Treatment Of C6 Glioma Cells Research Articles

Apigenin from Croton betulaster Müll restores the immune profile of microglia against glioma cells.

The flavonoid apigenin, extracted from the Brazilian plant Croton betulaster Müll. has demonstrated the ability to inhibit proliferation, induce differentiation, and modify the inflammatory profile of glioma cells. The aim of the present study was to evaluate the effect of apigenin on chemotaxis and regulation of inflammatory cytokines of microglia cells and these impacts on glioma cell growth. In cultures of isolated rat microglia, it was observed that apigenin induced changes in Iba1-positive cells to an ameboid phenotype, associated to an increase in the expression of the activated M1 profile marker OX-42 and iNOS and a reduction in the expression of the M2 profile marker CD206. Besides, apigenin modulated the tumor necrosis factor and IL-10 release by microglia. Treatment of C6 glioma cells with conditioned medium of microglia treated with apigenin-induced reduction of tumor migration and viability, associated with significant reduction in IL-6 levels. On the other hand, treatment of C6 cells with apigenin-induced microglia chemotaxis to glioma in vitro. Moreover, apigenin treatment of microglia/C6 co-cultures induced preferentially reduction in the viability of C6 cells and increased microglia-activated phenotype, associated with a change in the balance of TNF/IL-10 levels. Together, these results demonstrated that the flavonoid apigenin restores the immune profile of microglia against glioma cells.

Comparative evaluation of hesperetin loaded nanoparticles for anticancer activity against C6 glioma cancer cells

The aim of this study was to evaluate anti-cancer properties of hesperetin (Hsp) and hesperetin-loaded poly(lactic-co-glycolic acid) nanoparticles (HspNPs) for glioblastoma treatment. Nanoparticles prepared by single emulsion method had a size of less than 300 nm with 70.7 ± 3.9% reaction yield and 26.4 ± 1.1% Hsp loading capacity. Treatment of C6 glioma cells with HspNPs for 24 and 48 h resulted in dose- and time-dependent decrease in cell viability, with approximate IC50 of 28 and 21 μg/mL, respectively (p = .036 for 24 h, p = .025 for 48 h). The percentage of PCNA positive cells decreased to 20% and 10%, respectively, for Hsp- and HspNP-treated cells at concentration of 100 µg/mL. Treatment with increasing concentrations of HspNPs (25, 50, 75 and 100 µg/mL) resulted in 9.1-, 7-, 12.5- and 12.7-fold in increase in apoptotic cell number. Optimum doses of Hsp and HspNPs were found to increase oxidative damage in C6 glioma cells. MDA levels, an indicator of lipid peroxidation, were found to be significantly elevated at 75 and 100 µg/mL exposure concentration of HspNPs with (p = .002) and (p = .018), respectively for 48-h treatment. The results obtained with this study showed biocompatible polymeric nanoparticle systems has great advantages to enhance anti-cancer activity and poor solubility of therapeutic agents. Overall our findings suggest that Hsp-loaded PLGA nanoparticle systems showed significant anti-cancer activity and HspNPs could be used as promising novel anti-cancer agent.

Cold atmospheric-pressure air plasma treatment of C6 glioma cells: effects of reactive oxygen species in the medium produced by the plasma on cell death

An atmospheric-pressure air plasma is employed to treat C6 glioma cells in vitro. To elucidate on the mechanism causing cell death and role of reactive species (RS) in the medium produced by the plasma, the concentration of the long-lived RS such as hydrogen peroxide, nitrate, and ozone in the plasma-treated liquid (phosphate-buffered saline solution) is measured. When vitamin C is added to the medium as a ROS quencher, the viability of C6 glioma cells after the plasma treatment is different from that without vitamin C. The results demonstrate that reactive oxygen species (ROS) such as H2O2, and O3 constitute the main factors for inactivation of C6 glioma cells and the reactive nitrogen species (RNS) may only play an auxiliary role in cell death.

Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gαs

Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify Gαs localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of Gαs to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics.

Anti-neoplastic and Calcium modulatory action of Caffeic acid phenethyl ester and Dasatinib in C6 glial cells: A Therapeutic Perspective

Gliomas are often recognized as highly heterogeneous cancerous phenotype. They are perpetually recurrent, obstinately resistant to treatment and hence almost incurable. Drug development studies to date have revealed only modest effect in attenuating growth of these tumors. The present study was aimed at elucidating the potential of targeting glioma through a novel combination of drugs in comparison to single agent. Here, we show that the combined administration of Caffeic acid phenethyl ester [CAPE] and Dasatinib exerts a strong antitumor action on C6 glioma cells. Combinational treatment inhibits proliferation, induces apoptosis, modulates astrocytic phenotype and decreases cell density. Results suggest that combinational therapy inhibits migration and invasiveness, decreases cell survival fraction and hence clonogenic property of C6 cells. The Nitric oxide [NO] levels were significantly reduced by combination treatment at all time points and effect was persistent over the time in comparison to single drug treatment. Atomic Absorption Spectroscopy [AAS] analysis of intracellular and extracellular calcium revealed that the treatment with CAPE and Dasatinib strongly modulates the calcium [Ca(2+)] levels. Herein, we demonstrate that treatment of C6 glioma cells with CAPE and Dasatinib significantly decrease the activity of catalase [CAT]. The results in totality suggest that the combinational therapy remarkably reduces the proliferation of glioma cells possibly through different mechanisms, targeting multiple pathways involved in tumor growth, proliferation and development implicating the relevance of using these drugs in combination therapy for effective treatment of glioma. In vitro results suggest that CAPE and Dasatinib cotreatment could be therapeutically exploited for the management of gliomas.

Apoptosis induced by sonodynamic therapy with hematoporphyrin monomethyl ether in C6 glioma cells in vitro

Objective To study apoptotic effect and mechanisms of sonodynamic therapy (SDT) with hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. Methods Time of ultrasound irradiation was optimized by methyl thiazol tetrazolium (MTT) . Apoptotic rate( AR) generation of reactive oxygen species (ROS) ,mitochondrial membrane potential (MMP) were detected by flow cytometry ,and expressions of apoptosis proteins were measured by Western blotting after SDT treatment. Results 60 s is the optimal time irradiation by MTT when the ultrasound frequency was maintained in 1 mHz and the average intensity was kept in 1. 0 W/cm2. To compare with the control,HMME and ultrasound group,treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis(34. 7 ±1.9)% .which was associated with the production of ROS ( 36. 4 ± 3. 1) % and loss of MMP (44. 9 ± 3. 4) %. The results showed that significantly increased expression in bax,Caspase-9 ,-3 , decreased in bcl-2 after SDT treatment. The results also showed apparent release of cytochome c in the SDT group versus to the other three groups. Conclusion Our results suggested that SDT mediated by HMME could induce apoptosis in C6 glioma cells.The mitochondrial signal pathway may play a pivotal role in the apoptosis. Key words: Glioma; Sonodynamic therapy; Hematoporphyrin monomethy lether; Apoptosis

Post-injury treatment with lipopolysaccharide or lipooligosaccharide protects rat neuronal and glial cell cultures

Traumatic brain injury (TBI) is a major cause of disability in civilians and military personnel worldwide that is caused by the acceleration force of a primary shockwave, blast wind or the force of a direct contact. Following the primary injury, secondary injury is caused by activation of the immune response due to an influx of neuro-inflammatory cells, increased production of inflammatory cytokines, and edema. In ischemia models pre-conditioning with lipopolysaccharide (LPS) has been shown to be neuroprotective, and post-injury conditioning with LPS was found to be protective in a spinal cord and an acute brain injury model. In this study, we utilized an in vitro scratch model of TBI to assess the effect of post-injury treatment with Escherichia coli LPS and Neisseria meningitidis lipooligosaccharide (LOS) on cell death and cytokine induction by assessing the level of lactate dehydrodgenase released from cells and rat multiplex cytokine assays. Our results showed that post-injury treatment of C6 glioma cells with either the LPS or the LOS reduced cell death when compared to scratched controls treated with media only. Post-injury treatment of the primary mixed neuronal cultures with LPS reduced cell death and resulted in a significant up-regulation in IL-10 when compared to controls. With LOS post-scratch treatment of the primary cell cultures, we found that IL-1α, IL-1β, IL-6, and TNF-α were significantly upregulated in addition to IL-10 compared to the media-only controls. The results strongly support additional testing of the neuroprotective ability of post-injury treatment with LPS or LOS in models of TBI.

NADPH treatment decreases C6 glioma cell survival by increasing oxidative stress

NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) plays pivotal roles in antioxidation and reductive biosynthesis. However, the effect of NADPH treatment on cell survival is unknown. In this study, we determined the effect of NADPH treatment on the survival of glioma cells. Treatment of C6 glioma cells with as low as 1 μM NADPH for 24 hrs induced a significant decrease in the survival of the glioma cells, while NADPH treatment had no effect on the survival of primary astrocyte cultures. We also found that NADPH treatment increased intracellular oxidative stress. Three antioxidants and the NADPH oxidase inhibitor, apocynin, attenuated the effect of NADPH. Poly(ADP-ribose) polymerase (PARP) activation appears to be a downstream effector of the oxidative stress, since PARP inhibitors reduced the effect of NADPH. Calcium chelator, BAPTA-AM, also attenuated the effect of NADPH. Collectively, these data indicate a novel property of NADPH: NADPH decreases glioma cell survival by inducing the NADPH oxidase-dependent increase in oxidative stress and by PARP activation. These results also suggest a potential therapeutic effect of NADPH on gliomas.

Therapeutic efficacy of VP_(22) and cytosine deaminase gene modified rat neural stem cell in treatment of C6 glioma in vitro

Objective To explore the therapeutic efficacy of rat neural stem cells genetically engineered by VP_(22) and cytosine deaminase (CD) gene in the treatment of C6 glioma cells in vitro. Method The encoding CD gene fragment was got from pAdTrack-CMV-CD by polymerase chain reaction (PCR),and cloned into pHIV-EGFP and pHIV-VP_(22)-EGFP to yield pHIV-CD-EGFP and pHIV-VP_(22)-CD-EGFP, the constructors were confirmed by the restriction enzyme method and PCR. The three-plasmid system containing constructors of pHIV-EGFP plasmid, helper plasmid and envelope plasmid were co-transfected into 293T cells. The viral particles were collected, and infected rat neural stem cells(NSCs) which were cultured from pregnant SD rat brain. The expression of CD transcripts in NSCs/CD -EGFP and NSCs/VP_(22)-CD-EGFP were identified by RT-PCR. The NSCs transfected by lentivirus-EGFP, lcntivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were co-cultured with C6 glioma cells at a ratio of 1:1 respectively. 5-FC was added to the mixed cell cultures for 3days at the final concentration of 100 mg/L and cell viability was performed by MTT assay. Results The cloning sites of the encoding CD fragment in pHIV-EGFP and pHIV-VP_(22)-EGFP were confirmed by the restriction enzyme method and PCR. In the presence of 5-FC, the cell survival rates of C6 glioma cells co-cultrued with the rat NSCs transfected by lentivirus-EGFP, lentivirus-CD-EGFP, lentivirus-VP_(22)-EGFP or lentivirus-VP_(22)-CD-EGFP were (95.57±4.83)%, (49.96±7.19)%, (94.25±4.32)% and (28.06±6.26)% respectively, showed significant growth inhibition (P<0.01) in the presence of CD gene,and the presence of VP_(22)-CD gene were much more sensitive to 5-FC than the presence of CD gene only(P<0.01). Conclusions VP_(22) increases the cell-killing effect of neural stem cells modified by CD gene in C6 glioma in vitro. Key words: Lentivirus vector; VP_(22) shuttle protein; Neural stem cells; C6 glioma cell

Apoptotic effect of sonodynamic therapy mediated by hematoporphyrin monomethyl ether on C6 glioma cells in vitro

Sonodynamic therapy (SDT) is a new method for treating cancer by inducing cell necrosis and/or apoptosis. However, the molecular mechanisms of cell apoptosis after SDT treatment remain unclear. In this study we investigated the apoptotic effect and mechanisms of SDT mediated by hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. The time of ultrasound irradiation was optimized by MTT. The apoptotic rate, generation of ROS (reactive oxygen species), and mitochondrial membrane potential (MMP) were detected by flow cytometry, and expressions of apoptosis proteins were measured by Western blot after SDT treatment. The results displayed that treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis, which was associated with the production of ROS and loss of MMP. The results also showed that protein expression of Bax, caspase-9, and caspase-3 significantly increased, protein expression of Bcl-2 and Fas-L significantly decreased, and Fas protein expression was unchanged after SDT treatment. Our results suggested that the mitochondrial signal pathway may play a pivotal role in the apoptosis induced by SDT in C6 glioma cells.

Shadows across μ‐Star? Constitutively active μ‐opioid receptors revisited

Constitutively active μ‐opioid receptors (μ* receptors) are reported to be formed following prolonged agonist treatment of cells or whole animals. μ* receptors signal in the absence of activating ligand and a blockade of μ* activation of G‐proteins by naloxone and naltrexone has been suggested to underlie the profound withdrawal syndrome precipitated by these antagonists in vivo. In this issue of the Journal, Divin et al. examined whether treatment of C6 glioma cells with μ‐opioid receptor agonists produced constitutively active μ‐opioid receptors or other commonly reported adaptations to prolonged agonist treatment. Adenylyl cyclase superactivation was readily apparent following agonist treatment but there was no evidence of the formation of constitutively active μ‐opioid receptors. This result challenges the notion that prolonged agonist exposure inevitably produces μ* receptors, and is consistent with many studies of adaptations in neurons produced by chronic agonist treatment. The investigators provide no explanation of their failure to see μ* receptors in C6 cells, but this is perhaps understandable because the molecular nature of μ* receptors remains elusive, and the precise mechanisms that lead to their formation are unknown. Without knowing exactly what μ* receptors are, how they are formed and how they signal, understanding their role in cellular adaptations to prolonged opioid treatment will remain impossible. Studies such as this should refocus attention on establishing the molecular mechanisms that underlie that phenomenon of μ* receptors.

Dexamethasone inhibits proliferation and stimulates ecto-5′-nucleotidase/CD73 activity in C6 rat glioma cell line

Malignant gliomas are the most common and devastating primary tumors of the adult central nervous system. Dexamethasone, a synthetic glucocorticoid, is commonly co-administered to control edema in the management of brain tumors during chemotherapy and radiotherapy. In the present study, the effect of dexamethasone on proliferation and ectonucleotidase activities in rat C6 glioma cell line was investigated. Dexamethasone concentrations ranging from 0.001 to 10 microM induced a time- and concentration-dependent inhibition of C6 rat glioma cell proliferation after 24, 48 and 72-h treatment. The tetrazolium reduction assay (MTT) indicated a reduction of in cell viability (44 +/- 7.6%) after 48-h treatment with 1 microM dexamethasone. Pretreatment with 10 microM of RU38486, an antagonist of glucocorticoid receptors, abolished the effect of 1 microM dexamethasone by 78 +/- 9.8% after 48 h of treatment, indicating that this action is mediated via the glucocorticoid receptor. Members of the E-NTPDase family and ecto-5'-nucleotidase/CD73 can modulate extracellular ATP degradation and adenosine formation, both of which have been described as proliferation factors. Treatment of C6 glioma cells for 48 h with 1 microM dexamethasone increased in 38 +/- 8.09% the AMP hydrolysis and in 3.7-fold the ecto-5'-nucleotidase/CD73 expression, suggesting an increase in adenosine formation and, therefore, a possible modulatory role in the elicitation of cell death responses. In addition, pretreatment with 5 microM GF 109203X, a protein kinase C (PKC) inhibitor, abolished the effect of dexamethasone on cell proliferation and on ecto-5'-NT activity, suggesting that dexamethasone could exert this action via PKC. The alterations in the catabolism of extracellular purines induced by dexamethasone treatment in glioma C6 cells could be related to its pharmacological effects.

Rho/ROCK pathway as a target of tumor therapy

This study emphasizes the importance of Rho/ROCK pathway in lovastatin-induced apoptosis as replenishment with exogenous isoprenoid, geranylgeranylpyrophosphate (GGPP), resulted in inhibition of apoptosis in cultured tumor cells. Treatment of C6 glioma cells with Toxin B and exoenzyme C3 resulted in cell death suggesting the role of geranylgeranylated protein(s) in the survival of glioma cells. Relative apoptotic death observed in cells transfected with dominant negative constructs of RhoA, Rac, and cdc42 imply Rho A as playing the major role in cell survival. Furthermore, the inhibition of Rho A kinase (ROCK), a direct downstream effector of Rho A, by Y-27632 or dominant negative of ROCK, induced apoptosis in glioma cells. These findings indicate that RhoA/ROCK pathway is involved negatively in the regulation of glioma cell death pathway. Moreover, in vivo studies of lovastatin treatment in animals implanted with C6 glioma cell tumors also resulted in smaller tumor size and induced apoptosis in the tumor tissue. The implantation of stably transfected C6 glioma cells with expression vector of C3 exoenzyme, dominant negative of RhoA and ROCK, resulted in significant smaller tumor mass, further establishing the importance of geranylgeranylated proteins, specifically RhoA and its downstream effecter ROCK, in cell survival and tumor genesis.

The Role of Protein Kinase C and Cyclic AMP in the Ammonia-Induced Shift of the Taurine Uptake/Efflux Balance Towards Efflux in C6 Cells

A previous study showed that treatment of C6 glioma cells with 10 mM ammonium chloride monia") for 24 h decreases taurine uptake and evokes sodium-dependent taurine efflux, indicating reversal of the taurine transporter (TauT)-mediated transport as an underlying mechanism. Consistent with the involvement of TauT we now show that the ammonia-induced changes in Tau uptake and efflux are inhibited by the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). Ammonia treatment of C6 cells resulted in increased intracellular accumulation of cAMP. Incubation of the cells with dibutyryl cAMP (dbcAMP) mimicked the effects of ammonia on both taurine uptake and efflux. The effects of dbcAMP on taurine uptake and efflux were additive to the effects of ammonia. Collectively, the results suggest that the effects of ammonia on taurine uptake and efflux may be partly mediated by cAMP. Consistent with this mechanism, the adenyl cyclase inhibitor, miconazole reduced the stimulation of efflux by ammonia.

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline.

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.

Ceramide in Nitric Oxide Inhibition of Glioma Cell Growth: EVIDENCE FOR THE INVOLVEMENT OF CERAMIDE TRAFFIC

The treatment of C6 glioma cells with the nitric oxide donor, PAPANONOate ((Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate), resulted in a dose-dependent inhibition of cell proliferation. This was associated to a rapid and significant increase of ceramide levels and was mimicked by treatments that augment cellular ceramide. Metabolic experiments with radioactive sphingosine, serine, and choline showed that nitric oxide strongly reduced the utilization of ceramide for the biosynthesis of both sphingomyelin and glucosylceramide. Nevertheless, nitric oxide did not modify the activity of different enzymes of ceramide metabolism. The possibility that nitric oxide impairs the availability of ceramide for sphingolipid biosynthesis was then investigated. The metabolism of N-hexanoyl-[(3)H]sphingosine demonstrated that nitric oxide did not affect the biosynthesis of N-hexanoyl-[(3)H]sphingolipids but inhibited the metabolic utilization of long chain [(3)H]ceramide, synthesized in the endoplasmic reticulum (ER) from the recycled [(3)H]sphingosine. Moreover, results obtained with fluorescent ceramides, brefeldin A, ATP depletion, as well as in a ceramide transport assay indicate that nitric oxide impairs the traffic of ceramide from ER to Golgi apparatus. All this supports that, in glioma cells, the modulation of ceramide traffic can contribute to the regulation of its intracellular levels and participate in the nitric oxide-activated signaling pathway involved in the control of cell proliferation.

Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac) 5-GP, was produced by acetylation of a glycoside, isolated from an extract of Gardenia fructus. Previously, we have reported that C6 glioma cells could be inhibited in culturing as well as in bearing rats by treating with (Ac) 5-GP. In this study, the effect and action of (Ac) 5-GP on inducing cell death was examined in rat C6 glioma cells. Treatment of C6 glioma cells with (Ac) 5-GP caused cell death, chromatin condensation, and internucleosomal DNA ladder. Also, cell cycle arrest at G 0/G 1 phase revealed that (Ac) 5-GP-induced cell death appears to be mediated by apoptosis. In addition, the results also showed that p53 and c-Myc increased due to treatment of (Ac) 5-GP in a dose–response and time-dependent manner. Concomitant with the expression of p53 and c-Myc, decreased level of Bcl-2 and increased level of Bax protein were observed. These results suggest that cell death caused by (Ac) 5-GP through apoptosis and cell cycle arrest at G 0/G 1 may be associated with the induction of p53, c-Myc and may be mediated with apoptosis-related Bcl-2 family proteins.

Lovastatin induced cytokine‐mediated inducible nitric oxide synthase (INOS) expression in brain transformed cell lines

Nitric oxide (NO) produced by inducible nitric‐oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of neurodegenerative diseases. The present study underlines the involvement of RhoA protein in the expression of iNOS via NF‐κB in transformed brain cell lines [rat C6 glioma, human astrocytoma (T98G and A172) and immortal rat astrocytes]. Treatment of C6 glioma cells with lovastatin resulted in the induction of iNOS mRNA and increased NO production in a dose and time dependent manner. Addition of mevalonate and geranylgeranylpyrophosphate reversed the lovastatin‐mediated effect. A selective inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further enhanced the lovastatin as well as cytokine‐mediated iNOS expression suggesting the involvement of geranylgeranylated proteins in the down regulation of iNOS expression. Inactivators of Rho A family (bacterial toxin B), Rho A protein (C3 toxin from C. botulinum) and Rho A associated kinases (Y‐27632) increased the iNOS expression. Lovastatin induced NO production mediated by increasing IkB? degradation and NF‐κB translocation via inactivation of Rho A protein. Co‐transfection studies using dominant negative form of RhoA along with iNOS luciferase and NF‐κB luciferase reporter constructs further supported these observations. Taken together, these studies document a role for Rho A in lovastatin mediated induction of iNOS expression in brain glial cell lines.Acknowledgements: Supported by NIH grants NS‐22576, NS‐34741 and NS‐37766.

Mechanisms of ligand-induced desensitization of the 5-hydroxytryptamine(2A) receptor.

We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)(2A) receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT(2A) receptor antagonist ketanserin resulted in an attenuation in 5-HT(2A) receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT(2A) receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants beta-arrestin (319-418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT(2A) receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT(2A) receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [(125)I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([(125)I]DOI) and the ability of 5'-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT(2A) receptor to couple to G protein is impaired.

Advanced glycosylation end products induce nitric oxide synthase expression in C6 glioma cells: Involvement of a p38 MAP kinase-dependent mechanism

The mitogen-activated protein kinase (MAPK) pathway is believed to function as an important mediator of inducible nitric oxide synthase (iNOS) expression. In the present study, we investigated the role of the p38 MAPK signaling pathway in advanced glycosylation end products (AGEs)-induced iNOS expression in C6 glioma cells. AGEs caused a dose-dependent increase of nitrite accumulation in C6 glioma cells. The AGEs-stimulated nitrite production from C6 glioma cells was inhibited by actinomycin D, cyclohexamide, and the NO synthase inhibitor, N ω-nitro- L-arginine methyl ester (L-NAME), suggesting that the increase of AGEs-induced nitrite release is due to iNOS up-regulation. Consistently, treatment of C6 glioma cells with AGEs induced iNOS protein expression. AGEs-stimulated nitrite production was inhibited by pretreatment of C6 glioma cells with anti-AGEs antibodies (1:100 or 1:50). The tyrosine kinase inhibitor (genistein and tyrphostin), the Ras-farnesyl transferase inhibitor (FPT inhibitor-II), or the p38 MAPK inhibitor (SB203580) suppressed AGEs-induced iNOS expression and nitrite release from C6 glioma cells. AGEs activated p38 MAPK in C6 glioma cells, and this effect was blocked by genistein (20 μM), tyrphostin (30 μM), FPT inhibitor-II (20 μM), and SB203580 (10 μM). Taken together, our data suggest that AGEs may activate the pathways of tyrosine kinase and Ras to induce p38 MAPK activation, which in turn induces iNOS expression and NO production in C6 glioma cells.