Abnormalities of the p53 gene are the most common molecular change in human cancer. In the central nervous system, mutant p53 gene is frequently identified in the tumors with astrocytic differentiation. To investigate the relation between histologic subtypes and p53 protein overexpression, we examined 81 cases of astrocytic neoplasms (24 benign astrocytoma, 28 anaplastic astrocytoma and 29 glioblastoma multiforme) using the standard immunohistochemical method. All were formalin-fixed and paraffin-embedded tissue. The p53 immunoreactivity was found in 4/24 benign astrocytoma, 18/28 anaplastic astrocytoma, 22/29 glioblastoma multiforme. The degree of immunoreactivity closely correlated with histologic subtypes (p< 0.001). Overall p53 protein expression was most frequently detected in glioblastoma multiforme, but strong immunoreactivity (3+) was more frequently found in the anaplastic astrocytoma than in glioblastoma multiforme. Although the frequency of p53 protein expression is low, 4 benign astrocytoma showed distinct nuclear staining. In conclusion the malignant progression of astrocytic neoplasms may be associated with increasing expression of p53 protein.
Astrocytoma/*metabolism ; Brain Neoplasms/*metabolism ; Glioblastoma/*metabolism ; Human ; Immunohistochemistry ; Protein p53/*metabolism

Glioblastoma ; genetics ; metabolism ; Humans ; Mutation ; Oncogene Proteins ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism

The aim of this study was to investigate the functional characteristics and in vitro specific killing effect of EGFRvIII CAR-T cells co-expressing interleukin-15 and chemokine CCL19, in order to optimize the multiple functions of CAR-T cells and improve the therapeutic effect of CAR-T cells targeting EGFRvIII on glioblastoma (GBM). The recombinant lentivirus plasmid was obtained by genetic engineering, transfected into 293T cells to obtain lentivirus and infected T cells to obtain the fourth generation CAR-T cells targeting EGFRvIII (EGFRvIII-IL-15-CCL19 CAR-T). The expression rate of CAR molecules, proliferation, chemotactic ability, in vitro specific killing ability and anti-apoptotic ability of the fourth and second generation CAR-T cells (EGFRvIII CAR-T) were detected by flow cytometry, cell counter, chemotaxis chamber and apoptosis kit. The results showed that compared with EGFRvIII CAR-T cells, EGFRvIII-IL-15-CCL19 CAR-T cells successfully secreted IL-15 and CCL19, and had stronger proliferation, chemotactic ability and anti-apoptosis ability in vitro (all P < 0.05), while there was no significant difference in killing ability in vitro. Therefore, CAR-T cells targeting EGFRvIII and secreting IL-15 and CCL19 are expected to improve the therapeutic effect of glioblastoma and provide an experimental basis for clinical trials.
Humans ; Receptors, Chimeric Antigen/metabolism* ; Glioblastoma/metabolism* ; Interleukin-15/metabolism* ; Chemokine CCL19/metabolism* ; Cell Line, Tumor ; T-Lymphocytes/metabolism*

Patients with glioblastoma (GBM) generally have a bad prognosis and short overall survival after being treated with surgery, chemotherapy or radiotherapy due to the histological heterogeneity, strong invasive ability and rapid postoperative recurrence of GBM. The components of GBM cell-derived exosome (GBM-exo) can regulate the proliferation and migration of GBM cell via cytokines, miRNAs, DNA molecules and proteins, promote the angiogenesis via angiogenic proteins and non-coding RNAs, mediate tumor immune evasion by targeting immune checkpoints with regulatory factors, proteins and drugs, and reduce drug resistance of GBM cells through non-coding RNAs. GBM-exo is expected to be an important target for the personalized treatment of GBM and a marker for diagnosis and prognosis of this kind of disease. This review summarizes the preparation methods, biological characteristics, functions and molecular mechanisms of GBM-exo on cell proliferation, angiogenesis, immune evasion and drug resistance of GBM to facilitate developing new strategies for the diagnosis and treatment of GBM.
Humans ; Glioblastoma/genetics* ; Exosomes/metabolism* ; MicroRNAs/metabolism* ; Prognosis ; Cell Proliferation ; Brain Neoplasms/genetics* ; Cell Line, Tumor

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD⁺) metabolism. However, the functional role of NAD⁺ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD⁺ levels affect the characteristics of glioma-driven SSEA1⁺ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD⁺ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD⁺ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.
Glioblastoma* ; Lactic Acid ; Metabolism ; Mitochondria ; NAD ; NADP Transhydrogenases ; Phenotype ; Tics ; Up-Regulation*

Objective To investigate the relationship between the expression of glutathione peroxidase(GPX)genes and the clinical prognosis in glioma patients,and to construct and evaluate the model for predicting the prognosis of glioma. Methods The clinical information and GPX expression of 663 patients,including 153 patients of glioblastoma(GBM)and 510 patients of low-grade glioma(LGG),were obtained from The Cancer Genome Atlas(TCGA)database.The relationship between GPX expression and patient survival was analyzed.The key GPX affecting the prognosis of glioma was screened out by single- and multi-factor Cox's proportional-hazards regression models and validated by least absolute shrinkage and selection operator(Lasso)regression.Finally,we constructed the model for predicting the prognosis of glioma with the screening results and then used concordance index and calibration curve respectively to evaluate the discrimination and calibration of model. Results Compared with those in the control group,the expression levels of GPX1,GPX3,GPX4,GPX7,and GPX8 were up-regulated in glioma patients(all P<0.001).Moreover,the expression levels of other GPX except GPX3 were higher in GBM patients than in LGG patients(all P<0.001).The Kaplan-Meier curves showed that the progression-free survival of GBM with high expression of GPX1(P=0.013)and GPX4(P=0.040),as well as the overall survival,disease-specific survival,and progression-free survival of LGG with high expression of GPX1,GPX7,and GPX8,was shortened(all P<0.001).GPX7 and GPX8 were screened out as the key factors affecting the prognosis of LGG.The results were further used to construct a nomogram model,which suggested GPX7 was the most important variable.The concordance index of the model was 0.843(95%CI=0.809-0.853),and the calibration curve showed that the predicted and actual results had good consistency. Conclusion GPX7 is an independent risk factor affecting the prognosis of LGG,and the nomogram model constructed with it can be used to predict the survival rate of LGG.
Brain Neoplasms ; Glioblastoma ; Glioma/diagnosis* ; Glutathione Peroxidase/metabolism* ; Humans ; Peroxidases ; Prognosis ; Proportional Hazards Models

OBJECTIVETo study the clinical and pathologic features of gliosarcoma of cerebral hemispheres.

METHODSThe clinicopathologic features of 10 cases of gliosarcoma involving cerebral hemispheres were reviewed. Immunohistochemical study was carried out using EnVision method.

RESULTSThe mean age of the patients was 54 years and the male-to-female ratio was 6 to 4. Clinical symptoms included headache (6/10), nausea/vomiting (5/10), and sensory or motor impairment (4/10). Nine of the cases were primary gliosarcoma, with maximum diameter ranging from 2.4 to 5.5 cm (mean = 4.2 cm). The remaining case represented secondary gliosarcoma involving skull base and extracranial tissues. Histologic examination showed a biphasic pattern in all cases. Regarding the glial component, there were 9 cases of pleomorphic glioblastoma and 1 case of giant cell glioblastoma. Reticulin stain was positive in all cases. Immunohistochemical study showed that the tumor cells variably expressed GFAP (10/10), p16 (4/10), EGFR (1/10), CD68 (1/10) and p53 (6/10). The Ki-67 index ranged from 15% to 70% (mean = 34%). Six patients had follow-up data available. One patient was disease-free for 45 months and 5 patients died of the disease at 3 to 17 months after the operation (mean duration of survival = 9 months).

CONCLUSIONSGliosarcoma is a highly aggressive tumor, often locates in the deeper part cerebral hemispheres and has a relatively short duration of symptoms. It carries a poor prognosis. GFAP immunostain and reticulin stain are helpful in confirming the diagnosis. p53 and p16 are also expressed in some cases.

Adult ; Brain Neoplasms ; metabolism ; pathology ; Cerebrum ; pathology ; Female ; Glioblastoma ; metabolism ; pathology ; Gliosarcoma ; metabolism ; pathology ; Humans ; Male ; Middle Aged ; Neuroglia ; pathology

BACKGROUNDRecent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

METHODSA series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

RESULTSIn the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

CONCLUSIONSThese data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Line, Tumor ; Glioblastoma ; metabolism ; pathology ; Glioma ; metabolism ; pathology ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Peptides ; metabolism ; rac1 GTP-Binding Protein ; metabolism

BACKGROUNDSite A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.

METHODSFifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.

RESULTSThe IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate.

CONCLUSIONSMutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

Catalytic Domain ; genetics ; Glioblastoma ; genetics ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Isocitrates ; metabolism ; Mutagenesis ; Mutation ; Protein Binding ; Structure-Activity Relationship

BACKGROUND: The Nuclear Dbf2-related (NDR1) kinase is a member of the NDR/LATS family, which was a supplementary of Hippo pathway. However, whether NDR1 could inhibit glioblastoma (GBM) growth by phosphorylating Yes-associated protein (YAP) remains unknown. Meanwhile, the role of NDR1 in GBM was not clear. This study aimed to investigate the role of NDR1-YAP pathway in GBM. METHODS: Bioinformation analysis and immunohistochemistry (IHC) were performed to identify the expression of NDR1 in GBM. The effect of NDR1 on cell proliferation and cell cycle was analyzed utilizing CCK-8, clone formation, immunofluorescence and flow cytometry, respectively. In addition, the xenograft tumor model was established as well. Protein interaction was examined by Co-immunoprecipitation and immunofluorescence to observe co-localization. RESULTS: Bioinformation analysis and IHC of our patients' tumor tissues showed that expression of NDR1 in tumor tissue was relatively lower than that in normal tissues and was positively related to a lower survival rate. NDR1 could markedly reduce the proliferation and colony formation of U87 and U251. Furthermore, the results of flow cytometry showed that NDR1 led to cell cycle arrest at the G1 phase. Tumor growth was also inhibited in xenograft nude mouse models in NDR1-overexpression group. Western blotting and immunofluorescence showed that NDR1 could integrate with and phosphorylate YAP at S127 site. Meanwhile, NDR1 could mediate apoptosis process. CONCLUSION In summary, our findings point out that NDR1 functions as a tumor suppressor in GBM. NDR1 is identified as a novel regulator of YAP, which gives us an in-depth comprehension of the Hippo signaling pathway.
Animals ; Cell Nucleus/metabolism* ; Cell Proliferation ; Glioblastoma ; Humans ; Mice ; Phosphorylation ; Protein-Serine-Threonine Kinases/metabolism* ; Signal Transduction