 months) significantly increased the endometrial mRNA levels of FOXO1, ATG3, and UV radiation resistance-associated gene. These data suggest that increased androgen availability in PCOS is associated with metformin-sensitive transcriptional downregulation of endometrial autophagy.

Effects of IL-33/ST2 pathway in acute inflammation on tissue damage, antioxidative parameters, magnesium concentration and cytokines profile.

AIM: The aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue. MATERIAL AND METHODS: Male mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. RESULTS: Presence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I. CONCLUSION: The results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, ß-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and ß-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.

Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling.

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-kinases (S6K) in the hypothalamus is thought to be involved in nutrient sensing and control of food intake. Given the anatomical proximity of this pathway to circuits for the hormone ghrelin, we investigated the potential role of the mTORC1/S6K pathway in mediating the metabolic effects of ghrelin. We found that ghrelin promoted phosphorylation of S6K1 in the mouse hypothalamic cell line N-41 and in the rat hypothalamus after intracerebroventricular administration. Rapamycin, an inhibitor of mTORC1, suppressed ghrelin-induced phosphorylation of hypothalamic S6K1 and increased food intake and insulin in rats. Chronic peripheral administration of ghrelin induced a significant increase in body weight, fat mass and food efficiency in wild-type and S6K2-knockout but not in S6K1-knockout mice. We therefore propose that ghrelin-induced hyperphagia, adiposity and insulin secretion are controlled by a central nervous system involving the mTORC1/S6K1 pathway.

Arylpiperazine-mediated activation of Akt protects SH-SY5Y neuroblastoma cells from 6-hydroxydopamine-induced apoptotic and autophagic death.

We investigated the ability of 19 recently synthesized arylpiperazine compounds to protect human SH-SY5Y neuroblastoma cells from the neurotoxin 6-hydroxydopamine (6-OHDA). The compound with the most potent neuroprotective action was N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), which reduced 6-OHDA-induced apoptotic death through stabilization of mitochondrial membrane and subsequent prevention of superoxide production, caspase activation and DNA fragmentation. 6-OHDA-triggered autophagic response was also reduced by 6b, which prevented inactivation of the main autophagy repressor mTOR, upregulation of proautophagic beclin-1, conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, as well as intracytoplasmic acidification induced by 6-OHDA. The inhibition of autophagy using LC3ß gene silencing or pharmacological autophagy blockers 3-methyladenine or bafilomycin A1, mimicked the cytoprotective effect of 6b. While the treatment with 6b had no effect on the phosphorylation of proapoptotic MAP kinases ERK and JNK, it markedly increased the phosphorylation of the prosurvival kinase Akt in 6-OHDA-treated cells. Akt inhibitor DEBC or RNA interference-mediated Akt silencing reduced the ability of 6b to block 6-OHDA-triggered apoptotic and autophagic responses, thus confirming their dependency on Akt activation. The cytoprotective effect of 6b was also observed in 6-OHDA-treated neuronal PC12 cells, but not in SH-SY5Y or PC12 cells exposed to 1-methyl-4-phenylpyridinium, indicating that the observed neuroprotection was dependent on the cytotoxic stimulus. Because of the ability to prevent 6-OHDA induced apoptotic/autophagic cell death through activation of Akt, the investigated arylpiperazines could be potential candidates for treatment of neurodegenerative diseases.

Therapeutic improvement of glucoregulation in newly diagnosed type 2 diabetes patients is associated with a reduction of IL-17 levels.

We explored the effect of therapeutic glucoregulation on the blood levels of proinflammatory T helper (Th)17 cytokines interleukin (IL)-17 and IL-23, and Th1 cytokines interferon (IFN)-γ and IL-12 in newly diagnosed type 2 diabetes patients. The investigated group consisted of 23 subjects (17 men and 6 women, age 26-64). The cytokine serum levels, glycated hemoglobin (HbA1c) as a marker of glucoregulation, homeostasis model assessment index as a measure of insulin resistance (HOMA-IR), and body mass index (BMI) were determined before and after 12 weeks of therapy consisting of standard lifestyle modification and metformin (1000 mg b.i.d.). The levels of Th17 and Th1 cytokines before treatment did not correlate with age, BMI or HOMA-IR. The patients with poor glucoregulation (HbA1c>7%, n=12), compared to those with good glucoregulation (HbA1c≤7%, n=11), had higher serum levels of Th17 and Th1 cytokines, but only the differences in IL-17 (median 21.2 pg/ml vs. 4.8 pg/ml) and IFN-γ 5 (0.6 pg/ml vs. 27.7 pg/ml) reached statistical significance (p=0.003 and p=0.012, respectively). The reduction of HbA1c values (from 8.6 to 5.9%, p=0.000) observed upon treatment in patients with poor glucoregulation was associated with a significant decrease in the concentration of IL-17 (from 21.2 to 12.9 pg/ml, p=0.020), but not IFN-γ (50.6 vs. 52.3, p=0.349). These data indicate that therapeutic improvement of glucoregulation might contribute to a reduction of IL-17 levels in newly diagnosed type 2 diabetes patients.

Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells.

We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-II, upregulation of proautophagic beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3ß, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Akt inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis.

Chloroquine-mediated lysosomal dysfunction enhances the anticancer effect of nutrient deprivation.

PURPOSE: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. METHODS: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. RESULTS: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. CONCLUSION: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.

Intracerebroventricular administration of metformin inhibits ghrelin-induced Hypothalamic AMP-kinase signalling and food intake.

BACKGROUND/AIMS: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. METHODS: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. RESULTS: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. CONCLUSION: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.

Arylpiperazine dopamineric ligands protect neuroblastoma cells from nitric oxide (NO)-induced mitochondrial damage and apoptosis.

The protective ability of novel arylpiperazine-based dopaminergic ligands against nitric oxide (NO)-mediated neurotoxicity is investigated. The most potent neuroprotective arylpiperazine identified during the study was N-{4-[2-(4-phenyl-piperazin-1-yl)ethyl]-phenyl}picolinamide, which protected SH-SY5Y human neuron-like cells from the proapoptotic effect of NO donor sodium nitroprusside (SNP) by decreasing oxidative stress, mitochondrial membrane depolarization, caspase activation and subsequent phosphatydilserine externalization/DNA fragmentation. The protective effect was associated with the inhibition of proapoptotic (JNK, ERK, AMPK) and activation of antiapoptotic (Akt) signaling pathways, in the absence of interference with intracellular NO accumulation. The neuroprotective action of arylpiperazines was shown to be independent of dopamine receptor binding, as it was not affected by the high-affinity D1/D2 receptor blocker butaclamol. These results reported support the further study of arylpiperazines as potential neuroprotective agents.

Immunomodulatory actions of central ghrelin in diet-induced energy imbalance.

We investigated the effects of centrally administered orexigenic hormone ghrelin on energy imbalance-induced inflammation. Rats were subjected for four weeks to three different dietary regimes: normal (standard food), high-fat (standard food with 30% lard) or food-restricted (70%, 50%, 40% and 40% of the expected food intake in 1st, 2nd, 3rd and 4th week, respectively). Compared to normal-weight controls, starved, but not obese rats had significantly higher levels of proinflammatory cytokines (TNF, IL-1ß, IFN-γ) in the blood. When compared to normally fed animals, the hearts of starved and obese animals expressed higher levels of mRNAs encoding proinflammatory mediators (TNF, IL-1ß, IL-6, IFN-γ, IL-17, IL-12, iNOS), while mRNA levels of the anti-inflammatory TGF-ß remained unchanged. Intracerebroventricular (ICV) injection of ghrelin (1 µg/day) for five consecutive days significantly reduced TNF, IL-1ß and IFN-γ levels in the blood of starved rats, as well as TNF, IL-17 and IL-12p40 mRNA expression in the hearts of obese rats. Conversely, ICV ghrelin increased the levels of IFN-γ, IL-17, IL-12p35 and IL-12p40 mRNA in the heart tissue of food-restricted animals. This was associated with an increase of immunosuppressive ACTH/corticosterone production in starved animals and a decrease of the immunostimulatory adipokine leptin both in food-restricted and high-fat groups. Ghrelin activated the energy sensor AMP-activated protein kinase (AMPK) in the hypothalamus and inhibited extracellular signal-regulated kinase (ERK) in the hearts of obese, but not starved rats. Therefore, central ghrelin may play a complex role in energy imbalance-induced inflammation by modulating HPA axis, leptin and AMPK/ERK signaling pathways.

Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin.

The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR), a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3ß shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-dependent autophagic response might sensitize glioma cells to statin-induced apoptotic death.

In vitro and in vivo anti-melanoma action of metformin.

The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G(2)/M cell cycle arrest associated with apoptotic death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. Treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21- and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats

No disponible

The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 ìl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin (AU)