Federica Susta

Dietary alpha-linolenic acid reduces COX-2 expression and induces apoptosis of hepatoma cells.

Fatty acid synthetase (FAS) is overexpressed in various tumor tissues, and its inhibition and/or malonyl-CoA accumulation have been correlated to apoptosis of tumor cells. It is widely recognized that both ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) depress FAS expression in liver, although epidemiological and experimental reports attribute antitumor properties only to ω-3 PUFA. Therefore, we investigated whether lipogenic gene expression in tumor cells is differently regulated by ω-6 and ω-3 PUFAs. Morris hepatoma 3924A cells were implanted subcutaneously in the hind legs of ACI/T rats preconditioned with high-lipid diets enriched with linoleic acid or α-linolenic acid. Both-high lipid diets depressed the expression of FAS and acetyl-CoA carboxylase in tumor tissue, this effect correlating with a decrease in the mRNA level of their common sterol regulatory element binding protein-1 transcription factor. Hepatoma cells grown in rats on either diet did not accumulate malonyl-CoA. Apoptosis of hepatoma cells was induced by the α-linolenic acid-enriched diet but not by the linoleic acid-enriched diet. Therefore, in this experimental model, apoptosis is apparently independent of the inhibition of fatty acid synthesis and of malonyl-CoA cytotoxicity. Conversely, it was observed that apoptosis induced by the α-linolenic acid-enriched diet correlated with a decrease in arachidonate content in hepatoma cells and decreased cyclooxygenase-2 expression.

Mortalin inhibition in experimental Parkinson's disease†

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinsons disease. In the present work a rat model of Parkinsons disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinsons disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6‐hydroxydopamine rat model of Parkinsons disease. The electrophysiological effects of MKT‐077, a rhodamine‐123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham‐operated, and 6‐hydroxydopamine–denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6‐hydroxydopamine–treated rats in comparison with sham‐operated animals. MKT‐077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT‐077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinsons disease.

Protein expression changes induced in murine peritoneal macrophages by Group B Streptococcus

Protein expression changes induced in thioglycolate‐elicited peritoneal murine macrophages (MΦ) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control MΦ and MΦ incubated 2 h with live or heat‐inactivated GBS were separated by 2‐DE. Proteins whose expression was significantly different in infected MΦ, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in MΦ by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS‐infected MΦ. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose‐cycle enzymes being down‐regulated in MΦ infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.

Identification of differentially expressed proteins in papillary thyroid carcinomas with V600E mutation of BRAF

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS‐regulated MAPK pathway. V600E mutation of BRAF protein is the most common genetic alteration occurring in papillary thyroid carcinomas and is prognostic of poor clinicopathological outcomes. Protein expression in the subclass of PTC bearing the BRAFV600E mutation was investigated by using 2‐DE and MS/MS techniques and compared to that of matched normal thyroid tissues from seven patients. 2‐D gel image analysis revealed that the expression of eight polypeptide spots, corresponding to five proteins, were significantly underexpressed in PTC bearing BRAFV600E mutation whereas 25 polypeptides, representing 19 distinct proteins, were significantly upregulated in tumour tissue, as compared to normal thyroid. Among the differentially expressed polypeptides, mitochondrial proteins, ROS‐scavenger enzymes, apoptosis‐related proteins as well as proteins involved in tumour cell proliferation were identified. Although dissimilarities between the present results and those previously reported can be ascribed to the use of different 2‐DE techniques, the possibility that BRAFV600E mutation is responsible for changes in protein expression distinct from those induced by other oncogenes cannot be ruled out.

3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells

Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. SIGNIFICANCE Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug.