Apoptosis related microRNAs and MGMT in glioblastoma cell lines submitted to treatments with ionizing radiation and temozolomide

AimTo evaluate the effect of radiotherapy and temozolomide on the expression of miRNAs apoptotic (miRNAs-21, -221, -222 (anti-apoptotic) and miRNAs-15a, -16 (pro-apoptotic)) and the gene MGMT in glioblastoma cell lines.BackgroundThe limited knowledge of the molecular biology of malignant gliomas may hinder the development of therapeutic modalities. In this scenario, one of the greatest advances of recent years was the identification of microRNAs. These molecules have an important role in biological processes involving cancer, including glioblastoma.Materials and methodsTrypan blue was used to verify the cell viability, and real time PCR to quantify the expression of microRNAs and gene 24, 48 and 120 h after exposure to treatments.ResultsThere was a statistically significant decrease of expression of miR-15a between 48 and 120 h in line T98 G treated with radiation, increased expression of miR-15a between 24 and 120 h in line U251 treated with radiation and temozolomide, and increased expression of miR-16 between 24 and 120 h in line U251 treated with radiation alone and when combined with temozolomide. There was a decrease in MGMT gene expression, between 24 and 48 h in U343 cells treated with temozolomide.ConclusionsIonizing radiation and temozolomide modified the expression of miRNAs studied and MGMT

Apoptosis in Glioma Cells Treated with PDT

Background: Despite significant advances in neurosurgical techniques, the median survival time of patients with glioblastoma has improved little over the past 50 years and remains less than one year. Photodynamic therapy (PDT) is presently established as a widely accepted modality for the treatment of a variety of solid tumors. Objectives: This study evaluated the effect of PDT-Photogem (R) on five glioma cell lines (U87, U138, U251, U343, and T98G). Methods: The experiments were carried out in 25-cm(3) flasks with different groups of cells seeded at a density of 1 x 10(5) cells per flask. After 3 h, the medium was removed, and the cells were incubated for 4 h with Photogem (5 mu g/mL). After the incubation time, the photosensitizer-containing medium was removed and the cells were irradiated with LED (630 nm, 25 mW/cm(2), 25 J/cm(2)) devices for 17 min. For the final steps of the PDT, the cells were returned to the incubator and kept at 37 degrees C with 5% CO(2) for 24 h, the cell viability assay was assessed using the trypan blue method, and the expression of Caspase 3 mRNA levels was assessed by real-time quantitative PCR. Results: Upon PDT-Photogem (R) treatment, viable cells, as evaluated by the trypan blue dye-exclusion method, decreased in two cell lines (U87 and U138) but not in the other three. Apoptosis, as assessed by the expression of caspase-3 mRNA levels, was at least partly involved in the death mechanism of the cell lines. Conclusions: Collectively, our results indicated that PDT-Photogem (R) can act in glioma cells, thus encouraging new experiments in this field.FAEPA-Fundacao de Apoio ao Ensino, Pesquisa e Assistencia do Hospital das Clinicas da Faculdade de Medicina de Ribeirao Preto da Universidade de Sao Paulo (USP

Ethanol intake-induced apoptosis in glial cells and axonal disorders in the cerebellar white matter of UChA rats (voluntary ethanol consumers)

Ethanol intake may cause alterations in cellular metabolism altering motricity, learning and cognition. The cerebellum is one of the most susceptible organs to ethanol-related disorders during development, and is associated with oxidative stress-induced apoptosis being crucial for pathogenic consequences. The UChA variety is a special strain of Wistar rat genetically selected and represents a rare model for the studies related to genetic, biochemical, physiological, nutritional, and pharmacological effects of ethanol. We evaluated the structure and apoptosis in the cerebellar white matter of UChA rats. There were two groups of 09 rats: a control group that did not consume ethanol, and an experimental group of UChA rats that consumed ethanol at 10% (v/v) (<2 g ethanol/kg body weight/day). At 120 days old, rats were anaesthetized followed by decapitation, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for Caspase-3 and XIAP and transmission electron microscopy (TEM). The UChA group showed more glial cells immunoreactive for caspase-3 and less for XIAP than control group. Alcohol consumption affected myelin integrity. Severe ultrastructural damages in UChA group were observed such as disruption of the myelin sheath, disorganization and deformation of its components, and an increase in the interaxonal spaces. In conclusion, our data demonstrated that ethanol induced apoptosis in the glial cells and promoted an intense change in the myelin sheath of UChA rats, which may cause functional disorders.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Apoptosis of Purkinje and Granular Cells of the Cerebellum Following Chronic Ethanol Intake

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10 % (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electrondense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Expression of microRNAs miR-21 and miR-326 associated with HIF-1α regulation in neurospheres of glioblastoma submitted to ionizing radiation treatment

Background: Glioblastoma is an incurable neoplasm. Its hypoxia mechanism associated with cancer stem cells (CSCs) demonstrates hypoxia-inducible factor 1α (HIF-1α) expression regulation, which is directly related to tumor malignancy. The aim of this study was to identify a possible tumor malignancy signature associated with regulation of HIF-1α by microRNAs miR-21 and miR-326 in the subpopulation of tumor stem cells which were irradiated  by ion in primary culture of patients diagnosed with glioblastoma. Materials and methods: We used cellular cultures from surgery biopsies of ten patients with glioblastoma. MicroRNA expressions were analyzed through real-time polymerase chain reaction (PCR) and correlated with mortality and recurrence. The ROC curve displayed the cutoff point of the respective microRNAs in relation to the clinical prognosis, separating them by group. Results: The miR-21 addressed high level of expression in the irradiated neurosphere group (p = 0.0028). However, miR-21 was not associated with recurrence and mortality. miR-326 can be associated with tumoral recurrence (p = 0.032) in both groups; every 0.5 units of miR-326 increased the chances of recurrence by 1,024 (2.4%). Conclusion: The high expression of miR-21 in the irradiated group suggests its role in the regulation of HIF-1α and in the radioresistant neurospheres. miR-326 increased the chances of recurrence in both groups, also demonstrating that positive regulation from miR-326 does not depend on ionizing radiation treatment