Search

Guar gum-carboxymethyl cellulose derived piperine nanocomposite (GG-CMC@PIP) was synthesized to develop a phytochemical-based nanomaterial by following a simple synthetic procedure. In this study, piperine, an alkaloid prodrug was nanonized by entrapping within guar gum-carboxymethyl cellulose derived nanocomposite (GG-CMC). GG-CMC@PIPs were of (i) size (25 ± 3) nm by TEM, (ii) zeta potential (−) 33.1 ± 0.73 mV and (iii) entrapment efficiency 86 ± 0.46 %. High loading percentage of piperine in polymeric nanocomposites revealed excellent incorporation efficiency. In addition, pH responsive release behaviour of piperine from nanocomposites exhibited sustained but faster release pattern in pH 5.5. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of piperine loaded nanocomposite on Gram positive Enterococcus faecalis and Gram-negative Escherichia coli , Pseudomonas aeruginosa were in the range of 200–400 μg/ml. IC 50 value of GG-CMC@PIP was found to be around 500 μg/ml against C6 glioma cell line. Experimental outcome confirmed excessive accumulation of reactive oxygen species (ROS) mediated cytotoxicity, mitochondrial depolarization and higher degree of nuclear damage followed by increased mortality of C6 glioma cell after treatment with piperine-loaded nanocomposite. [Display omitted] • Synthesis of piperine incorporated guar gum-carboxymethyl cellulose derived bio nanocomposite using nostabilizer. • High drug loading efficiency and sustained release behaviour of piperine from nanocomposite is observed. • Antibacterial activity of nanocomposite was observed due to oxidative stress-mediated loss of cellular integrity. • Depolarized mitochondrion, elevated ROS level, nuclear damage revealed prominent cytotoxic activity [ABSTRACT FROM AUTHOR]

Anesthetics are documented to affect tumors; therefore, we studied the antiglioma effect of propofol on proliferation and invasiveness of glioma cells and explored the underlying mechanism. C6 glioma cells were cultured and treated with propofol, and cell viability, invasiveness, and migration were measured. Glutamate release was measured using an enzyme-catalyzed kinetic reaction. xCT protein and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR2 subunit protein expression was assessed with Western blot analysis and immunofluorescent staining. We observed that propofol significantly inhibited C6 glioma cell viability, invasiveness, and migration and decreased glutamate release. An agonist of the cystine/glutamate antiporter system (system x c − ), N -acetylcysteine (NAC), reversed propofol's effects, and propofol could inhibit C6 glioma cell proliferation by adding excess exogenous glutamate (100 μM). Finally, propofol increased the surface expression of GluR2, but decreased surface expression of xCT. The effects of propofol on surface expression of GluR2 and xCT could be rescued by (R, S)-AMPA, an agonist of Ca 2+ permeable AMPA receptor (CPAR). Thus, propofol can inhibit cell viability, invasiveness, and migration of C6 glioma cells, and the CPAR-system x c − pathway contributes to these events. [ABSTRACT FROM AUTHOR]

2,3-Dihydro-7-hydroxy-2R*,3R*-dimethyl-2-[4,8-dimethyl-3(E),7-nonadienyl]-furo[3,2-c]coumarin (named DAW22), a sesquiterpene coumarin isolated from the roots of Ferula ferulaeoides (Steud.) Korov., has been reported to bear anti-proliferative activities toward different types of cancer cells. In this study, we demonstrated that DAW22 induced apoptosis in C6 glioma cells. Subsequently, we found that DAW22-induced apoptosis in C6 glioma cells occurred via the mitochondria-mediated and death-receptor pathways. Moreover, we found a massive cytoplasmic vacuolization, a dramatic change of endoplasmic reticulum (ER), up-regulation of CHOP and cleavage of caspase-12, suggesting that DAW22-induced apoptosis is involved in ER stress. In addition, we revealed that DAW22 treatment induced the activation of PERK, ATF6α and IRE1α. We further found that knockdown of CHOP affected DAW22-induced apoptosis, and DAW22-stimulated down-regulation of Bcl-2, caspase-8 activation and PARP cleavage were inhibited. Taken together, these results demonstrate that DAW22 induces apoptosis by ER stress and mitochondrial/death-receptor pathways, which may provide a new clue for exploiting this compound as a potential anti-neoplastic drug in future glioma cancer therapy. [ABSTRACT FROM AUTHOR]

Introduction Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms. Methods We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert -butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate assays. Results There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI). Discussion The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements. [ABSTRACT FROM AUTHOR]

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6 + 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion. [ABSTRACT FROM AUTHOR]

Abstract: Short/branched chain acyl-CoA dehydrogenase (SBCAD) deficiency is an autosomal recessive disorder of isoleucine metabolism biochemically characterized by accumulation of 2-methylbutyrylglycine (2MBG) and 2-methylbutyric acid (2MB). Affected patients present predominantly neurological symptoms, whose pathophysiology is not yet established. In the present study, we investigated the in vitro effects of 2MBG and 2MB on important parameters of oxidative stress in cerebral cortex of young rats and C6 glioma cells. 2MBG increased thiobarbituric acid-reactive species (TBA-RS), indicating an increase of lipid oxidation. 2MBG induced sulfhydryl oxidation in cortical supernatants and decreased glutathione (GSH) in these brain preparations, as well as in C6 cells, indicating a reduction of nonenzymatic brain antioxidant defenses. In contrast, 2MB did not alter any of these parameters and 2MBG and 2MB did not affect carbonyl formation (protein damage). In addition, 2MBG-induced increase of TBA-RS levels and decrease of GSH were prevented by free radical scavengers, implying that reactive species were involved in these effects. Furthermore, the decrease of GSH levels caused by 2MBG was not due to a direct oxidative action since this metabolite did not alter sulfhydryl content from a commercial solution of GSH. Nitric oxide production was not altered by 2MBG and 2MB, suggesting that reactive oxygen species possibly underlie 2MBG effects. Finally, we verified that 2MBG did not induce cell death in C6 cells. The present data show that 2MBG induces lipid oxidative damage and reduces the antioxidant defenses in rat brain. Therefore, it may be postulated that oxidative stress induced by 2MBG is involved, at least in part, in the pathophysiology of the brain damage found in SBCAD deficiency. [Copyright &y& Elsevier]

In this study we investigated the anti-inflammatory effects of chronic ethanol (EtOH) treatment on lipopolysaccharide (LPS)-stimulated C6 glioma cells. The cells were chronically treated with 200 mM EtOH; coincubation with LPS and EtOH was obtained upon addition of 2 μg/ml LPS to the incubation medium in the last 24 h of EtOH exposure. We found that EtOH prevented the LPS-induced production of tumor necrosis factor α (TNFα) without decreasing cell viability. Either LPS treated or EtOH plus LPS treated cells presented upregulated glial fibrillary acidic protein (GFAP) and downregulated vimentin levels characterizing a program of reactive astrogliosis. Also, EtOH plus LPS stimulation greatly increased the oxidative stress generation evaluated by DCF-DA measurement, while either EtOH alone or LPS alone was unable to induce oxidative stress. Western blot analysis indicated that either EtOH, LPS or EtOH plus LPS treatments are unable to affect Akt/GSK3β signaling pathway. However, LPS alone and EtOH plus LPS co-treatment inhibited Erk phosphorylation. A dramatic loss of stress fibers was found in EtOH exposed cells, evaluated by cytochemistry using phalloidin-fluorescein. However, LPS alone was not able to disrupt actin organization. Furthermore, cells co-incubated with LPS and EtOH presented reversion of the disrupted stress fibers provoked by EtOH. Supporting this action, RhoA and vinculin immunocontent were upregulated in response to EtOH plus LPS. Interestingly, EtOH suppresses the inflammatory cascade (TNFα production) in response to LPS. Concomitantly it sustains Erk inhibition, increases oxidative stress generation and induces reactive astrogliosis in the presence of LPS, conditions associated with neurotoxicity. The effects observed were not supported by actin reorganization. Altogether, these findings suggest that Erk signaling inhibition could play a role in both suppressing TNFα production and inducing oxidative stress generation and astrogliosis, therefore modulating a dual action of EtOH plus LPS in glial cells. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the susceptibility of the group II metabotropic glutamate receptor mGlu2 to agonist-induced desensitization, the receptor was stably expressed in Chinese hamster ovary (CHO-mGlu2) or C6 glioma cells (C6-mGlu2). Exposure of CHO-mGlu2 cells to the group II mGlu receptor agonist (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (LCCG-1; 10μM) for up to 15h did not affect the subsequent ability of LCCG-1 to inhibit forskolin-stimulated cAMP accumulation. Similarly, in C6-mGlu2 cells, prolonged exposure to LCCG-1 also did not affect the subsequent ability of LCCG-1 to inhibit cAMP formation. In contrast, exposure of CHO-mGlu2 cells to the protein kinase C activator phorbol myristate acetate (PMA) suppressed the ability of LCCG-1 to inhibit cAMP formation. Using an in vitro model of group II mGlu receptor activity, the hemisected neonatal rat spinal cord preparation, the ability of the selective group II agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC) to depress the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) did not desensitize when applied for up to 2h. Together these results indicate that in contrast to most G protein-coupled receptors, the mGlu2 receptor is resistant to agonist-induced homologous desensitization, and that in vitro data suggests that resistance to desensitization is a physiologically relevant property of this mGlu receptor subtype. [ABSTRACT FROM AUTHOR]

Abstract: Neurons and glial cells in the brain are surrounded by a fractal environment. A fractal alkylketene dimmer (AKD) surface was shown to provide such a biomimetic environment for glial cell culture. However, little is known about the effects of fractal surface on the complexity of cell morphology. In particular, whether fractal surface induces glial cell differentiation remains to be elucidated. The present work, thus determined the fractal dimension (FD) of cell complexity with a geometrically calculational parameter, the expressions of GFAP gene and protein in C6 glioma cells on fractal AKD, non-fractal AKD and PLL-coated surfaces. Fractal surface suppressed the proliferation of glioma cell, and significantly increased the length and number of cell process. Furthermore, the enhanced values of FD were accompanied with the expressions of GFAP gene and protein, especially that of gene. However, cells on non-fractal and PLL surface proliferated gradually along with the culture time, showing the fibroblast-like morphology, and accompanied with the consistent expressions of GFAP gene and protein. These results suggested that C6 glioma cell differentiation can be induced by fractal AKD surface. [Copyright &y& Elsevier]

Abstract: Although there is ample evidence that dexamethasone (DEX) has an antiproliferative effect on C6 glioma cells, the molecular mechanism remains elusive. Src suppressed C kinase substrates (SSeCKS), as a member of PKC substrates, have been implicated to be a negative regulator of cell proliferation. In this study, we provided novel evidence that DEX induced the expression of SSeCKS mRNA and protein in a time- and dose-dependent manner, and translocation of SSeCKS from the cytosol to the membrane. The glucocorticoid receptor antagonist, RU486, significantly decreased DEX-induced SSeCKS expression, inhibited SSeCKS translocation and actin cytoskeleton reorganization after DEX challenge. Knock-down of SSeCKS expression by RNA interference inhibited DEX-induced actin cytoskeleton reorganization and reversed DEX-induced growth arrest. We also presented the novel observation that knock-down of SSeCKS expression elevated the expression of cyclin D1 and the phosphorylation of extracellular signal-regulated Kinase 1/2, indicating that SSeCKS is involved in the regulation of cell cycle related proteins and is essential for DEX induced growth arrest. [Copyright &y& Elsevier]

Abstract: Polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate-80 have been extensively studied for delivery of drugs into the animal models; however, 1% polysorbate-80 coated gemcitabine PBCA nanoparticles (GCTB-PBCA-NPs) remain unknown. In this study, we investigated the inhibitory effects of brain targeted 1% polysorbate-80 coated GCTB-PBCA-NPs on C6 glioma cells in vitro and in vivo. GCTB-PBCA-NPs were prepared by emulsion polymerization and freeze drying. C6 glioma cells treated with 1% polysorbate-80 coated GCTB-PBCA-NPs showed poor growth with less cell density and increased detachment. Cell morphology was also greatly altered with nuclear vacuoles, ruptured cells and dead cells. Meanwhile, by flow cytometry, the numbers of cells treated with 1% polysorbate-80 coated GCTB-PBCA-NPs showed increase in G0/G1 phase and decreased in the S phase (P <0.01) compared with the blank control. CCK-8 assay also showed that GCTB could significantly inhibited cell proliferation in a concentration dependent manner. Finally, various preparations were injected (90 mg preparation per kg body weight) into the brain tumor model, which was produced after inoculating C6 glioma cells into Sprague Dawley (SD) rats for 14 days, it was shown that 1% polysorbate-80 coated GCTB-PBCA-NPs could significantly extend the survival time compared with the saline control (P <0.05). Taken together, 1% polysorbate-80 coated GCTB-PBCA-NPs can effectively inhibit the growth of C6 glioma cells in vitro and enhance antitumor activity on brain tumor in vivo. [Copyright &y& Elsevier]

Abstract: Different cell types response differently to toxic insult. In a previous study, it was demonstrated that the C6 glioma cell is more sensitive to Cd induced oxidative stress than the HepG2 cells. To explain the difference between the two cell lines in their response to oxidative stress, it was hypothesized that the activity of glutathione metabolizing enzymes may be different. The objective of this study is to determine the activities of glutathione peroxidase (GPx) and glutathione reductase (GR) in the two cell lines and to explain how these differences may affect the susceptibility of the two cells to oxidative stress. In the HepG2 cells, the activity of GPx was 2.24±0.18μmol/mgprotein/min and that for GR was 5.63±0.58μmol/mgprotein/min. For the C6 glioma cells, GPx and GR activities were 1.29±0.14 and 1.07±0.11μmol/mgprotein/min, respectively. Using the kinetic equilibrium: K eq =([GSSG]×[NADPH]×[H+])/([GSH]2 ×[NADP+]), and the GSH/GSSG previously published (HepG2: 2.6 and C6 glioma: 3.6), resting NADPH/NADP+ for the cell lines were calculated. The results showed that NADPH/NADP+ for HepG2 cells (17.8) is higher than that in the C6 glioma cells (10.8). These data supported the notion that the reducing power (NADPH/NADP+) in the HepG2 cells is higher than that in the C6 glioma cell and thus, the later would be more susceptible to oxidative stress. The results also suggested that besides GSH/GSSG, the activities of GPx and GR are important in predicting tissue redox state. Applying this hypothesis to animal tissues, the ratio of the activities of the two enzymes in mouse liver, cerebral cortex, hippocampus and cerebellum were measured. It was demonstrated that the activities of GPx and GR were different in the different tissues studied. The possible correlation between enzymatic activities and the redox state in the different tissues were discussed. [Copyright &y& Elsevier]

Abstract: Caffeic acid phenethyl ester (CAPE), a component of honeybee propolis, has been reported to hold various biochemical responses. In the preliminary study, we found that CAPE inhibited the growth of C6 glioma cells in a dose dependent and time dependent manner as shown by the results of trypan blue dye exclusion assay and cell proliferation assay. In addition, the cell number percentage of the G0/G1 phase increased to 85% after the treatment with 50μM of CAPE for 24h. After treatment with CAPE (50μM) for 6h, it demonstrated that the protein level of hyperphosphorylated pRb decreased, and cyclin dependent kinase inhibitors p21, p27, and p16 were marked up-regulated. The association of CDK2 and cyclin E that affects the CDK2 activity decreased. When C6 cells were grown as xenografts in nude mice, treatment with CAPE (1–10mg/kg; ip) induced a significant dose dependent decrease in tumor growth by evaluating tumor volume and tumor weight. Histochemical and immunohistochemical analysis revealed that CAPE treatment significantly reduced the number of mitotic cells and proliferating cell nuclear antigen (PCNA)-positive cells in C6 glioma. These results suggest that CAPE presents an antitumor potential for glioma by inhibiting the growth of tumor cells. [Copyright &y& Elsevier]

Abstract: Cp is an acute phase reactant protein that also acts as a ferroxidase, and thus indirectly decreases the production of the reactive oxygen species hydroxyl radical. Ceruloplasmin (Cp) expression is induced by a variety of central nervous system injuries, but the mechanism by which this occurs is unclear. Based on the fact that peripheral nerve injury induces interleukin-6 (IL-6) expression and that there are three IL-6 response elements in the upstream region of the Cp gene, we studied their role in transcriptional regulation of Cp in astrocytic C6 glioma cells, using transfection of a rat Cp-luciferase construct, followed by sequential and simultaneous mutation of the IL-6 response elements. We found that 0.8 kb of sequence upstream to the rat ceruloplasmin start site was sufficient to drive luciferase expression in C6 glioma cells. Cells transfected with Cp-luc and treated with 100 ng/ml rat IL-6 induced 216.8% ± 4.6% of control activity. Mutagenesis of the IL-6 response elements decreased luciferase activity, with the maximal decline (9.7 ± 0.7% of wild-type) after mutation of the second site. Mutagenesis of multiple sites decreased activity beyond mutagenesis of single sites with mutation of all three sites decreasing activity to 5.3 ± 0.4% of wild-type. Gel shift and supershift assays indicated that activation of Cp in these cells was not via STAT-3. These results are consistent with a signaling process via IL-6 response elements for Cp upregulation. [Copyright &y& Elsevier]

Heme oxygenase-1 (HO-1) is a 32-kDa stress induced enzyme that degrades heme to carbon monoxide (CO) and biliverdin. By employing RT-PCR and Western blotting techniques, we have examined the HO-1 induction in C6 glioma cells that were treated with cadmium chloride (CdCl2) or spermine NONOate (SPER/NO). By employing a cell viability assay, we have also examined the cytoprotective effect of HO-1 induction against the cytotoxicity caused by toxic dose of CdCl2. In C6 glioma cells exposed to CdCl2, expression of HO-1 (mRNA and protein) was increased in a dose- and time-dependent manner. Nitric oxide (NO) generated from SPER/NO very rapidly increased HO-1 mRNA expression in the C6 glioma cells. The induction of HO-1 by SPER/NO protected the cells from toxic dose of CdCl2. The up-regulation of HO-1 mRNA expression by CdCl2 was inhibited by a pre-incubation of the cells with actinomycin D, a potent inhibitor of mRNA transcription. Upon the inhibition of elevated HO-1 mRNA expression by the use of zinc protoporphyrin IX (ZnPP), an inhibitor of HO activity, the change of HO-1 mRNA expression by ZnPP was not observed. Thus, the glial cell may respond to CdCl2 toxicity by enhancing the HO-1 expression in its effort to minimize the CdCl2-derived oxidative damage, and to survive. In the glioma cells, when the HO-1 expression was elevated by a prior incubation with SPER/NO, the cell viability against the cytotoxicity of CdCl2 was significantly increased. When the results of our experiment are taken together, we discovered that NO provided a rapid enhancement of HO-1 expression, and it provided a protective effect against CdCl2-derived oxidative injury in the C6 rat glioma cells. [Copyright &y& Elsevier]

The energy and redox states of the HepG2 hepatoma and the C6 glioma cells were studied by quantifying the levels of ATP, ADP, AMP, GSH, and GSSG. These values were used to calculate the energy charge potential (ECP = [ATP + 0.5ADP]/TAN), total adenosine nucleotides (TAN = ATP + ADP + AMP), total glutathione (TG = [GSH + GSSG]/TAN), and the redox state (GSH/GSSG ratio). For comparison between cell types, the level of each energy metabolite (ATP, ADP, and AMP) was normalized against TAN of the respective cell. The results showed that ATP:ADP:AMP ratio was 0.76:0.11:0.13 for the HepG2 cells and 0.80:0.11:0.09 for the C6 glioma cells. ECP was 0.81 ± 0.01 and 0.85 ± 0.01 for the HepG2 and the C6 glioma cells, respectively. GSH/GSSG ratio was 2.66 ± 0.16 and 3.63 ± 0.48 for HepG2 and C6 glioma cells, respectively. TG was 3.2 ± 0.54 for the HepG2 cells and 2.43 ± 0.18 for the C6 glioma cells, indicating that the level of total glutathione is more than two to three times higher than the total energy metabolites in these cell lines. Following a 3-h incubation in medium containing different concentrations of Cd, there was a dose-dependent decrease in cell viability. The 3-h LC50 for the HepG2 cells was 0.5 mM and that for the C6 glioma cells was 0.4 mM. Cellular TAN decreased with a decrease in cell viability. Upon careful analysis of the energy state, there was a significant increase in relative amount of ATP and decrease in ADP and AMP in both cells as Cd concentration increased from 0 to 0.1, 0.2, and 0.6 mM. However, ECP in both cell lines increased, which indicated that the level of high energy phosphate was adequate. There was also a significant increase in TG and a significant decrease in GSH/GSSG in the C6 glioma cells when cells were exposed to as low as 0.1 mM Cd, which suggested that the cellular redox state was compromised. The HepG2 cells, on the other hand, showed no significant change in both TG and GSH/GSSG level until Cd concentration reached 0.6 mM. Results of the present study also showed that there were differences between the two cells in response to the same level of Cd exposure. The C6 glioma cells were more sensitive to Cd-induced injuries. Although there was a decrease in total amount of high energy phosphate as cell viability decreased, the surviving cells were not devoid of high energy phosphates. The relative abundance of ATP amongst the adenosine nucleotide pool and the increase in ECP could be interpreted as a way the cells signal the conservation of energy utilization in response to the damaged mitochondrial function. This move for energy conservation might be the cause of eventual cell death through the process of apoptosis. [Copyright &y& Elsevier]

There is considerable debate at present as to whether the uptake of anandamide (AEA) into cells is by a facilitated transport process or by passive diffusion driven by fatty acid amide hydrolase (FAAH). The possibility that both processes occur, but to different extents depending upon the cell type used, has been difficult to investigate pharmacologically since available compounds show little selectivity between inhibition of AEA uptake and inhibition of FAAH. Recently, three compounds, UCM707 [N-(Fur-3-ylmethyl)arachidonamide], OMDM-1 and OMDM-2 [the 1′-(S)- and 1′-(R)-enantiomers of the 1′-4-hydroxybenzoyl analogue of oleoylethanolamide], selective for the uptake process, have been described and we have used these compounds, together with AM404 [(N-(4-hydroxyphenyl) arachidonoyl amide)] and VDM11 [(5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide]), with the initial aim of determining which mechanism of uptake predominates in C6 glioma and RBL-2H3 cells. AM404 and VDM11 were both found to decrease the uptake of 2 μM AEA into cells (IC50 values 6–11 μM), but they also inhibited rat brain FAAH (IC50 values 1–6 μM). However, when using a different FAAH assay protocol, VDM11 was a much less potent FAAH inhibitor (IC50>50 μM) regardless of the cell type and animal species used. In contrast, we confirmed that UCM707, OMDM-1 and OMDM-2 were weak inhibitors of FAAH (IC50 values >50 μM) under all conditions used. However, their potency as inhibitors of AEA cellular accumulation appears to be largely dependent on the cell type and assay conditions used. In particular, the potency of UCM707 (IC50 value ≥25 μM) was considerably lower than the submicromolar potency previously reported for U937 cells. It is concluded that the cause/effect relationship between AEA uptake and hydrolysis cannot be investigated uniquely by using supposedly selective inhibitors of each process. [Copyright &y& Elsevier]

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidant, anti-inflammation, antiviral action, and anticancer effect. Our previous studies showed that CAPE exhibited significant cytotoxicity in oral cancer cells. Herein we further investigated the cytotoxicity potential of CAPE and the mechanism of its action in C6 glioma cells. The data exhibited that C6 glioma cells underwent internucleosomal DNA fragmentation 24 hr after the treatment of CAPE (50 μM). The proportion of C6 glioma cells with hypodiploid nuclei was increased to 24% at 36 hr after the exposure. Further results showed that CAPE induced the release of cytochrome c from mitochondria into cytosol, and the activation of CPP32. CAPE application also enhanced the expression of p53, Bax, and Bak. Finally, the potential signaling components underlying CAPE induction of apoptosis were elucidated. We found that CAPE activated extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in C6 glioma cells. More importantly, p38 kinase formed a complex with p53 after the treatment of CAPE for 0.5 hr. The expression of p53, phospho-serine 15 of p53, and Bax, and inactivate form of CPP32 was suppressed by a pretreatment of a specific p38 MAPK inhibitor, SB203580. The resultant data suggest that p38 MAPK mediated the CAPE-induced p53-dependent apoptosis in C6 glioma cells. [Copyright &y& Elsevier]

Time-course effects of lipopolysaccharide (LPS) with phorbol-12-myristate-13-acetate (PMA) were quantitatively examined using an atomic force microscope (AFM) cantilever with a type I collagen-coated spherical bead as a probe. LPS/PMA affects the expression level of fibronectin (FN), increasing the adhesion strength for collagen type I about 1.7 times. After 4 days, the adhesion strength for collagen increased 1.6 times. Our method is suitable for quantitative studies of cell adhesion phenomena on the single cell level. [Copyright &y& Elsevier]

C6 glioma cells were treated for hours with 100 μM l-glutamate, quisqualate or trans-ACPD. In all cases, phospholipase C-coupled metabotropic glutamate receptors (mGluRs) present in these cells are down-regulated after this agonist treatment. Cell surface metabotropic glutamate receptor density was minimum at 6 h of agonist treatment and reached near control values after 30 h of treatment. This recovery was associated with a progressive increase in mGluR1 and mGluR1a mRNA level between 6 and 24 h and was not due to agonist removal. Specific l-[3H]glutamate or [3H](±)trans-ACPD binding decrease detected in C6 cells after 6 h of 100 μM l-glutamate treatment was associated with a remarkable increase of specific l-[3H]glutamate binding detected in clathrin-coated vesicles isolated from these treated cells. Moreover, this decrease was blocked in the presence of 0.5 M sucrose or 1 μM phenylarsine oxide, suggesting that desensitization and down-regulation of mGluR can be due to an endocytosis process through clathrin-coated pits and vesicles. [Copyright &y& Elsevier]