Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Toxic effects of phytol and retinol on human glioblastoma cells are associated with modulation of cholesterol and fatty acid biosynthetic pathways.

Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.

Characteristics of sulfasalazine-induced cytotoxicity in C6 rat glioma cells.

Glioblastoma is the most aggressive primary brain tumor. Surgical resection, radiotherapy and temozolomide (TMZ), an alkylating agent, is the standard of care. Glioma cells may synthetize the antioxidant glutathione by importing cystine through a cystine/glutamate antiporter, which is inhibited by sulfasalazine (SAS). C6 rat glioma cells are largely used in in vitro and in vivo models for developing new glioblastoma treatment strategies. We treated C6 cells with 25µM TMZ and/or 0.25mM or 0.5mM SAS for 1, 3 or 5days and evaluated viability, apoptosis, total glutathione levels and metalloproteinase MMP2 and MMP9 activities. TMZ treatment slightly reduced cell viability by 9.5% compared with vehicle treatment (0.1% dimethyl sulfoxide) only after 5days. In addition, TMZ did not modify apoptosis, glutathione content or MMP2/MMP9 activities. The 0.25mM SAS treatment reduced cell viability by 31.1% and 19.4% after the first and third days, respectively. This effect was not sustained after the fifth day of treatment. In contrast, 0.5mM SAS caused a reduction in cell viability by nearly 100%, total glutathione depletion and apoptosis induction. Moreover, the effect of 0.5mM SAS was greater than that of TMZ in terms of cell viability reduction, total glutathione depletion and apoptosis induction. MMP9 activity was reduced by 40% after 5days of 25µM TMZ and 0.5mM SAS co-administration. Considering previous data from our group, we verified that the cellular viability results differed between rat and human cells; C6 cells were more vulnerable to 0.5mM SAS than human A172 and T98G glioblastoma lineages. We propose that C6 cells may not be appropriate for studying human glioblastoma and that the results obtained using these cells should be interpreted with caution.

Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

JAK2 inhibition is neuroprotective and reduces astrogliosis after quinolinic acid striatal lesion in adult mice.

Quinolinic acid (QA) striatal lesion in rodents induces neuronal death, astrogliosis and migration of neuroblasts from subventricular zone to damaged striatum. These phenomena occur in some human neurodegenerative illnesses, but the underlying mechanisms are unknown. We investigated the effect of AG490, a Janus-kinase 2 (JAK2) inhibitor, on astrogliosis, neuronal loss and neurogenesis in the striatum of adult mice after unilateral infusion of QA (30 nmol). Animals were given subcutaneous injections of AG490 (10 mg/kg) or vehicle immediately after lesion and then once daily for six days. Brain sections were used for neuronal stereological quantification, immunohistochemical and Western Blotting analyses for GFAP and doublecortin, markers of astrocytes and neuroblasts, respectively. The total area of doublecortin-positive cells (ADPC) and the number of neurons (NN) in the lesioned (L) and contralateral (CL) sides were evaluated. Neurogenesis index (NI=ADPC(L)/ADPC(CL)) and neuronal ratio (NR=NN(L)/NN(CL)) were calculated. After QA administration, blotting for GFAP showed an ipsilateral decrease of 19% in AG490- vs vehicle-treated animals. NR was 25% higher in mice given AG490 vs controls given vehicle. NI showed a decrease of 21% in AG490- vs vehicle-treated mice. Our results indicate that JAK2 inhibition reduces QA lesion and suggest that astrogliosis may impair neuronal survival in this model.