Rat Astrocytoma Cells Research Articles

Effect of Aster tataricus on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells

Neuroinflammation is the commonest cause of neurodegenerative diseases such as Alzheimer’s disease. Present investigation evaluates the inhibitory effect of ethanolic root extract of Aster tataricus (AS) on inflammatory mediators production in lipopolysaccharide (LPS) stimulated C6 cells. C6 cells were treated with AS (20and40mg/kg) and nimesulide (NSL, 1.5μg/ml) for 1day. Thereafter various parameters such as production of ROS, release of nitrite, MDA, glutathione level and NF-κB translocation were estimated in C6 cell lines. Effect of AS was estimated on the expressions of tumor necrosis factor α (TNF-α) of human monocytic leukemia cell line (THP-1). It was observed that AS (20and40mg/ml) treated group shows significant (p<0.01) decrease in production of ROS, Nitrite release and MDA level in LPS activated C6 cell lines compared to negative control group. Moreover, treatment with it decreases glutathione level and inhibits the translocation of NF-κB in LPS activated C6 cell lines compared to negative control group. There were significant (p<0.01) decreases in expression of TNF-α in AS treated group compared to negative control group in THP-1 cell lines. Present investigation concludes the anti neuroinflammatory effect of ethanolic extract of AS root by decreasing oxidative stress and attenuates the cytokine.

Sulforaphane Ameliorates Okadaic Acid-Induced Memory Impairment in Rats by Activating the Nrf2/HO-1 Antioxidant Pathway.

Okadaic acid (OKA) causes memory impairment and attenuates nuclear factor erythroid 2-related factor 2 (Nrf2) along with oxidative stress and neuroinflammation in rats. Sulforaphane (dietary isothiocyanate compound), an activator of Nrf2 signaling, exhibits neuroprotective effects. However, the protective effect of sulforaphane in OKA-induced neurotoxicity remains uninvestigated. Therefore, in the present study, the role of sulforaphane in OKA-induced memory impairment in rats was explored. A significant increased Nrf2 expression in the hippocampus and cerebral cortex was observed in trained (Morris water maze) rats, and a significant decreased Nrf2 expression in memory-impaired (OKA, 200ng icv) rats indicated its involvement in memory function. Sulforaphane administration (5 and 10mg/kg, ip, days 1 and 2) ameliorates OKA-induced memory impairment in rats. The treatment also restored Nrf2 and its downstream antioxidant protein expression (GCLC, HO-1) and attenuated oxidative stress (ROS, nitrite, GSH), neuroinflammation (NF-κB, TNF-α, IL-10), and neuronal apoptosis in the cerebral cortex and hippocampus of OKA-treated rats. Further, to determine whether modulation of Nrf2 signaling is responsible for the protective effect of sulforaphane, in vitro, Nrf2 siRNA and its downstream HO-1 inhibition studies were carried out in a rat astrocytoma cell line (C6). The protective effects of sulforaphane were abolished with Nrf2 siRNA and HO-1 inhibition in astrocytes. The results suggest that Nrf2-dependent activation of cellular antioxidant machinery results in sulforaphane-mediated protection against OKA-induced memory impairment in rats. Graphical Abstract ᅟ.

P.2.a.004 The role of the catecholaminergic system in the antidepressant-like effect of the nociceptin antagonist BAN ORL 24

Impaired insulin signaling, amyloid pathology and neuroinflammation are closely associated with neurodegenerative disorder like Alzheimer's disease (AD). Our earlier studies showed that intracerebroventricular streptozotocin (STZ) induces insulin receptor (IR) signaling defect in the hippocampus, which is associated with memory impairment in rats. Astrocytes are the most abundant cells in the brain and play a major role in neuroinflammation. However, involvement of astrocytes in STZ induced IR dysfunction has not received much attention. Therefore, the present study was planned to explore the effect of STZ on IR signaling, proinflammatory markers and amyloidogenesis in rat astrocytoma cell line, (C6). STZ (100 μM) treatment in astrocytes (n = 3) for 24 h, resulted significant decrease in IR mRNA and protein expression, phosphorylation of IRS-1, Akt, GSK-3α and GSK-3β (p < 0.01). Further STZ induced amyloidogenic protein expression as evidenced by the increase in APP, BACE-1 and Aβ1-42 expression (p < 0.05) in astrocytes. STZ also significantly induced astrocytes activation as evidenced by increased expression of GFAP and p-P38 MAPK (p < 0.05). STZ treatment caused enhanced translocation of p65 NF-kB, triggered over expression of TNF-α, IL-1β, COX-2, oxidative/nitrosative stress and caspase activation (p < 0.05) in astrocytes. Insulin (25–100 nM) pretreatment (n = 3) significantly prevented changes in IR signaling, amyloidogenic protein expression and levels of proinflammatory markers (p < 0.05) in STZ treated astroglial cells. In the present study, the protective effect of insulin suggests that, IR dysfunction along with amyloidogenesis and neuroinflammation may have played a major role in STZ induced toxicity in astrocytes which are relevant to AD pathology.

Protection of streptozotocin induced insulin receptor dysfunction, neuroinflammation and amyloidogenesis in astrocytes by insulin

Impaired insulin signaling, amyloid pathology and neuroinflammation are closely associated with neurodegenerative disorder like Alzheimer's disease (AD). Our earlier studies showed that intracerebroventricular streptozotocin (STZ) induces insulin receptor (IR) signaling defect in the hippocampus, which is associated with memory impairment in rats. Astrocytes are the most abundant cells in the brain and play a major role in neuroinflammation. However, involvement of astrocytes in STZ induced IR dysfunction has not received much attention. Therefore, the present study was planned to explore the effect of STZ on IR signaling, proinflammatory markers and amyloidogenesis in rat astrocytoma cell line, (C6). STZ (100 μM) treatment in astrocytes (n = 3) for 24 h, resulted significant decrease in IR mRNA and protein expression, phosphorylation of IRS-1, Akt, GSK-3α and GSK-3β (p < 0.01). Further STZ induced amyloidogenic protein expression as evidenced by the increase in APP, BACE-1 and Aβ1-42 expression (p < 0.05) in astrocytes. STZ also significantly induced astrocytes activation as evidenced by increased expression of GFAP and p-P38 MAPK (p < 0.05). STZ treatment caused enhanced translocation of p65 NF-kB, triggered over expression of TNF-α, IL-1β, COX-2, oxidative/nitrosative stress and caspase activation (p < 0.05) in astrocytes. Insulin (25–100 nM) pretreatment (n = 3) significantly prevented changes in IR signaling, amyloidogenic protein expression and levels of proinflammatory markers (p < 0.05) in STZ treated astroglial cells. In the present study, the protective effect of insulin suggests that, IR dysfunction along with amyloidogenesis and neuroinflammation may have played a major role in STZ induced toxicity in astrocytes which are relevant to AD pathology.

Comments on the business of genetic testing

Evidence has shown that extracellular adenosine induces apoptosis in variety of cancer cells, mainly through two pathways: an intrinsic pathway relative to adenosine uptake into the cells, and an extrinsic pathway involving the adenosine receptors. We elucidated the mechanisms underlying the adenosine-induced anticancer effects.Extrinsic pathway analysis showed that extracellular adenosine induces apoptosis in CW2 human colon cancer and RCR-1 rat astrocytoma cells through the A1 adenosine receptor; in Caco-2 human colon cancer and HepG2 human hepatoma cells through the A2a adenosine receptor; and through the A3 adenosine receptor in A549, Lu-65, and SBC-3 human lung cancer cells, RCC4-VHL human renal cancer cells, 5637 human bladder cancer cells, and human malignant pleural mesothelioma cells. In the intrinsic pathways, intracellularly transported adenosine induces apoptosis in GT3-TKB human lung cancer cells, human malignant pleural mesothelioma cells, HuH-7 and HepG2 human hepatoma cells, and MCF-7 human breast cancer cells by a) activating AMPK, b) upregulating p53, c) downregulating c-FLIP expression, d) neutralizing caspase-3 inhibition due to inhibition of apoptosis protein (IAP) in cooperation with DIABLO, e) accumulating AMID in the nucleus, f) regulating apoptosis-related gene transcription, or g) promoting GATA-2-regulated p53 gene transcription.Adenosine exerts its anticancer action on a wide variety of cancer cell types through diverse signaling pathways. Therefore, adenosine and its signaling cascades can be useful as possible targets in the development of promising anticancer therapies.

Adenosine exerts potent anticancer effects through diverse signaling pathways

Abstract Purpose Evidence has shown that extracellular adenosine induces apoptosis in variety of cancer cells, mainly through two pathways: an intrinsic pathway relative to adenosine uptake into the cells, and an extrinsic pathway involving the adenosine receptors. We elucidated the mechanisms underlying the adenosine-induced anticancer effects. Study section and results Extrinsic pathway analysis showed that extracellular adenosine induces apoptosis in CW2 human colon cancer and RCR-1 rat astrocytoma cells through the A 1 adenosine receptor; in Caco-2 human colon cancer and HepG2 human hepatoma cells through the A 2a adenosine receptor; and through the A 3 adenosine receptor in A549, Lu-65, and SBC-3 human lung cancer cells, RCC4-VHL human renal cancer cells, 5637 human bladder cancer cells, and human malignant pleural mesothelioma cells. In the intrinsic pathways, intracellularly transported adenosine induces apoptosis in GT3-TKB human lung cancer cells, human malignant pleural mesothelioma cells, HuH-7 and HepG2 human hepatoma cells, and MCF-7 human breast cancer cells by a) activating AMPK, b) upregulating p53, c) downregulating c-FLIP expression, d) neutralizing caspase-3 inhibition due to inhibition of apoptosis protein (IAP) in cooperation with DIABLO, e) accumulating AMID in the nucleus, f) regulating apoptosis-related gene transcription, or g) promoting GATA-2-regulated p53 gene transcription. Conclusions Adenosine exerts its anticancer action on a wide variety of cancer cell types through diverse signaling pathways. Therefore, adenosine and its signaling cascades can be useful as possible targets in the development of promising anticancer therapies.

Effect of Pluchea lanceolata bioactives in LPS-induced neuroinflammation in C6 rat glial cells

Neuroinflammation plays a significant role in various chronic and acute pathological conditions of the central nervous system. In the Indian system of medicine, Pluchea lanceolata is used to treat the neurological disorders. We investigated the effect of major pentacyclic triterpene and its naturally occurring acetate derivative isolated from P. lanceolata on lipopolysaccharide (LPS)-stimulated neuroinflammatory condition associated to inflammatory cytokine production in rat astrocytoma cell line (C6). The log concentration dependence of Pluchea bioactive taraxasterol (Tx) significantly (p < 0.05) attenuates the release of pro-inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, while its in situ produced acetyl derivative, i.e., taraxasterol acetate (TxAc), did not inhibit the LPS-induced IL-6 production at lower concentration (p > 0.05). Surflex-Dock molecular modeling study was performed to simulate the binding capacity of compounds into the active site of the TNF-α (2AZ5), tumor protein P53 (2VUK), and NF-kappa-B (1RAM). The differential inhibition of cytokines by Tx and TxAc was further confirmed by high docking scores showing the high affinity to target proteins. Findings of the study demonstrated the comparatively greater role of Pluchea triterpene than its in situ produced acetate derivate in neuroinflammation-associated disorders.

Evaluation of amentoflavone isolated from Cnestis ferruginea Vahl ex DC (Connaraceae) on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells

Ethnopharmacological relevanceCnestisferruginea (CF) Vahl ex DC (Connaraceae) is a shrub widely used in traditional African medicine for the treatment of various psychiatric illness and inflammatory conditions. Aim of the studyThis study was carried out to investigate the effect of amentoflavone isolated from methanolic root extract of CF on lipopolysaccharide (LPS)-induced neuroinflammatory cascade of events associated to the oxidative and nitrative stress, and TNF-α production in rat astrocytoma cell line (C6) and human monocytic leukemia cell line (THP-1), respectively. Materials and methodsRat astrocytoma cells (C6) were stimulated with LPS (10μg/ml) alone and in the presence of different concentrations of amentoflavone (0.1–3μg/ml) for 24h incubation period. Nitrite release, reactive oxygen species (ROS), malondialdehyde (MDA) and reduced-glutathione (GSH) in C6 cells were estimated; while the TNF-α level was estimated in THP-1 cell lysate. In vivo analgesic activity was evaluated using mouse writhing and hot plate tests while the anti-inflammatory effect was investigated using carrageenan-induced oedema test. ResultsLPS (10μg/ml) significantly (P<0.05) stimulated C6 cells to release nitrite, ROS, MDA, and TNF-α generation while GSH was down regulated in comparison to control. However, amentoflavone significantly (P<0.05) attenuated nitrite, ROS, MDA and TNF-α generation and also up regulated the level of GSH. Amentoflavone per se did not have any significant effect on C6 and THP-1 cells. Amentoflavone (6.25–50mg/kg) significantly (P<0.05) reduced number of writhes and also increase pain threshold in hot plate test. It produced time course significant (P<0.05) decrease in oedema formation in rodents. Discussion and conclusionFindings in this study demonstrate the anti-neuroinflammatory and antinoceptive effects of amentoflavone which may suggest its beneficial roles in neuroinflammation associated disorders.

The effect of guggulipid and nimesulide on MPTP-induced mediators of neuroinflammation in rat astrocytoma cells, C6

Oxidative stress plays an important role in the pathophysiology of Parkinson’s disease (PD) but its mechanism is still not properly explored. Cyclooxygenase-2 (COX-2) inhibition has also been known a major neuroprotective strategy in the various 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) induced models of Parkinson’s disease (PD) but its role in astrocytes is still not properly understood. The present study demonstrated that, guggulipid and nimesulide (preferentially selective COX-2 inhibitor) treatment of rat astrocytoma cells, C6 for 24h significantly decreased MPTP (400μM) induced nitrative and oxidative stress and intracellular calcium ion (Ca2+) level. Guggulipid and nimesulide also deactivated MPTP-induced P-p38 MAPK (Phosphorylated p38 mitogen-activated protein kinase) and down regulated expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and CHOP (C/EBP, homologous protein 10). At transcriptional level of inflammatory cytokine genes, guggulipid and nimesulide down regulated MPTP-induced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA expressions with up regulations in interleukin-6 (IL-6) and interleukin-1α (IL-1α) mRNA expressions. In addition to this, guggulipid and nimesulide inhibited translocation of nuclear factor kappa-B (NF-κB) from cytosol to nucleus. In conclusion, our findings elucidated the potential antioxidant and anti-neuroinflammatory effect of guggulipid and nimesulide in rat astrocytoma cells C6, which may suggest the use of these drugs in the management of neuroinflammation associated pathophysiology of PD.

Melatonin attenuated mediators of neuroinflammation and alpha-7 nicotinic acetylcholine receptor mRNA expression in lipopolysaccharide (LPS) stimulated rat astrocytoma cells, C6

Melatonin has been known to affect a variety of astrocytes functions in many neurological disorders but its mechanism of action on neuroinflammatory cascade and alpha-7 nicotinic acetylcholine receptor (α7-nAChR) expression are still not properly understood. Present study demonstrated that treatment of C6 cells with melatonin for 24 hours significantly decreased lipopolysaccharide (LPS) induced nitrative and oxidative stress, expressions of cyclooxigenase-2 (COX-2), inducible nitric-oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP). Melatonin also modulated LPS-induced mRNA expressions of α7-nAChR and inflammatory cytokine genes. Furthermore, melatonin reversed LPS-induced changes in C/EBP homologous protein 10 (CHOP), microsomal prostaglandin E synthase-1(mPGES-1) and phosphorylated p38 mitogen activated protein kinase (P-p38). Treatment with pyrrolidine dithiocarbamate (PDTC) inhibited α7-nAChR mRNA expression in LPS-induced C6 cells. Our findings explored anti-neuroinflammatory action of melatonin, which may suggests its beneficial roles in the neuroinflammation associated disorders.

Curcumin inhibits LPS-induced CCL2 expression via JNK pathway in C6 rat astrocytoma cells.

The important role of neuroinflammation in many chronic and acute pathological conditions of the central nervous system is widely recognized. Curcumin is a major component of turmeric and reportedly has anti-inflammatory and anti-oxidant effects. This study investigated the inhibitory effect of curcumin on lipopolysacharide (LPS)-induced chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1) production and whether the effect is mediated by mitogen-activated protein kinases (MAPKs) in the rat astrocytoma cell C6. We observed that LPS (1 μg/ml) induced the upregulation of CCL2 mRNA and protein in C6. Treatment with curcumin (2.5, 10, and 25 μM) decreased the expression of CCL2 mRNA and protein in a dose-dependent manner under treatment with LPS. Additionally, the c-jun N-terminal kinase (JNK) inhibitor (SP600125) dose-dependently inhibited LPS-induced CCL2 upregulation, whereas the MAPK kinase (MEK) inhibitor (PD98059) only had a mild effect and the p38 MAPK inhibitor (SB203580) had no effect. Finally, western blot showed that LPS induced rapid JNK activation and curcumin reduced LPS-induced phosphoJNK (pJNK) expression at 30 min after LPS stimulation. These data suggest that the anti-neuroinflammatory effect of curcumin relates to the downregulation of CCL2 expression through the JNK pathway in astrocytoma cells, which indicates a possible benefit from the use of curcumin in the treatment of neuroinflammation-associated disorders.

The expression of neuroglobin in astrocytoma

Neuroglobin (NGB) is a characterized heme globin that is widely expressed in the vertebrate central and peripheral nervous system as well as retina and endocrine tissues. However, to date, it is not determined whether functional NGB is expressed in cells of glia origin. In this study, we aimed to explore the detailed expression of NGB in a rat astrocytoma cell line (C6) and human astrocytoma cell line (U251) by reverse transcription-polymerase chain reaction, immunofluorescence, and Western blotting, and to detect the expression of NGB in human astrocytoma tissues by an immunohistochemical method. We found that NGB was present in a rat astrocytoma cell line (C6), human astrocytoma cell line (U251), and human astrocytoma tissues. The expression and potential roles of NGB in astrocytomas may provide insight into the mechanisms of tumor cells to adapt and survive in hypoxic microenvironments and also represent a novel therapeutic approach to astrocytomas.

Guggulipid and nimesulide differentially regulated inflammatory genes mRNA expressions via inhibition of NF-kB and CHOP activation in LPS-stimulated rat astrocytoma cells, C6.

Neuroinflammation is an integral part of neurodegenerative diseases. Lipo-polysacharide (LPS) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of neurological diseases, but its mechanism of action is still not properly known. The effect of guggulipid and nimesulide on LPS-induced neuroinflammatory changes is also not properly understood. This work demonstrated the mechanism of actions of guggulipid and nimesulide on inflammatory genes expressions in LPS-stimulated rat astrocytoma cells, C6. We observed that LPS (10μg/ml) treatment of rat astrocytoma cells, C6, for 24h significantly increased intracellular Ca(2+) ion and expression of inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-kB), C/EBP homologous protein 10 (CHOP), c-fos, and c-jun proteins. At transcriptional stage, LPS upregulated mRNA levels of cyclooxygenase-2 and IL-6 with downregulation in IL-1α, IL-1β, and microsomal prostaglandin E synthase-1 (mPGES-1) through activating NF-kB translocation. Treatment with guggulipid reversed these LPS-induced changes in rat astrocytoma cells. Treatment with nimesulide also attenuated LPS-induced Ca(2+) ion, iNOS, NF-kB, and c-fos expressions, but does not significantly influence CHOP, c-jun protein expressions, and mRNA levels of IL-6, IL-1α, IL-1β, and mPGES-1 genes. In conclusion, our findings elucidated the molecular mechanism of neuroinflammation in response to LPS and its modulation by guggulipid and nimesulide in rat astrocytoma cells (C6), which suggest the use of these drugs in the treatment of neuroinflammation-associated disorders.

The mechanism of action of MPTP-induced neuroinflammation and its modulation by melatonin in rat astrocytoma cells, C6

The 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of Parkinson's disease (PD), but its mechanism of action on astrocytes is not understood. The effect of melatonin on MPTP-induced neuroinflammation in astrocytes is also not known. The present study demonstrated that MPTP treatment of rat astrocytoma cells, C6 for 24 h significantly increased nitrative and oxidative stress and intracellular calcium (Ca2++) level. MPTP also activated phosphorylated p38 mitogen activated protein kinase (P-p38 MAPK) and up-regulated expressions of inflammatory proteins. Moreover, MPTP modulated mRNA expressions of pro-inflammatory cytokine genes via activating nuclear factor kappa-B (NF-kB) translocation. Treatment of melatonin with MPTP reversed all these MPTP-induced changes. Study with deprenyl demonstrates that MPTP is inducing neuroinflammation in astrocytoma cells. The present findings elucidated the molecular mechanism of MPTP-induced neuroinflammation and its modulation by melatonin in astrocytoma cells (C6).

Expression pattern and sub‐cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U‐73122 in rat astrocytoma cells

Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.

Evaluation of guggulipid and nimesulide on production of inflammatory mediators and GFAP expression in LPS stimulated rat astrocytoma, cell line (C6)

Aim of the study The present study was designed to investigate effect of guggulipid, a drug developed by CDRI and nimesulide on LPS stimulated neuroinflammatory changes in rat astrocytoma cell line (C6). Materials and methods Rat astrocytoma cells (C6) were stimulated with LPS (10 μg/ml) alone and in combinations with different concentrations of guggulipid or nimesulide for 24 h of incubation. Nitrite release in culture supernatant, ROS in cells, expressions of COX-2, GFAP and TNF-α in cell lysate were estimated. Results LPS (10 μg/ml) stimulated C6 cells to release nitrite, ROS generation, up regulated COX-2 and GFAP expressions at protein level and TNF-α at mRNA level. Both guggulipid and nimesulide significantly attenuated nitrite release, ROS generation and also down regulated expressions of COX-2, GFAP and TNF-α. Guggulipid and nimesulide per se did not have any significant effect on C6 cells. Conclusion Results demonstrate the anti-inflammatory effect of guggulipid comparable to nimesulide which suggest potential use of guggulipid in neuroinflammation associated conditions in CNS disorders.

FGF-1-Induced Reactions for Biogenesis of apoE-HDL are Mediated by Src in Rat Astrocytes

Fibroblast growth factor-1 (FGF-1) is released from astrocytes in stress and stimulates MEK/ERK and PI3K/Akt pathways in autocrine fashion to increase synthesis of cholesterol and 25-OH-cholesterol, and to induce transport and secretion of apoE, respectively. FGF-1-induced phosphorylation of Src, and phosphorylation of MEK, ERK and Ark was inhibited by Src inhibitors in rat astrocytes. Src inhibitors also suppressed FGF-1-induced increase of biosynthesis and release of cholesterol and increase of apolipoprotein E (apoE) secretion. The results were reproduced in rat astrocytoma cells transfected by rat apoE and in 3T3-L1 cells. Down-regulation of Src expression reduced FGF-1-induced phosphorylation of the signalling protein and subsequent reactions. Increase by FGF-1 of messages of apoE and HMG-CoA reductase was not influenced by Src inhibitors or by its down-regulation. We conclude that FGF-1 activates Src for activation of MEK/ERK and PI3K/Akt pathways, while Src may not be involved in enhancement of transcription of the cholesterol-related genes.

Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes

Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3.his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription.

Cis-Parinaric Acid Effects, Cytotoxicity, c-Jun N-terminal Protein Kinase, Forkhead Transcription Factor and Mn-SOD Differentially in Malignant and Normal Astrocytes

Cis-parinaric acid (c-PNA), a natural four conjugated polyunsaturated fatty acid, increases free radical production and it is preferentially cytotoxic to malignant glial cells compared to normal astrocytes in-vitro. In order to explain the increased cytotoxicity of c-PNA in malignant glial cells, we compared the effects of c-PNA on the oxidative stress-dependent signal transducing events in 36B10 cells, a malignant rat astrocytoma cell line, and in fetal rat astrocytes. Our results show that c-PNA treatment in 36B10 cells caused a persistent activation of c-Jun N-terminal protein kinase (JNK) at RNA and protein levels. Specific inhibitors of the kinase significantly reversed the cytotoxicity of c-PNA. Additionally, c-PNA caused the phosphorylated inactivation of forkhead transcription factor-3a (FKHR-L1, FOXO3a) and drastically decreased the activity of mitochondrial superoxide dismutase (Mn-SOD) that protects cells from oxidative stress. On the other hand, identical c-PNA treatments in normal astrocytes increased the dephosphorylated activation of FKHR-L1, maintained activity of Mn-SOD and failed to phosphorylate JNK. Taken together, the results imply that a selective activation of JNK and the opposite regulation of FKHR-L1 and Mn-SOD contribute to the differential cytotoxicity of c-PNA in malignant and normal glial cells.

Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.