Background:Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues.Methods:To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines.Results:Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells.Conclusion:These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.

Background:Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs.Methods:Neurospheres and CD133+ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines.Results:Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133+ cells compared with CD133− cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2.Conclusion:Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific small interfering RNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-PCR. c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.

These data suggest that ligand-dependent EphB1 signaling negatively regulates glioma cell invasion, identifying EphB1 as a favorable prognostic factor in malignant glioma.

Our results give the evidence that the increase of O(6)-methylguanine-DNA methyltransferase mRNA expression caused by methylation changes in recurrence may be associated with chemoresistance in the recurrent glioma.

A 55-year-old man was admitted to our hospital complaining of left hemiparesis. Magnetic resonance imaging (MRI) showed a smooth ring-like enhanced cystic tumor in the right parietal lobe. He underwent gross total resection of the tumor under neuronavigation and 5-aminolevulinic acid (5-ALA) fluorescence guiding method. Histopathological examination of the tumor showed small cells formed epithelioid solid nests with some focus of duct-like structure. On the basis of the MRI and operative and histological findings, this tumor was diagnosed as a metastatic poorly differentiated carcinoma, although the primary cancer could not be detected by metastatic work-ups. Afterward, this tumor recurred repeatedly. Histopathological examination of specimen from the fourth surgery indicated that the tumor was a glioblastoma (GBM). In the review of the histology and immunohistochemistry of the first tumor, atypical fibrillary cells were seen between solid nests and positive for glial fibrillary acidic protein, therefore the tumor was retrospectively diagnosed as epithelioid GBM. We assessed whether the changes in histopathology were accompanied by changes in the methylation status of O6-methylguanine methyltransferase (MGMT) promoter and the status of 5-ALA fluorescence. The methylation status of the MGMT promoter was found to have changed from methylated to unmethylated and 5-ALA fluorescence became positive along with the histological change.

Ginsenoside Rg3 is a natural active ingredient that is extracted from Korean red ginseng root. It elevates the therapeutic effect of radiotherapy and chemotherapy, but previous studies found that the application of Rg3 is heavily limited by its low bioavailability and poor absorption via oral administration.To overcome these problems, Rg3-loaded PEG-PLGA-NPs (Rg3-NPs) were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the physicochemical characteristics of Rg3-NPs were investigated. We treated primary glioblastoma with 50 mM Rg3-NPs for 48h. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of real-time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. The results showed that the Rg3-NPs are slick and uniform, the average diameter of the nanoparticles is 75-90 nm, and their entrapment efficiency is 89.7 + 1.7%. MTT showed that the growth of cells can be significantly inhibited by Rg3-NPs in a dose-dependent manner. FCM testing showed Rg3-NPs can be released from the conjugate nanoparticle and react with the genes in the cell nuclei, causing changes in the gene molecules. We also found that cancer cells treated with Rg3-NPs undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes BUB1, CDC20, TTK, and CENPE, as determined by microarray analysis and verified by real-time PCR. Furthermore, Rg3-NPs induced the expression of the apoptotic and antimigratory protein p53 in cell lines. The results of the present study, together with the results of earlier studies, show that Rg3-NPs target genes involved in the progression of the M-phase of the cell cycle. It is associated with several important pathways, which include apoptosis (p53). Rg3-NPs may be a potent cell-cycle regulation drug targeting the M-phase in glioblastoma cell lines.