Amphiregulin is a factor for resistance of glioma cells to cannabinoid-induced apoptosis.

Gliomas, one of the most malignant forms of cancer, exhibit high resistance to conventional therapies. Identification of the molecular mechanisms responsible for this resistance is therefore of great interest to improve the efficacy of the treatments against these tumors. Delta9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the ability of these compounds to induce apoptosis of tumor cells. By analyzing the gene expression profile of two sub-clones of C6 glioma cells with different sensitivity to cannabinoid-induced apoptosis, we found a subset of genes with a marked differential expression in the two sub-clones. Furthermore, we identified the epidermal growth factor receptor ligand amphiregulin as a candidate factor to mediate the resistance of glioma cells to cannabinoid treatment. Amphiregulin was highly overexpressed in the cannabinoid-resistant cell line, both in culture and in tumor xenografts. Moreover, in vivo silencing of amphiregulin rendered the resistant tumors xenografts sensitive to cannabinoid antitumoral action. Amphiregulin expression was associated with increased extracellular signal-regulated kinase (ERK) activation, which mediated the resistance to THC by blunting the expression of p8 and TRB3-two genes involved in cannabinoid-induced apoptosis of glioma cells. Our findings therefore identify Amphirregulin as a factor for resistance of glioma cells to THC-induced apoptosis and contribute to unraveling the molecular bases underlying the emerging notion that targeted inhibition of the EGFR pathway can improve the efficacy of antitumoral therapies.

Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes.

Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Oleic Acid and Hydroxytyrosol Inhibit Cholesterol and Fatty Acid Synthesis in C6 Glioma Cells.

Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives for a healthier brain. Here, we investigated the effects of oleic acid (OA) and hydroxytyrosol (HTyr), two important extra virgin olive oil compounds, on lipid synthesis in C6 glioma cells. OA and HTyr inhibited both de novo fatty acid and cholesterol syntheses without affecting cell viability. The inhibitory effect of the individual compounds was more pronounced if OA and HTyr were administered in combination. A reduction of polar lipid biosynthesis was also detected, while triglyceride synthesis was marginally affected. To clarify the lipid-lowering mechanism of these compounds, their effects on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase-ACC and fatty acid synthase-FAS) and cholesterologenesis (3-hydroxy-3-methylglutaryl-CoA reductase-HMGCR) were investigated in situ by using digitonin-permeabilized C6 cells. ACC and HMGCR activities were especially reduced after 4 h of 25 µM OA and HTyr treatment. No change in FAS activity was observed. Inhibition of ACC and HMGCR activities is corroborated by the decrease of their mRNA abundance and protein level. Our results indicate a direct and rapid downregulatory effect of the two olive oil compounds on lipid synthesis in C6 cells.

Stimulation of myelin proteolipid protein gene expression by eicosapentaenoic acid in C6 glioma cells.

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 microM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways.

Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells.

Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-(14)C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced ( approximately 80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.