Human U373 MG Astrocytoma Cells Research Articles

JDP2 is directly regulated by ATF4 and modulates TRAIL sensitivity by suppressing the ATF4-DR5 axis.

Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.

Nitric Oxide/Cyclic GMP-Dependent Calcium Signalling Mediates IL-6- and TNF-\u03b1-Induced Expression of Glial Fibrillary Acid Protein

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca2+]i) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca2+]i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca2+]i, respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes.

Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 μM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.

Gnetin C suppresses double-stranded RNA-induced C-C motif chemokine ligand 2 (CCL2) and CCL5 production by inhibiting Toll-like receptor 3 signaling pathway.

The innate immune system is a prerequisite for biophylactic ability, but its dysregulation can cause inflammatory and autoimmune diseases. To determine a safe method of controlling inflammatory reactions in the brain, we examined the effects of gnetin C, a natural resveratrol dimer, on C-C motif chemokine ligand 2 (CCL2) and CCL5 (pro-inflammatory chemokines) production observed after treatment with polyinosinic-polycytidylic acid [poly IC; a synthetic analog of dsRNA as a Toll-like receptor 3 (TRL3) ligand, 30 μg/mL] in cultured human astrocytoma U373MG and neuroblastoma SH-SY5Y cells. The addition of gnetin C (10 μM) to the media moderately reduced the CCL2 production and markedly suppressed CCL5 production in both cells. In the TLR3-interferon (IFN)-β-phosphorylated-STAT1 (signal transducer and activator of transcription protein 1)RIG-I (retinoic acid-inducible gene-I) pathway that mediates CCL2 and CCL5 production, gnetin C first inhibits IFN-β expression in SH-SY5Y cells and primarily inhibits STAT1 phosphorylation in U373MG cells. In any case, gnetin C attenuated the dsRNA-activated TLR3 signaling resulting in CCL2 and CCL5 production, thus, may be useful for controlling TLR3-mediated inflammation in the brain.

Anti‑inflammatory actions of gabapentin and pregabalin on the substance P‑induced mitogen‑activated protein kinase activation in U373 MG human glioblastoma astrocytoma cells.

Gabapentin (GBP) and pregabalin (PGB) exert antinociceptive effects on chronic nociceptive responses with neuropathic or inflammatory conditions. Furthermore, it is considered that GBP and PGB exhibit anti‑inflammatory effects by modulating the substance P (SP)‑mediated neurokinin‑1 receptor (NK1R; a SP receptor) response. Thus, in the present study, the effects of GBP and PGB on SP‑induced activation were investigated in the human glioblastoma astrocytoma U373 MG cell line, which expresses high levels of functional high‑affinity NK1R, and produces interleukin (IL)‑6 and IL‑8 in response to SP. The results indicated that GBP and PGB suppressed the SP‑induced production of IL‑6, and IL‑8 in U373 MG cells. Furthermore, GBP and PGB inhibited the SP‑induced phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB, and the nuclear translocation of NF‑κB in U373 MG cells. Together, these observations suggest that GBP and PGB likely prevent SP‑induced IL‑6 and IL‑8 production in U373 MG cells via the inhibition of signaling molecules, including p38 MAPK and NF‑κB, thereby exhibiting antineuroinflammatory effects.

Interferon (IFN)-induced protein 35 (IFI35) negatively regulates IFN-β-phosphorylated STAT1-RIG-I-CXCL10/CCL5 axis in U373MG astrocytoma cells treated with polyinosinic-polycytidylic acid

Interferon (IFN)-stimulated genes (ISGs) exert multiple functions in immune system. IFN-induced protein 35 (IFI35) is a member of ISGs, and has been suggested to regulate innate immune reaction. However, the physiological functions and pathological roles of IFI35 in the central nervous system are not characterized well. In the present study, we found that the expression of IFI35 was induced by a Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid (poly IC) in U373MG human astrocytoma cells. Knockdown of IFI35 using RNA interference resulted in increased expression of IFN-β, phosphorylated STAT1 (P-STAT1), retinoic acid-inducible gene-I (RIG-I), CXCL10 and CCL5 induced by poly IC. Poly IC-induced expression of CXCL10 and CCL5 was decreased by knockdown of RIG-I. These results suggest that IFI35 may negatively regulate the TLR3-IFN-β-P-STAT1-RIG-I-CXCL10/CCL5 axis in U373MG cells, and IFI35 may play a role at least partially in the regulation of innate immune reactions in astrocytes.

Castanea sativa Mill. Bark Extract Protects U-373 MG Cells and Rat Brain Slices Against Ischemia and Reperfusion Injury.

Ischemic brain injury is one of the most important causes of death worldwide. The use of one-drug-multi-target agents based on natural compounds is a promising therapeutic option for cerebral ischemia due to their pleiotropic properties. This study assessed the neuroprotective properties of Castanea sativa Mill. bark extract (ENC) in human astrocytoma U-373 MG cells subjected to oxygen-glucose deprivation and reperfusion and rat cortical slices subjected to ischemia-like conditions or treated with glutamate or hydrogen peroxide. Neuroprotective effects were determined by assessing cells or slices viability (MTT assay), ROS formation (DCFH-DA assay), apoptosis (sub G0/G1 peak), nuclear fragmentation and chromatin condensation (DAPI staining) as well as changes in lysosomes and mitochondria morphology (Acridine Orange and Rhodamine123 staining, respectively). ENC treatment before injury on U-373 MG cells (5-50 μg/ml) and cortical slices (50-100 μg/ml) provided neuroprotection, while lower or higher concentrations (100 μg/ml U-373 MG cells, 200 μg/ml brain slices) were ineffective. ENC addition during reperfusion or after the injury was not found to be effective. The results suggest that ENC might hold potential as preventive neuroprotective agent, and indicate the importance of further studies exploring its mechanism of action. J. Cell. Biochem. 118: 839-850, 2017. © 2016 Wiley Periodicals, Inc.

Ligand anchored poly(propyleneimine) dendrimers for brain targeting: Comparative in vitro and in vivo assessment

The present investigation was aimed at developing various ligands-anchored dendrimers and comparing their brain targeting potential at one platform. Sialic acid (S), glucosamine (G) and concanavalin A (C) anchored poly(propyleneimine) (PPI) dendritic nanoconjugates were developed and evaluated for delivery of anti-cancer drug, paclitaxel (PTX) to the brain. MTT assay on U373MG human astrocytoma cells indicated IC50 values of 0.40, 0.65, 0.95, 2.00 and 3.50μM for PTX loaded SPPI, GPPI, CPPI, PPI formulations, and free PTX, respectively. The invivo pharmacokinetics and biodistribution studies in rats showed significantly higher accumulation of PTX in brain as compared to free PTX. The order of targeting potential of various ligands under investigation was found as sialic acid>glucosamine>concanavalin A. Thus, it can be concluded that sialic acid, glucosamine and Con A can be used as potential ligands to append PPI dendrimers for enhanced delivery of anticancer drugs to the brain for higher therapeutic outcome.

Desferrioxamine, an iron chelator, induces CXCL8 expression in U373MG human astrocytoma cells

Desferrioxamine, an iron chelator, induces CXCL8 expression in U373MG human astrocytoma cells

Interferon-stimulated gene (ISG) 60, as well as ISG56 and ISG54, positively regulates TLR3/IFN-β/STAT1 axis in U373MG human astrocytoma cells

Treatment of cells with interferons (IFNs) induces the phosphorylation of signal transducer and activator of transcription 1 (STAT1), leading to the expression of hundreds of IFN-stimulated genes (ISGs). ISGs exert various antiviral and pro-inflammatory reactions. We have previously reported that ISG56 and ISG54 are induced by polyinosinic–polycytidylic acid (poly IC), an authentic agonist for Toll-like receptor 3 (TLR3), in U373MG human astrocytoma cells. ISG56 and ISG54 are also named as IFN-induced proteins with tetratricopeptide repeats (IFIT) 1 and IFIT2, respectively. In the present study, we demonstrated that poly IC induces the expression of ISG60, also named as IFIT3, in U373MG cells. RNA interference experiments showed that the induction of ISG60 by poly IC was mediated by TLR3, IFN-β, ISG56 and ISG54, whereas ISG60 is involved in poly IC-induced expression of ISG56, ISG54 and a chemokine CXCL10. The level of phosphorylated STAT1 was enhanced by poly IC, and it was inhibited by knockdown of ISG56, ISG54 or ISG60. These results suggest that there is a positive feedback loop between phosphorylated STAT1 and these ISGs.

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells.

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

Desferrioxamine, an iron chelator, inhibits CXCL10 expression induced by polyinosinic–polycytidylic acid in U373MG human astrocytoma cells

Although iron is essential in physiological processes, accumulation of iron in central nervous system is associated with various neurological diseases including Alzheimer's disease and Parkinson's disease. Innate immune reactions are involved in the pathogenesis of those diseases, but roles of iron in innate immunity are not known well. In the present study, pretreatment of U373MG human astrocytoma cells with an iron chelator desferrioxamine (DFX) inhibited the expression of CXCL10 induced by a Toll-like receptor 3 (TLR3) agonist polyinosinic–polycytidylic acid (poly IC). Induction of interferon-β (IFN-β) was not affected, but phosphorylation of signal transducer and transcription 1 (STAT1) was decreased by DFX. We have previously reported that various IFN-stimulated genes (ISGs) are involved in CXCL10 induction by poly IC. Pretreatment with DFX also decreased the expression of these ISGs. Pretreatment of cells with FeSO4 counteracted inhibitory effects of DFX on ISG56, retinoic acid-inducible gene-I (RIG-I), CXCL10 and phosphorylation of STAT1. These results suggest that iron may positively regulate STAT1 phosphorylation and following signaling to express ISG56, RIG-I and CXCL10 in U373MG cells treated with poly IC. Iron may contribute to innate immune and inflammatory reactions elicited by the TLR3 signaling in astrocytes, and may play an important role in neuroinflammatory diseases.

Phenolic Content, Antioxidant and Astroprotective Response to Oxidative Stress of Ethanolic Extracts of Mentha longifolia from Sinai

The aerial parts ofMentha longifolia L. are used as herbal remedies for curing different diseases through traditional Bedouin medicine. The antioxidant activity of the ethanolic extracts of M longifolia was investigated measuring peroxyl radical-scavenging activity by ORAC assay, with Trolox (a water-soluble analogue of α-tocopherol) employed as reference compound. In addition, the total content of phenolic compounds estimated by the Folin-Ciocalteau method and the identification of the polyphenols using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) have been performed. Furthermore, the effect of these extracts on cell viability and intracellular ROS production was assayed using the U373-MG human astrocytoma cell line in a H2O2-induced oxidative stress model. Results showed that the major type of polyphenols found were benzoic acids, cinnamic acids, flavones and flavanones. The total phenolic content was 37.7 mg gallic acid/g sample and the ORAC value was 1.355 .mol TE/mg sample. The data obtained in cellular assays demonstrated that these ethanolic extracts protected H2O2-induced astrocyte damage by increasing cell viability and inhibiting production of intracellular ROS. These results suggest that the investigated extracts obtained from the aerial parts of M longifolia have antioxidant potential related to their phenol content which have important beneficial health effects, especially in those disease associated with ROS.

Compositional Analysis and in vitro Protective Activity against Oxidative Stress of Essential Oils from Egyptian Plants Used in Traditional Medicine

The Sinai desert in Egypt contains great variability in plants extensively used for traditional medicines such as Achillea fragrantissima, Chiliadenus montanus, Mentha longifolia and Haplophyllum tuberculatum. The essential oils extracted by hydrodistillation from the aerial parts have been analyzed. Subsequently, their potential protective activity against oxidative stress has been evaluated, employing H2O2 as oxidant inductor and astrocytes as the cell model. The chemical composition of the essential oils was analyzed by GC/MS. Most of the compounds identified in A. fragrantissima and M. longifolia samples were oxygenated monoterpene derivatives, whereas for H. tuberculatum they were monoterpenes hydrocarbons and oxygenated compounds, and for C. montanus oxygenated monoterpenes and oxygenated sesquiterpenes predominated. The in vitro evaluation of antioxidant properties, using ORAC assay, revealed that M. longifolia essential oil possessed the highest scavenging activity against peroxyl radicals, following by H. tuberculatum, A. fragrantissima and C. montanus. Under oxidative stress conditions, M. longifolia and H. tuberculatum essential oils were the only ones that protected human astrocytoma U373-MG cells against H2O2 damage. Both essential oils prevented cell death and inhibited ROS production caused by H2O2. M. longifolia essential oil was the most active, suggesting an interesting prevention role in those CNS disorders associated with oxidative stress.

ISG54 and ISG56 are induced by TLR3 signaling in U373MG human astrocytoma cells: Possible involvement in CXCL10 expression

Toll-like receptor (TLR) 3 is a pattern recognition receptor that recognizes double-stranded RNA (dsRNA). TLR3 signaling in astrocytes leads to the expression of interferon-β (IFN-β), and IFN-β regulates immune and inflammatory reactions by inducing IFN-stimulated genes (ISGs). We demonstrated in the present study that polyinosinic-polycytidylic acid (poly IC), an authentic dsRNA, up-regulated the expression of ISG54 and ISG56 in U373MG human astrocytoma cells. This reaction was confirmed to be mediated via the TLR3/IFN-β pathway. We also found that ISG56 positively regulates the expression of ISG54, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). In addition, positive feedback loops were found between ISG54 and ISG56, and also between ISG54 and RIG-I. RNA interference experiments revealed that all of ISG54, ISG56, RIG-I and MDA5 were involved in the poly IC-induced expression of a chemokine CXCL10. These results suggest that ISG54 and ISG56 are involved in the induction of CXCL10 in TLR3/IFN-β signaling at least partly by co-operating with RIG-I and MDA5. ISG54 and ISG56 may contribute to immune and inflammatory reactions elicited by the TLR3/IFN-β signaling pathway in astrocytes, and may play an important role both in antiviral immunity and in neuroinflammatory diseases.

Carnosic acid suppresses the production of amyloid-β 1-42 and 1-43 by inducing an α-secretase TACE/ADAM17 in U373MG human astrocytoma cells

Amyloid beta (Aβ) peptides are key molecules in the pathogenesis of Alzheimer's disease (AD). The sequential cleavage of amyloid precursor protein (APP) by the β- and γ-secretases generates Aβ peptides; however, the alternate cleavage of APP by the α- and γ-secretases decreases Aβ production. We previously reported that carnosic acid (CA), a phenolic diterpene compound found in the labiate herbs rosemary and sage, suppresses Aβ (1-40 and 1-42) production by activating α-secretase in cultured SH-SY5Y human neuroblastoma cells (Neurosci. Res. 2013; 75: 94–102). Here, we investigated the effect of CA on the production of Aβ peptides (1-40, 1-42 and 1-43) in U373MG human astrocytoma cells. The treatment of cells with CA suppressed Aβ40/42/43 release (55–71% decrease at 50μM). CA treatment enhanced the mRNA expressions of an α-secretase TACE (tumor necrosis factor-α-converting enzyme, also called a disintegrin and metalloproteinase-17, ADAM17); however, the β-secretase BACE1 (β-site APP-cleaving enzyme-1) was not increased by CA. Knockdown of TACE by siRNA reduced soluble-APPα release enhanced by CA and partially recovered the CA-suppressed Aβ40/42/43 release. These results suggest that CA reduces Aβ production, at least partially, by activating TACE in human astroglial cells. The use of CA may have a potential in the prevention of Aβ-mediated diseases.

Analog of Somatostatin Vapreotide Exhibits Biological Effects in vitro via Interaction with Neurokinin-1 Receptor

Objectives: Vapreotide, a synthetic analog of somatostatin, has analgesic activity most likely mediated through the blockade of neurokinin-1 receptor (NK1R), the substance P (SP)-preferring receptor. The ability of vapreotide to interfere with other biological effects of SP has yet to be investigated. Methods: We studied the ability of vapreotide to antagonize NK1R in three different cell types: immortalized U373MG human astrocytoma cells, human monocyte-derived macrophages (MDM) and a human embryonic kidney cell line, HEK293. Both U373MG and MDM express endogenous NK1R while HEK293 cells, which normally do not express NK1R, are stably transformed to express human NK1R (HEK293-NK1R). Results: Vapreotide attenuates SP-triggered intracellular calcium increases and nuclear factor-κB activation in a dose-dependent manner. Vapreotide also inhibits SP-induced interleukin-8 and monocyte chemotactic protein-1 production in HEK293-NK1R and U373MG cell lines. Vapreotide inhibits HIV-1 infection of human MDM in vitro, an effect that is reversible by SP pretreatment. Conclusions: Our findings indicate that vapreotide has NK1R antagonist activity and may have a potential application as a therapeutic intervention in HIV-1 infection.

MDA5 and ISG56 mediate CXCL10 expression induced by Toll-like receptor 4 activation in U373MG human astrocytoma cells

Toll-like receptor (TLR) 4 is a pattern recognition receptor, and recognizes not only bacterial lipopolysaccharide (LPS) but also endogenous danger-associated molecular patterns released from dying or injured cells. It has been reported that TLR4 signaling in astrocytes plays an important role in various neurological diseases. However, details of TLR4 signaling in astrocytes are not fully elucidated. In the present study, we demonstrated that TLR4 signaling, induced by LPS, increases the expression of melanoma differentiation-associated gene 5 (MDA5) and interferon (IFN)-stimulated gene 56 (ISG56) in U373MG human astrocytoma cells. We also found that nuclear factor-κB, p38 mitogen-activated protein kinase and IFN-β are involved in the expression of MDA5 and ISG56 induced by LPS. RNA interference experiments revealed that MDA5 and ISG56 positively regulate the LPS-induced expression of a chemokine CXCL10, but not CCL2. In addition, it was suggested that MDA5 and ISG56 constitute a positive feedback loop. These results suggest that MDA5 and ISG56 may contribute not only to physiological inflammatory reactions but also to the pathogenesis of various neurological diseases elicited by TLR4 in astrocytes, at least in part, by regulating the expression of CXCL10.

Mitochondrial-Targeted Protective Properties of Isolated Diterpenoids from Sideritis spp. in Response to the Deleterious Changes Induced by H2O2

Mitochondrial impairment and oxidative stress are considered widely to be central events in many forms of neurodegenerative disease. The current study has evaluated for the first time the potential protective role of three diterpenoids [andalusol (1), conchitriol (2), and lagascatriol (3)] in response to the deleterious H2O2-induced changes on mitochondrial function. U373-MG human astrocytoma cells and PC12 rat adrenal pheochromocytoma cells were used as models for evaluating the cytoprotective potential of these compounds. In the absence of diterpenoids 1-3, H2O2 compromised mitochondrial function, decreasing mitochondrial membrane potential and ATP levels, increasing caspase-3 activity, and disrupting cytosolic and mitochondrial calcium homeostasis. However, treatment with the diterpenoids, prior to H2O2, prevented these mitochondrial perturbations. In particular, 1 and 3 were the most effective compounds in protecting mitochondrial function against H2O2-induced oxidative stress in U373-MG, whereas all three diterpenoids studied were significantly active against PC12 cells. Since consistent evidence has demonstrated the contribution of H2O2 on both progression and pathological development of several human diseases associated with mitochondrial function and oxidative stress responses, compounds 1-3 are worthy of further investigation.

P2‐373: The anti‐rheumatic gold drug auranofin could be beneficial in Alzheimer's disease by inhibiting astrocyte‐mediated neuroinflammation

Alzheimer's disease (AD) is characterized by chronic inflammation in brain areas affected by the disease process, which may contribute to the neuronal loss characteristic of AD. It is thought that by reducing this inflammation, the neuronal loss observed in AD could be decreased. Astrocytes support neurons by regulating their external chemical environment, but when activated by pathogenic stimuli, these cells can secrete pro-inflammatory and neurotoxic substances that contribute to neuroinflammation and neuronal death. There are currently no effective therapeutic agents directed at astrocyte neurotoxicity or neuroinflammation in general. Auranofin is a potent anti-inflammatory drug which has been used in the treatment of rheumatoid arthritis for many years and has been shown to inhibit several inflammatory pathways such as those involving p38 mitogen activated protein kinases and thioredoxin reductase. However, a potential for auranofin to reduce neuroinflammatory reactions has not been reported. Therefore, the effectiveness of auranofin and several other gold-containing compounds as inhibitors of astrocyte pro-inflammatory functions was investigated. Effects of gold compounds on the following activation parameters of interferon (IFN)-γ stimulated human U373-MG and U118-MG astrocytoma cells as well as primary human astrocytes were studied: i) mRNA expression (RT-qPCR) of several inflammatory markers including interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1 and heme oxygenase (HOX)-1; ii) pro-inflammatory cytokine secretion (ELISA); and iii) toxicity towards SH-SY5Y neuroblastoma cells (MTT and LDH cell viability assays). In the low micromolar range (0.1-5 m M), auranofin inhibited toxicity of stimulated astrocytoma and human primary astrocytes towards SH-SY5Y neuroblastoma cells. Treating SH-SY5Y cells directly with 1 m M auranofin protected neuronal cells from toxicity induced by supernatants from stimulated astrocytes. 1 mM auranofin upregulated expression of HOX-1 in both stimulated and unstimulated astrocytes. It also affected the secretion of pro-inflammatory cytokines. Several other gold-based drugs and a gold salt had no effect in the above assays. Auranofin may decrease toxicity of stimulated astrocytes towards neurons by up-regulating expression of the protective enzyme HOX-1. As an effective inhibitor of neuroinflammatory reactions, auranofin should be tested in animal models and clinically for its potential to slow neurodegeneration observed in AD.