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Glioblastoma multiforme (GBM) is a highly malignant primary brain tumour with a poor prognosis in adults. Identifying biomarkers that can aid in the molecular classification and risk stratification of GBM is critical. Here, we conducted a transcriptional profiling analysis of T-cell immunity in the tumour microenvironment of GBM patients and identified two novel T cell exhaustion (TEX)-related GBM subtypes (termed TEX-C1 and TEX-C2) using the consensus clustering. Our multi-omics analysis revealed distinct immunological, molecular and clinical characteristics for these two subtypes. Specifically, the TEX-C1 subtype had higher infiltration levels of immune cells and expressed higher levels of immune checkpoint molecules than the TEX-C2 subtype. Functional analysis revealed that upregulated genes in the TEX-C1 subtype were significantly enriched in immune response and signal transduction pathways, and upregulated genes in the TEX-C2 subtype were predominantly associated with cell fate and nervous system development pathways. Notably, patients with activated T-cell activity status in the TEX-C1 subgroup demonstrated a significantly worse prognosis than those with severe T cell exhaustion status in the TEX-C2 subgroup. Finally, we proposed a machine-learning-derived novel gene signature comprising 12 TEX-related genes (12TexSig) to indicate tumour subtyping. In the TCGA cohort, the 12TexSig demonstrated the ability to accurately predict the prognosis of GBM patients, and this prognostic value was further confirmed in two independent external cohorts. Taken together, our results suggest that the TEX-derived subtyping and gene signature has the potential to serve as a clinically helpful biomarker for guiding the management of GBM patients, pending further prospective validation., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Breast cancer patients with brain metastasis (BM) have a poor prognosis. This study aims to identify the risk factors of BM in patients with metastatic breast cancer (MBC) and establish a competing risk model for predicting the risk of brain metastases at different time points along the course of disease., Methods: Patients with MBC admitted to the breast disease center of Peking University First Hospital from 2008 to 2019 were selected and retrospectively analyzed to establish a risk prediction model for brain metastases. Patients with MBC admitted to eight breast disease centers from 2015 to 2017 were selected for external validation of the competing risk model. The competing risk approach was used to estimate cumulative incidence. Univariate Fine-Gray competing risk regression, optimal subset regression, and LASSO Cox regression were used to screen potential predictors of brain metastases. Based on the results, a competing risk model for predicting brain metastases was established. The discrimination of the model was evaluated using AUC, Brier score, and C-index. The calibration was evaluated by the calibration curves. The model was assessed for clinical utility by decision curve analysis (DCA), as well as by comparing the cumulative incidence of brain metastases between groups with different predicted risks., Results: From 2008 to 2019, a total of 327 patients with MBC in the breast disease center of Peking University First Hospital were admitted into the training set for this study. Among them, 74 (22.6%) patients developed brain metastases. From 2015 to 2017, a total of 160 patients with MBC in eight breast disease centers were admitted into the validation set for this study. Among them, 26 (16.3%) patients developed brain metastases. BMI, age, histological type, breast cancer subtype, and extracranial metastasis pattern were included in the final competing risk model for BM. The C-index of the prediction model in the validation set was 0.695, and the AUCs for predicting the risk of brain metastases within 1, 3, and 5 years were 0.674, 0.670, and 0.729, respectively. Time-dependent DCA curves demonstrated a net benefit of the prediction model with thresholds of 9-26% and 13-40% when predicting the risk of brain metastases at 1 and 3 years, respectively. Significant differences were observed in the cumulative incidence of brain metastases between groups with different predicted risks (P < 0.05 by Gray's test)., Conclusions: In this study, a competing risk model for BM was innovatively established, with the multicenter data being used as an independent external validation set to confirm the predictive efficiency and universality of the model. The C-index, calibration curves, and DCA of the prediction model indicated good discrimination, calibration, and clinical utility, respectively. Considering the high risk of death in patients with metastatic breast cancer, the competing risk model of this study is more accurate in predicting the risk of brain metastases compared with the traditional Logistic and Cox regression models., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Diffuse midline gliomas (DMGs) are pediatric high-grade brain tumors in the thalamus, midbrain, or pons; the latter subgroup are termed diffuse intrinsic pontine gliomas (DIPG). The brain stem location of these tumors limits the clinical management of DIPG, resulting in poor outcomes for patients. A heterozygous, somatic point mutation in one of two genes coding for the noncanonical histone H3.3 is present in most DIPG tumors. This dominant mutation in the H3-3A gene results in replacement of lysine 27 with methionine (K27M) and causes a global reduction of trimethylation on K27 of all wild-type histone H3 proteins, which is thought to be a driving event in gliomagenesis. In this study, we designed and systematically screened 2'- O -methoxyethyl phosphorothioate antisense oligonucleotides (ASOs) that direct RNase H-mediated knockdown of H3-3A mRNA. We identified a lead ASO that effectively reduced H3-3A mRNA and H3.3K27M protein and restored global H3K27 trimethylation in patient-derived neurospheres. We then tested the lead ASO in two mouse models of DIPG: an immunocompetent mouse model using transduced mutant human H3-3A cDNA and an orthotopic xenograft with patient-derived cells. In both models, ASO treatment restored K27 trimethylation of histone H3 proteins and reduced tumor growth, promoted neural stem cell differentiation into astrocytes, neurons, and oligodendrocytes, and increased survival. These results demonstrate the involvement of the H3.3K27M oncohistone in tumor maintenance, confirm the reversibility of the aberrant epigenetic changes it promotes, and provide preclinical proof of concept for DMG antisense therapy.

Objective: To evaluate the efficacy and influencing factors of surgery combined with neoadjuvant chemoradiotherapy in the treatment of children with non-orbital head and neck rhabdomyosarcoma (HNRMS). Methods: Information from 45 children diagnosed as non-orbital HNRMS and subjected to surgery combined with neoadjuvant chemoradiotherapy in Beijing Children's Hospital affiliated to Capital Medical University from August 2017 to July 2021 was analyzed. The patients included 25 males and 20 females, aged from 1 to 17 years old. The primary tumor site, pathological subtype, clinical stage, risk group, therapeutic regimen, resection range and outcome of all cases were also collected. The survival curves were made using the Kaplan-Meier method and the potential prognostic factors were investigated by Cox regression analysis. Results: Fifteen (33.3%) of 45 children achieved negative surgical margin under complete tumor resection. The postoperative pathological results showed that there were 20 cases of embryonic subtype, 19 cases of alveolar subtype and 6 cases of spindle sclerosis subtype. The postoperative follow-up time ranged from 4 to 71 months, with a median of 26 months. During the follow-up period, 13 children died, among whom brain metastasis was the most common cause of death, accounting for 7/13. The 3-year overall survival rate was 67.6%. Multivariate analysis showed that non-embryonic subtype ( HR =6.26, 95% CI : 1.52-25.87, P =0.011) and failure to reach R0 resection ( HR =9.37, 95% CI : 1.18-74.34, P =0.034) were independent risk factors affecting overall survival rate. Conclusion: Surgery combined with neoadjuvant chemoradiotherapy can offer a good efficacy for children with non-orbital HNRMS. Non-embryonic subtype and resection without negative operative microscopic margins are independent risk factors for poor prognosis, and brain metastasis is the main cause of death in these children.

As the tumor cell-centered treatment strategies cannot curb the malignant progression of glioblastoma effectively, the therapeutic effect of glioblastoma is still not satisfactory. In addition to glioma cells, glioma microenvironment (GME) comprises massive numbers of non-tumor cells and soluble cytokines. The non-tumor cells include endothelial cells, pericytes, microglia/macrophages, mesenchymal cells, astrocytes, neurons, etc. These non-tumor cell components, together with glioma cells, form one organism which regulates the progression of glioma. Considerable progress has been been in research on GME, which will be conducive to the development of non-tumor cell targeted therapies and and improvements in the prognosis of glioma patients. Herein, we summarized the interaction of glioma cells with endothelial cells, pericytes, microglia/macrophages, astrocytes, neurons and mesenchymal cells, a topic that has been extensively researched, as well as the corresponding translational studies. We also discussed the potential challenges and opportunities of developing glioma treatments based on tumor microenvironment., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Rationale: Brain metastasis in patients with lung cancer is life-threatening. However, the molecular mechanism for this catastrophic disease remains elusive, and few druggable targets are available. Therefore, this study aimed to identify and characterize proteins that could be used as therapeutic targets. Methods: Proteomic analyses were conducted to identify differentially expressed membrane proteins between brain metastatic lung cancer cells and primary lung cancer cells. A neuronal growth-associated protein, brain acid soluble protein 1 (BASP1), was chosen for further investigation. The clinical relevance of BASP1 in lung adenocarcinoma was first assessed. Tyrosine kinase activity assays and in vitro and in vivo functional assays were conducted to explore the oncogenic mechanisms of BASP1. Results: The protein levels of BASP1 were positively associated with tumor progression and poor prognosis in patients with lung adenocarcinoma. Membrane-bound BASP1 increased EGFR signaling and stabilized EGFR proteins by facilitating their escape from the ubiquitin-proteasome pathway. Reciprocally, activation of EGFR recruited more BASP1 to the plasma membrane, generating a positive feedback loop between BASP1 and EGFR. Moreover, the synergistic therapeutic effects of EGFR tyrosine kinase inhibitor and arsenic trioxide led to a reduction in the level of BASP1 protein observed in lung cancer cells with acquired resistance to EGFR inhibitors. Conclusions: The reciprocal interaction between BASP1 and EGFR facilitates EGFR signaling in brain metastatic lung cancer. Targeting the newly identified BASP1-EGFR interaction could open new venues for lung cancer treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objective: To identify novel epigenetic signatures that could provide predictive information that is complementary to promoter methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) gene for predicting temozolomide (TMZ) response, among glioblastomas (GBMs) without glioma-CpGs island methylator phenotype (G-CIMP) METHODS: Different cohorts of primary non-G-CIMP GBMs with genome-wide DNA methylation microarray data were included for discovery and validation of a multimarker signature, combined using a RISK score model. Different statistical analyses and functional experiments were performed for clinical and biological validation., Results: By employing discovery cohorts with radiotherapy (RT) and TMZ versus RT alone and a strict multistep selection strategy, we identified seven CpGs, each of which was significantly correlated with overall survival (OS) of non-G-CIMP GBMs with RT/TMZ, independent of age, MGMT promoter methylation status, and other identified CpGs. A RISK score signature of the 7 CpGs was developed and validated to distinguish non-G-CIMP GBMs with differential survival outcomes to RT/TMZ, but not to RT alone. The interaction analyses also showed differential outcomes to RT/TMZ versus RT alone within the RISK score-based subgroups. The signature could also improve the risk classification by age and MGMT promoter methylation status. Functional experiments showed that HSBP2 appeared to be epigenetically regulated by one identified CpG and was associated with TMZ resistance, but it was not associated with cell proliferation or apoptosis in GBM cell lines. The predictive value of the single CpG methylation of HSBP2 by pyrosequencing was observed in a local cohort of isocitrate dehydrogenase 1 (IDH1) R132H wild-type GBMs., Conclusions: This novel epigenetic signature might be a promising predictive (but not a general prognostic) biomarker and be helpful for refining the MGMT-based guiding approach to TMZ usage in non-G-CIMP GBMs.

Aims: We aimed to identify a clinically useful biomarker using DNA methylation-based information to optimize individual treatment of patients with glioblastoma (GBM)., Methods: A six-CpG panel was identified by incorporating genome-wide DNA methylation data and clinical information of three distinct discovery sets and was combined using a risk-score model. Different validation sets of GBMs and lower-grade gliomas and different statistical methods were implemented for prognostic evaluation. An integrative analysis of multidimensional TCGA data was performed to molecularly characterize different risk tumors., Results: The six-CpG risk-score signature robustly predicted overall survival (OS) in all discovery and validation cohorts and in a treatment-independent manner. It also predicted progression-free survival (PFS) in available patients. The multimarker epigenetic signature was demonstrated as an independent prognosticator and had better performance than known molecular indicators such as glioma-CpG island methylator phenotype (G-CIMP) and proneural subtype. The defined risk subgroups were molecularly distinct; high-risk tumors were biologically more aggressive with concordant activation of proangiogenic signaling at multimolecular levels. Accordingly, we observed better OS benefits of bevacizumab-contained therapy to high-risk patients in independent sets, supporting its implication in guiding usage of antiangiogenic therapy. Finally, the six-CpG signature refined the risk classification based on G-CIMP and MGMT methylation status., Conclusions: The novel six-CpG signature is a robust and independent prognostic indicator for GBMs and is of promising value to improve personalized management., (© 2018 John Wiley & Sons Ltd.)

BACKGROUND Red blood cell distribution width (RDW) is a parameter of the complete blood count (CBC) test. Recent evidence suggests that pretreatment RDW is associated with patient survival in various malignant tumors. We explored the association of pretreatment RDW and other red blood cell (RBC) parameters with clinical parameters and assessed their prognostic impact on overall survival (OS) in patients with glioblastoma (GBM). MATERIAL AND METHODS In total, 109 patients with newly diagnosed GBM were retrospectively reviewed. The Cox proportional hazards regression model and Kaplan-Meier method were used to examine the survival function of pretreatment RDW, mean cell volume (MCV), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC count, and hematocrit (HCT) values in patients with newly diagnosed GBM. RESULTS Univariate analysis showed that MCV, MCHC, and RDW were associated with overall survival (OS). However, only RDW remained significant in multivariate analysis. The Kaplan-Meier survival curves showed that patients belonging to the high-RDW group had a worse median OS (293 days versus 375 days, P=0.023) than those belonging to the low-RDW group. CONCLUSIONS The present study showed that pretreatment RDW was superior to MCV and MCHC as a prognostic predictor of clinical outcome in patients newly diagnosed with GBM. Pretreatment RDW was derived directly from the CBC test, which can be easily performed in clinical practice. Therefore, pretreatment RDW values can provide additional prognostic information for patients with GBM. Further larger and prospective studies are needed to confirm these findings and to investigate the mechanism by which of RDW is associated with prognosis in patients with GBM.

Objective: To investigate the miR-130b expression in patients with glioma and to analyze its role and underlying molecular mechanism on the carcinogenesis., Patients and Methods: The expression levels of miR-130b were detected with quantitative Real-time PCR. The relationship between miR-130b expression and clinicopathologic characteristics were analyzed. MiR-130b inhibitor was transfected into glioma cell lines to investigate its role in HCC. MTT assays were conducted to explore the impact of miR-130b down-expression on the proliferation of human glioma cells. Cell cycle and cell apoptosis assays were performed using flow cytometry. Levels of ERK/MAPK pathway related proteins were evaluated by Western blotting. Data were analyzed using the 2-ΔΔCT method through student's t-test via the GraphPad Prism software (La Jolla, CA, USA)., Results: The expression of miR-130b was markedly upregulated in glioma cell lines and tissues, and high miR-130b expression was significantly associated with advanced WHO grade (p = 0.022) and low Karnofsky performance score (p = 0.001). In addition, downregulation of miR-130b inhibited the proliferation of glioma cells and induced cell-cycle arrest and cells apoptosis in vivo. Importantly, ERK/MAPK pathway was found to be inactivated in the glioma cell lines after miR-130b knockout experiment., Conclusions: The current data indicated that miR-130b may play a critical role in the progression of glioma via ERK/MAPK signaling cascades, suggesting that it may be a useful therapeutic agent in glioma patients.

Background: Sichuan is a province in the west of China with a population of 81.4 million. This is the first statistical report of central nervous system (CNS) tumors surgically treated and histologically diagnosed in a large Chinese population., Methods: All the patient data were obtained from 86 medical facilities, which covered the Sichuan province population. Data from patients who underwent surgery between 2008 and 2013 and corresponding histology samples were re-reviewed in the major pathology centers. All the CNS tumors were categorized according to International Classification of Diseases (ICD)-10 and ICD-O-3 classifications and reviewed manually. The tumor distribution was analyzed and stratified by gender, age, race, and tumor sites. Tumors in some ethnic minorities, such as the Tibetan people, also were analyzed., Results: The final analytic dataset included 35,496 records. The top four histologic tumors were meningioma (28.51 %), pituitary adenoma (15.00 %), nerve sheath (13.77 %), and glioblastoma (11.82 %). There was a dramatically high incidence of malignant tumor in males. The median age at diagnosis ranged from 13 years (pineal region tumors) to 56 years (metastatic brain tumors). Most of the tumors in the insular lobe or cerebellum were low grade, whereas those in the thalamus or basal ganglia were likely to be high grade. The incidence of malignant tumors or high-grade gliomas in the Tibetans was significantly lower than in the Chinese Han population., Conclusion: This report is a preliminary statistical analysis of brain and spinal tumors in a large Chinese population and may serve as a useful resource for clinicians, researchers, and patients' families.

Increasing evidence indicates that genetic biomarkers play important roles in the development of glioma-associated seizures. Thus, we performed a systematic review to summarise biomarkers that are associated with seizures in glioma patients. An electronic literature search of public databases (PubMed, Embase and Medline) was performed using the keywords glioma, seizure and epilepsy. A totall of 26 eligible studies with 2224 cases were included in this systematic review of publications to 20 June, 2016. Genetic biomarkers such as isocitrate dehydrogenase 1 (IDH1) mutations, low expression of excitatory amino acid transporter 2 (EAAT2), high xCT expression, overexpression of adenosine kinase (ADK) and low expression of very large G-protein-coupled receptor-1 (VLGR1) are primarily involved in synaptic transmission, whereas BRAF mutations, epidermal growth factor receptor (EGFR) amplification, miR-196b expression and low ki-67 expression are associated with regulation of cell proliferation. However, there is limited evidence regarding the roles of RAD50 interactor 1 (RINT1) and olig2 in epileptogenesis among glioma patients. Glioma-related seizure was related to the dysfunction of tumor microenvironment. Our findings may provide new mechanistic insights into targeted therapy for glioma-related seizures and may result in the development of multi-target therapies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Accumulating evidence demonstrates that prognostic nutritional index(PNI) is linked to the clinical outcome of patients with malignant tumors, but few studies had investigated the clinical significance of PNI in glioblastoma multiforme(GBM). This study aimed to clarify the association between PNI and the clinical outcome of patients with GBM., Methods: The clinical data of 84 patients with GBM were retrospectively analyzed. PNI was calculated from the following formula: 10×serum albumin (g/dL)+0.005×total lymphocyte count (per mm 3 ). X-tile software was used to determine the cut-off of PNI and other hematological parameters. GBM patients were dichotomized as two groups based on the PNI cut-off., Results: The optimal PNI cut-off level was 44.4. There were 14 patients with a PNI<44.4 and 70 patients with a PNI≥44.4. The results showed that PNI score was associated with gender, serum albumin, and hemoglobin level. Univariate analysis suggested that age, extent of resection, adjuvant treatment, platelet count and PNI score were predictors of overall survival in patients with GBM. The 1- and 2-survival rates of patients with a PNI<44.4 were 28.60 and 0%, respectively, while the corresponding values for patients with a PNI≥44.4 were 52.90 and 5.70%, respectively. Based on multivariate analysis, a PNI≥44.4 (HR:0.479, 95% CI:0.235-0.975,p=0.042) remained an independent prognostic indicator for a favorable outcome of patients with GBM. Furthermore, patients with a PNI≥44.4 may have a better efficacy of adjuvant treatment than patients with a PNI<44.4 (HR:0.259, 95% CI:0.096-0.700, p=0.008)., Conclusion: A PNI>44.4 was an independent prognostic parameter of overall survival in patients with GBM and the efficacy of adjuvant treatment. Interventions aimed at correcting the nutritional and immune status of patients with GBM may, therefore, promote the effectiveness of adjuvant treatment and improve the survival outcomes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Previous studies have demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) hyperpermeability via both paracellular and transcellular pathways. In a recent study, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent signaling pathway is involved in EMAP-II-induced BTB hyperpermeability. This study further investigated the exact mechanisms through which the cAMP/PKA-dependent signaling pathway affects EMAP-II-induced BTB hyperpermeability. In an in vitro BTB model, low-dose EMAP-II (0.05 nM) induced a significant decrease in Rac1 activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to elevate intracellular cAMP concentration completely blocked EMAP-II-induced inactivation of Rac1. Besides, pretreatment with 6Bnz-cAMP to activate PKA partially attenuated EMAP-II-induced Rac1 inactivation. Moreover, 6Bnz-cAMP pretreatment significantly diminished EMAP-II-induced changes in BTB permeability, myosin light chain (MLC) phosphorylation, expression and distribution of ZO-1, and actin cytoskeleton arrangement in RBMECs. These effects of 6Bnz-cAMP were completely blocked in the presence of NSC-23766 (the specific inhibitor of Rac1). In conclusion, this study demonstrates that low-dose EMAP-II induces BTB hyperpermeability via the cAMP/PKA/Rac1 signaling pathway.

The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3'UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.

Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment.

After demonstrating bradykinin (BK) could increase the permeability of blood-tumor barrier (BTB) via opening the tight junction (TJ), and that the possible mechanism is unclear, we demonstrated that BK could increase the expressions of eNOS and nNOS and promote ZONAB translocation into nucleus. NOS inhibitors l-NAME and 7-NI could effectively block the effect of BK on increasing BTB permeability, decreasing the expressions of claudin-5 and occludin and promoting the translocation of ZONAB. Overexpression of ZONAB could significantly enhance BK-mediating BTB permeability. Meanwhile, chromatin immunoprecipitation verified ZONAB interacted with the promoter of claudin-5 and occludin respectively. This study indicated NOS/NO/ZONAB pathway might be involved in BK's increasing the permeability of BTB., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our previous studies demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) can selectively increase the permeability of blood-tumor barrier (BTB). In addition, low-dose EMAP-II significantly decreases the cyclic adenosine monophosphate (cAMP) concentration and the protein kinase A (PKA) expression level in tumor tissues in the rat C6 glioma model. In this study, an in vitro BTB model was used to investigate the potential role of cAMP/PKA signaling cascade in EMAP-II-induced BTB hyperpermeability. Our data revealed that low-dose EMAP-II (0.05 nM) induced a significant decrease in total intracellular cAMP concentration and PKA activity in rat brain microvascular endothelial cells (RBMECs). Pretreatment with forskolin to increase intracellular cAMP nearly completely blocked the EMAP-II-induced decrease in transendothelial electric resistance and increase in horseradish peroxidase flux across the BTB. Similar pretreatment completely prevented the EMAP-II-induced changes in RhoA/Rho kinase activity, expression and distribution of tight junction-associated protein ZO-1, and myosin light chain phosphorylation, as well as actin cytoskeleton arrangement in RBMECs. Pretreatment with 6Bnz-cAMP to activate PKA significantly attenuated these EMAP-II-induced alterations in RBMECs. In summary, our present study demonstrates that the cAMP/PKA signaling cascade works as a crucial signaling pathway in EMAP-II-induced BTB hyperpermeability.

Background: The aim of this study was to determine the influence of the BMI1 gene on chemotherapy sensitivity in human glioma cells., Material/methods: The expression of the BMI1 gene in 41 cases of human brain glioma was determined by quantitative real-time PCR. The silencing effect of RNA interference on the BMI1 gene was detected by Western blot. Methyl thiazolyl tetrazolium assay (MTT) and flow cytometry methods were used to determine the cell viability and apoptosis rate of the U251 cells with BMI1 silencing. After those U251 cells were treated with Cisplatin (DDP), the cell viability and apoptosis rate were further detected., Results: The BMI1 mRNA in glioma was remarkably up-regulated, 176.3% as much as that in peri-cancerous tissues (P<0.05). The siRNA-BMI1 significantly and effectually inhibited the expression of BMI1 protein (P<0.05). The cell viability decreased in U251 cells with BMI1 silenced, and the apoptosis rate upgraded significantly (P<0.05 for both). After treating with DDP at various concentrations (1, 3, and 5 μg/ml), the cell viability in the BMI1-slienced U251 cells was much lower than that in corresponding control U251 cells at each DDP concentration (P<0.05 for all), and the apoptosis rate showed the opposite changing trends (P<0.05 for all)., Conclusions: There is a notable relationship between the over-expression of BMI1 and the carcinogenesis of gliomas. The silence of BMI1 inhibited cell proliferation and enhanced the apoptosis of the U251 cells, and increased the chemotherapy sensitivity of U251 cells to DDP.

The present study was performed to examine whether Endothelial-monocyte-activating polypeptide II (EMAP II) could inhibit glioma growth by inducing rat brain glioma C6 cells apoptosis. The results revealed that the EMAP II decreased cell viability of rat C6 glioma cells in a time-dependent manner. Apoptotic proportion was increased gradually after EMAP II. EMAP II induced the decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. Meanwhile, EMAP II-induced apoptosis was accompanied by an increase of reactive oxygen species (ROS). The significant up-regulation in the expressions of Bax and Apaf-1 as well as down-regulation in the expression of Bcl-2 was observed. The time course change of ROS was prior to the changes of above investigated indexes. All of these results strongly suggest that EMAP II could induce rat C6 glioma cells apoptosis via the mitochondrial pathway, and ROS, Bax/Bcl-2 might be involved in this processing., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: This study was conducted to determine the influence of MACC1 expression on chemotherapy sensitivity in human U251 glioblastoma cells., Materials and Methods: Expression of the MACC1 gene in 49 cases of human brain glioma was determined by quantitative real-time PCR. Silencing effects of RNA interference on MACC1 was detected by Western-blotting. Flow cytometry methods and methyl thiazolyl tetrazolium assay (MTT) were used to determine the apoptosis and growth inhibitory rates of the U251 cells with MACC1 silencing. before and after treatment with cisplatin (DDP)., Results: MACC1 mRNA in gliomas was up-regulated remarkably, to 158.8% of that in peri-cancerous tissues (P<0.05). The siRNA-MACC1 could inhibit the expression of MACC1 protein significantly (p<0.05), associated with an increase in apoptosis rate from 2.57% to 5.39% in U251 cells and elevation of the growth inhibitory rate from 1.5% to 17.8% (p<0.05 for both). After treatment with DDP at various concentrations (1, 3, 5μg/ml), compared with control U251 cells, the apoptosis rate of MACC1-silenced U251 cells rose from 8.41%, 13.2% and 19.5% to 12.8%, 17.8% and 25.8%; the growth inhibitory rate increased from 16.2%, 19.3% and 24.5% to 23.7%, 28.4% and 36.3%., Conclusions: There is a notable relationship between over-expression of MACC1 and the characteristics of glioma cells. Silencing of MACC1 was found to enhance the apoptosis and growth inhibitory rates of U251 glioma cells, and thereby increase their sensitivity to DDP chemotherapy.

The purposes of this study were to investigate the possible molecular mechanisms of miR-18a regulating the permeability of blood-tumor barrier (BTB) via down-regulated expression and distribution of runt-related transcription factor 1 (RUNX1). An in vitro BTB model was established with hCMEC/D3 cells and U87MG cells to obtain glioma vascular endothelial cells (GECs). The endogenous expressions of miR-18a and RUNX1 were converse in GECs. The overexpression of miR-18a significantly impaired the integrity and increased the permeability of BTB, which respectively were detected by TEER and HRP flux assays, accompanied by down-regulated mRNA and protein expressions and distributions of ZO-1, occludin and claudin-5 in GECs. Dual-luciferase reporter assay was carried out and revealed RUNX1 is a target gene of miR-18a. Meanwhile, mRNA and protein expressions and distribution of RUNX1 were downregulated by miR-18a. Most important, miR-18a and RUNX1 could reversely regulate the permeability of BTB as well as the expressions and distributions of ZO-1, occludin and claudin-5. Finally, chromatin immunoprecipitation verified that RUNX1 interacted with "TGGGGT" DNA sequence in promoter region of ZO-1, occludin and claudin-5 respectively. Taken together, our present study indicated that miR-18a increased the permeability of BTB via RUNX1 mediated down-regulation of tight junction related proteins ZO-1, occludin and claudin-5, which would attract more attention to miR-18a and RUNX1 as potential targets of drug delivery across BTB and provide novel strategies for glioma treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

TRAP1/Hsp75 (tumor necrosis factor receptor-associated protein 1), a paralogue of the Hsp90 family, has been recently described as a molecular marker and novel therapeutic target in local and metastatic prostate cancer. It has been proved to be associated with tumor invasion and metastasis in various human malignancies. In our study, the protein expression level of TRAP1 in 236 cases of glioma is investigated by immunohistochemistry assay. Statistical analysis was utilized to evaluate the association of TRAP1 with clinicopathological characteristics and prognosis of patients. It was proved that TRAP1 protein expression was increased in glioma compared with that in normal brain tissue. Moreover, TRAP1 immunohistochemical staining was correlated with World Health Organization (WHO) grade and Karnofsky performance score (KPS). Strong positive TRAP1 staining is more frequently detected in glioma of advanced grade or low KPS. It is also demonstrated that TRAP1 could be an independent negative prognostic factor in glioma, for patients with glioma of strong TRAP1 staining tend to have high risk of death. These results proved that TRAP1 is associated with prognosis of glioma, which may also suggest the potential role of TRAP1 in glioma management.

Background: The aim of this study was to determine whether miR-210 can affect the apoptosis and proliferation of human U251 glioma cells from down-regulating SIN3A., Material and Methods: The expression of miRNA-210 was detected by quantitative real-time PCR in normal brain tissue and glioma samples. The apoptosis and proliferation ability of U251 cells were analyzed by MTT and flow cytometry assay after anti-miR-210 transfection. For the regulation mechanism analysis of miR-210, TargetScan, PicTar, and microRNA were selected to predict some potential target genes of miR-210. The predicted gene was identified to be the direct and specific target gene of miR-210 by luciferase activities assay and Western blot. RNA interference technology was used to confirm that the apoptosis and proliferation effects of miR-210 were directly induced by SIN3A., Results: The expression of miR-210 increased significantly in glioma in comparison with normal brain tissue. The silence of miR-210 expression could inhibit the proliferation of U251 cells and induce the apoptosis. Mechanism analysis revealed that SIN3A was a specific and direct target gene of miR-210. The siRNA-SIN3A could down-regulate the expression of SIN3A protein, which was up-regulated in U251 cells by anti-miR-210 transfection, and our experiments found that silence of SIN3A could inhibit the apoptosis and sharply increase the proliferation of U251 cells. The regulation effects of anti-miR-210 on apoptosis and proliferation can be reversed respectively by the expression silence of SIN3A., Conclusions: Aberrantly expressed miR-210 regulates human U251 glioma cells apoptosis and proliferation partly through directly down-regulating SIN3A protein expression. This might offer a new potential therapeutic stratagem for glioma.

Purpose: Our aim was to investigate the relationship between mutant p53 and the prognosis of malignant glioma treated with temozolomide, and the regulation of mutant TP53 induced drug resistance, by molecular experimentation and a clinical trial., Methods: Adult patients with newly surgical diagnosed glioblastoma were randomly assigned to receive either temozolomide or semustine after radiation treatment. The statuses of TP53 and expression of TP53 and O(6)-methylguanine DNA-methyltransferase (MGMT) were determined retrospectively in tumor tissue from enrolled patients. The primary end point was overall survival. Synthetic small interfering RNA was used to knock down mutant TP53 in T98G and U138 cells, which are human glioblastoma cells with a P53 mutation, by screening of exons 4-8. Viable cell survival was measured when these cells were exposed to temozolomide or semustine. Expression of MGMT at the messenger RNA level was also determined., Results: The overall survival was 34.3 % at 2 years, 22.9 % at 3 years, 11.4 % at 4 years, and 8.6 % at 5 years with temozolomide, versus 18.2, 12.1, 3.0, and 0 %, respectively, with semustine. TP53 mutation and expression of mutant TP53 and MGMT showed significant inverse correlations with overall survival. Knockdown of mutant TP53 led to a fivefold increase in chemosensitivity to temozolomide but not semustine. Mutant TP53 knockdown induced down-regulation of MGMT expression., Conclusions: Mutant TP53 is strongly associated with a poor prognosis for overall survival in patients with glioblastoma. Also, TP53 mutation may decrease the chemosensitivity of glioblastoma to temozolomide by increasing MGMT expression.

Objective: Pineal region tumors (PRTs) are uncommon, and treatments vary among neoplasm types. The authors report their experience with gamma knife surgery (GKS) as an initial treatment in a series of PRT patients with unclear pathological diagnoses., Method: Seventeen PRT patients with negative pathology who underwent GKS were retrospectively studied. Nine patients had further whole-brain and spinal cord radiotherapy and chemotherapy 6-9 months after GKS., Results: Sixteen of 17 cases were followed up over a mean of 33.3 months. The total response rate was 75%, and the control rate was 81.3%. No obvious neurological deficits or complications were attributable to GKS., Conclusion: The findings indicate that GKS may be an alternative strategy in selected PRT patients who have negative pathological diagnoses, and that good outcomes and quality of life can be obtained with few complications.

Background: Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy., Methods: We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines., Results: Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy-events that could be prevented by silencing ABCB6 via siRNA treatment., Conclusions: Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

The "gain of function" of mutant TP53 is an important determinant in human tumor development and progression. This study aimed to investigate the possible mechanism of mutant TP53 inducing temozolomide resistance in glioblastoma cells. Three established human glioma cell lines, T98G, U87, and U138, were chemoresistant cells. The mRNA of cells was sequenced to confirm the status of TP53. Synthetic small interfering RNA (siRNA) was used to knock down TP53 in cells. TP53 mRNA was detected "silenced" by reverse transcriptase-polymerase chain reaction (RT-PCR) in five consecutive days. Viable cell survival was measured when these cells were exposed to temozolomide or semustine in step-up concentrations. The expression of O(6)-methylguanine DNA-methyltransferase (MGMT) at mRNA level was also determined. T98G, U87, and U138 cells were resistant to temozolomide. T98G and U138 cells expressed mutant-type TP53 with positive MGMT, while U87 cell expressed wild-type TP53 with negative MGMT. TP53-siRNA knocked down TP53 effectively (P = 0.021) in five consecutive days. Knockdown of mutant TP53 in T98G and U138 cells led to a fivefold increase in chemosensitivity to temozolomide, but not semustine. Knockdown of wild TP53 in U87 cell did not affect the chemoresistance. In addition, mutant TP53 knockdown induced a dramatic decrease of MGMT expression (P = 0.0000034). TP53 mutation decreases the chemosensitivity of malignant gliomas to temozolomide. This "gain of function" in drug resistance may be obtained by increasing MGMT expression.

Altered microRNA-205 (miR-205) expression has been found in glioma tissue samples and cell lines; however, the clinical significance of this is unclear. The aim of this study was to confirm the miR-205 expression pattern in human glioma and to investigate its clinical relevance. Quantitative reverse-transcription polymerase chain reaction assays showed that miR-205 expression was significantly lower in glioma tissues than in non-neoplastic brain tissues (P<0.001). Statistical analysis revealed a significant correlation between low miR-205 expression and both high grade glioma (World Health Organization [WHO] criteria, P=0.008) and a low Karnofsky performance status score (P=0.02). Survival analysis demonstrated that the cumulative 5-year overall survival rate of patients with glioma in the high miR-205 expression group was significantly higher than that in the low miR-205 expression group (P<0.001). Multivariate Cox regression analysis further indicated that miR-205 expression (P=0.01) and WHO grade (P=0.01) were independent prognostic indicators of the overall survival of patients with glioma. Moreover, subgroup analyses revealed that the cumulative 5-year overall survival rate of patients with high grade (III-IV) glioma was significantly worse for the low miR-205 expression group than for the high miR-205 expression group (P<0.001), but no significant difference was found for patients with low grade (I-II) glioma (P=0.09). In conclusion, down-regulation of miR-205 was associated with glioma progression. Our data are the first to suggest that miR-205 holds potential as a prognostic factor for glioma, especially for patients with advanced disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Tumor surgery in brain motor functional areas remains challenging. Novel techniques are being developed to gain maximal and safe resection for brain tumor surgery. Herein, we assessed the magnetic resonance diffusion tensor imaging (MR-DTI) and fluorescein sodium dyeing (FLS) guiding technique for surgery of glioma located in brain motor functional areas., Methods: Totally 83 patients were enrolled according to our inclusion and exclusion criteria (56 patients in experimental group, 27 patients in control group). In the experimental group, the surgical approach was designed by DTI imaging, which showed the relationship between the tumor and motor tract. The range of resection in the operation was determined using the FLS-stained area, which recognized the tumor and its infiltrated tissue. The traditional routine method was used in the control group. Postoperatively, all patients underwent enhanced brain MRI within 72 hours to ascertain the extent of resection. Patients were followed in our outpatient clinic over 6 - 24 months. Neurological deficits and Karnofsky scoring (KPS) were evaluated., Results: There were no significant differences in balance test indexes of preoperative data (sex, age, lesion location and volume, and neurological deficits before operation) and diagnosis of histopathology between the two groups. There was a trend in the experimental group for greater rates of gross total resection (80.4% vs. 40.7%), and the paralysis rate caused by surgery was lower in experimental (25.0%) vs. control (66.7%) groups (P < 0.05). The 6-month KPS in the low-grade and high-grade gliomas was 91 ± 11 and 73 ± 26, respectively, in the experimental group vs. 82 ± 9 and 43 ± 27, respectively, in the control group (P < 0.05 for both)., Conclusions: MR-DTI and FLS dye guiding for surgery of glioma located in brain motor functional areas can increase the gross total resection rate, decrease the paralysis rate caused by surgery, and improve patient quality of life compared with traditional glioma surgery.

Objectives: To analyze the long-term survivors of glioblastoma and to identify any prognostic factors that potentially contribute to survival., Methods: Fifteen glioblastomas patients underwent surgery from June 2007 to April 2009 who survived longer than 3 years were enrolled in. Clinical characteristics such as age, location of tumor, extent of resection, and radiotherapy or chemotherapy were analyzed. The expressions of epidermal growth factor receptor (EGFR), tumor protein 53 (P53), phosphatase and tensin homolog (PTEN), O6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase 1 gene (IDH1), and neurofibromatosis type 1 (NF-1) in tumor samples were measured by immunohistochemical method, and the status of P53 and IDH1 were detected by direct DNA sequencing as well. And the patients who survived less than 1 year were set as control. Kaplan-Meier analysis was used to evaluate the prognostic factors., Results: The average age of patients at diagnosis was 45.6 years. And the overall survival time was 3-6 years (median survival time 3.5 years). Thirteen patients underwent a total resection, and 14 patients took orally temozolomide. The occurrence frequency of these molecular markers in long-term survivors was PTEN (13/15), IDH1 (13/15), IDH1 mutation (12/15), P53 (8/15), P53 mutation (7/15), EGFR (6/15), MGMT (4/15) and NF-1 (3/15). There was a good correlation between IDH1 protein expression and IDHI mutation, and between P53 protein expression and P53 mutation. And the survival analysis showed that age above 50 years at diagnosis (OR = 0.262, 95%CI: 0.102 - 0.672), total resection (OR = 0.372, 95%CI: 0.149 - 0.931) and combined oral temozolomide (OR = 0.131, 95%CI: 0.044 - 0.390) were favorable clinical prognostic factors. PTEN (OR = 0.201, 95%CI: 0.074 - 0.549) and IDH1 (OR = 0.151, 95%CI: 0.050 - 0.454) expression, IDH1 mutation (OR = 0.276, 95%CI: 0.108 - 0.709) in tumor cells contributed to a favorable prognosis., Conclusions: There is probably no single molecular marker that is responsible for long-term survival of patients with glioblastoma, may be a balance between all these molecular events result in a favorable outcome.

Objective: To investigate the relationship between isocitrate dehydrogenase 1 (IDH1) mutation and clinicopathological features in human supratentorial WHO grade II gliomas., Methods: Ninety five supratentorial WHO grade II glioma patients were treated in our department from January 2009 to January 2011. The clinical data and tumor samples of each patient were collected. IDH1 mutation in the tumor was measured by sequencing the IDH1 gene of tumor specimen. The relationship between IDH1 mutation and the clinicopathological features was analyzed., Results: There were 69 cases (72.6%) with IDH1 mutations were found, all of which were R132H type mutations. The mutation rates of diffuse astrocytoma, oligodendroglioma and oligoastrocytoma were 73.6%, 68.8% and 73.1% respectively. The mean ages of IDH1 mutant group and wild type group were significantly different [(39 6 +/- 7.4) yr. vs. (46.9 +/- 11.6) yr., P < 0.05). The mutant rates of patients aged > or = 50 years and < 50 years were 43.8% and 78.5% respectively (P < 0.05), while those of male patients and female patients were 68.6% and 77.3% respectively (P > 0.05). The mutant rates of patients whose tumor > or = 5 cm and < 5 cm were 60.0% and 90.0% respectively (P < 0.05), while those of monolobe involvement group and deepen structures involvement group were 93.3% and 32.0% respectively (P < 0.05)., Conclusion: The mutation of IDH1 in supratentorial WHO grade II glioma is common, and is associated with patients' age, tumor size and tumor location.

Low-frequency ultrasound (LFU) irradiation under certain acoustic intensity can increase blood-brain barrier permeability non-invasively and reversibly. The aim of this study was to find out the effect of LFU irradiation on blood-tumor barrier (BTB) permeability in rat C6 glioma model and the possible mechanism. In this research, Evans blue and H&E staining were used to evaluate the optimal parameter of LFU to open the BTB without damaging the normal brain tissue. Transmission electron microscopy was used to observe the changes of the number of pinocytotic vesicles in cerebral or glioma microvascular endothelial cells. The phosphorylation of tyrosine kinase Src, caveolin-1, and caveolin-2 was detected by western blot. The distribution and expressing levels of caveolae proteins, caveolin-1 and caveolin-2, were detected by immunohistochemical and immunofluorescent staining, RT-PCR, and western blot. Our research data showed that, in rat C6 glioma model, LFU irradiation at a frequency of 1 MHz, a power of 12 mW, and exposure time of 20 s induced the increase of BTB permeability temporally, which reached a peak at 1.5 h, then decreased and restored to normal level at 12 h after LFU irradiation. In the glioma microvascular endothelial cells of rat glioma model, LFU irradiation induced a significant increase of the pinocytotic vesicles' density. The phosphorylation of Src, caveolin-1, and caveolin-2 began to increase at 0.5 h and reached a maximum at 1 h. Immunohistochemical and immunofluorescent staining showed that caveolin-1 and caveolin-2 were co-localized in the glioma microvascular endothelial cells and glioma cells. The mRNA and protein expression levels of caveolin-1 and caveolin-2 were up-regulated, reached the peak value at 1.5 h, and re-normalized at 12 h after LFU irradiation. These results demonstrated that LFU irradiation increased BTB permeability by promoting transcellular transport in glioma microvascular endothelial cells. The phosphorylation of tyrosine kinase Src, caveolin-1, caveolin-2 and up-regulation of caveolin-1 and caveolin-2 were involved in LFU-induced caveolae-mediated endocytosis.

Our previous studies have demonstrated that both the RhoA/Rho kinase and the protein kinase C (PKC) signaling pathways are involved in the low-dose endothelial monocyte-activating polypeptide-II (EMAP-II)-induced blood-tumor barrier (BTB) opening. In the present study, an in vitro BTB model was used to investigate which isoforms of PKC were involved in this process as well as the interactions between the RhoA/Rho kinase and the PKC signaling pathways. Our results showed that EMAP-II-activated PKC-α, β, and ζ and induced translocations of them from the cytosolic to the membrane fractions of rat brain microvascular endothelial cells. The EMAP-II-induced alterations in BTB permeability and tight junction (TJ) protein expression were partially blocked by GÖ6976, the inhibitor of PKC-α/β, and PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI). Meanwhile, we observed that GÖ6976 partly inhibited the EMAP-II-induced rearrangement of actin cytoskeleton as well as phosphorylation of myosin light chain and cofilin, whereas PKC-ζ-PI had no effect on these above-mentioned changes induced by EMAP-II. Also, our data revealed that inhibition of RhoA or inhibition of Rho kinase significantly diminished the activities and the translocations of PKC-α and PKC-β induced by EMAP-II, whereas PKC-ζ was unaffected. However, inhibition of PKC-α/β or inhibition of PKC-ζ did not cause any changes in the RhoA and Rho kinase activities. The effects of EMAP-II on BTB permeability and TJ proteins expression were completely blocked by inhibition of both RhoA and PKC-ζ, whereas inhibition of both RhoA and PKC-α/β had an effect similar to that of inhibition of RhoA alone. In summary, this study demonstrates for the first time that three PKC isoforms, PKC-α, β, and ζ, are involved in the EMAP-II-induced BTB opening. It is PKC-α/β, but not PKC-ζ, which serves as the downstream target for RhoA and Rho kinase, suggesting that EMAP-II induces BTB opening via the RhoA/Rho kinase/PKC-α/β signaling pathways. However, PKC-ζ is involved in this process by other mechanisms.

DNA topoisomerase II alpha (Topo-IIα) is an essential nuclear enzyme with its up-regulation demonstrated in different tumors. This study is to evaluate the expression of Topo-IIα in glioblastoma cancer stem cells (CSCs) and its effects on cell proliferation and apoptosis. We observed the expression differences of Topo-IIα gene transcript in CSCs and non-CSCs in glioblastoma cell line U87 by quantitative real-time PCR and western blot; we silenced Topo-IIα expression in U87 CSCs using Topo-IIα-specific siRNA to investigate the role of Topo-IIα in cell proliferation, cell cycle, and apoptosis. We found higher expression of Topo-IIα in CSCs than in non-CSCs. After using Topo-IIα-specific siRNA to silence the expression of Topo-IIα gene, we observed decreasing cell proliferation, cell cycle arrest, and induction of apoptosis in CSCs, which suggest that Topo-IIα contributes to both cell proliferation and/or cell survival. Topo-IIα may be a novel marker of cell proliferation and/or survival factor in glioblastoma CSCs. It may be a potential target that will open up new opportunities to develop therapeutic strategies for gliomas., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Endothelial monocyte-activating polypeptide-II (EMAP-II) increases blood-tumor barrier (BTB) permeability by inducing alterations in the tight junction (TJ) complex between brain endothelial cells. In the present study, an in vitro BTB model was used to search for the interacting and functional cell surface molecule of EMAP-II as well as the signaling pathway involved in the EMAP-II-induced BTB hyperpermeability. Our results revealed that EMAP-II-induced increase in BTB permeability and down-regulation of TJ-related proteins occludin and ZO-1 were associated with its binding to ATP synthase α subunit (α-ATP synthase) on the surface of rat brain microvascular endothelial cells (BMECs). In addition, we observed that EMAP-II administration activated protein kinase C (PKC) and induced the translocation of PKC from the cytosolic to the membrane fraction of BMECs. The effects of EMAP-II on BTB permeability as well as expression levels of occludin and ZO-1 in BMECs were significantly diminished by H7, the inhibitor of PKC. In summary, these data suggest that EMAP-II increases BTB permeability through α-ATP synthase on the surface of BMECs, and PKC signaling pathway might be involved in this process.

The purpose of the present study was to determine the potential for RhoA/ROCK signaling to play a role in endothelial-monocyte-activating polypeptide (EMAP) II-induced increase in blood-tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). In the present study, we used an in vitro BTB model, a RhoA inhibitor (C3 exoenzyme) and a ROCK inhibitor (Y27632) to determine whether RhoA/ROCK pathway play a role in the process of TJ disassembly, stress fiber formation, MLC and cofilin phosphorylation, as well as increase of BTB permeability induced by EMAP II. The results revealed that BTB permeability was increased by EMAP II induction, and C3 exoenzyme or Y27632 could partially inhibit the EMAP II-induced increase of BTB permeability. The significant down-regulations in tight junction (TJ)-associated proteins occludin, claudin-5 and ZO-1 and stress fiber formation by EMAP II administration were observed, which were partly prevented by C3 exoenzyme or Y27632 pretreatment. Moreover, the significant increases in RhoA activity, myosin light chain (MLC) and cofilin phosphorylation by EMAP II administration were observed, MLC and cofilin phosphorylation were partly inhibited by C3 exoenzyme or Y27632 pretreatment. The present study demonstrates that the activation of RhoA/ROCK signaling in RBMECs was required for the increase of BTB permeability and these effects are related with the ability for RhoA/ROCK to mediate TJ disassembly and stress fiber formation by phosphorylating cofilin and MLC.

The purpose of this study is to determine whether the expressions of MMP-7 and MMP-14 are associated with the ERK 1/2 signaling pathway in human brain gliomas of different pathological grades. Immunohistochemistry and western blot methods were used to determine the expressions of MMP-7, MMP-14 and the phosphorylation status of ERK1/2 in 73 cases of human brain glioma specimens and two cases of normal brain tissues. Results indicated that the protein expression levels of MMP-7, MMP-14 and ERK1/2 phosphorylation level were all elevated with the increasing pathological grades in brain glioma tissues, and correlation assay indicated that the level of ERK1/2 phosphorylation was positively correlated with protein expression levels of MMP-7 and MMP-14 in gliomas of different pathological grades respectively. Moreover, the impact of ERK1/2 inhibitor U0126 on the expressions of MMP-7 and MMP-14 was examined in human U87 glioma cells by western blot analysis. The expressions of MMP-7 and MMP-14 were significantly decreased in human U87 glioma cells after treatment with ERK1/2 inhibitor U0126. The above results suggest that the expressions of MMP-7 and MMP-14 may be associated with activation of ERK1/2 signaling pathway in human brain gliomas of different pathological grades.

Stemness genes, including NANOG, which have been reported to play a significant role in embryonic stem cells (ESCs), are purported to be expressed in specific human tumor types. In the present study, we explored the expression of NANOG in gliomas to demonstrate its key roles in maintaining the undifferentiated state of glioma cells. Brain tumor stem cells (BTSCs) were isolated from the human glioma cell line U87 and cultured in simplified serum-free medium. Significantly higher NANOG mRNA and protein expression levels were demonstrated in U87 parental attached cells and suspended BTSCs as well as in 69 glioma specimens of different pathological grades. The relative levels of NANOG mRNA and protein expression were higher in the BTSCs as compared to those in the U87 parental attached cells and were significantly positively correlated with pathological grade. The coexpression and relationship of NANOG, CD133 and GFAP in situ in the cellular levels was determined through double-label immunohistochemical staining in the gliomas. A positive correlation of NANOG and CD133 expression with pathological grade of the samples was noted, while NANOG and GFAP expression correlated negatively with the pathological grade (P<0.01). Overexpression of NANOG in gliomas and its close relationship with the undifferentiated state of glioma cells in vivo and in vitro indicated that NANOG may contribute to the existence of BTSCs and may be related to tumorigenesis of the cerebrum by maintaining the undifferentiated state of glioma cells, which provides a foundation to further explore its role in the biological behavior of gliomas.

The first goal of this study was to determine the effect of vascular endothelial growth factor (VEGF) on permeability of the blood-tumor barrier (BTB). The second goal was to determine possible cellular mechanisms by which VEGF increases permeability of the BTB. In the rat C6 glioma model, the permeability of the BTB was significantly increased after VEGF injection at dose of 0.05 ng/g and reached its peak at 45 min. Meanwhile, we observed that the density of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB increased dramatically by transmission electron microscopy. The immunohistochemistry and western blot analysis revealed that the expression level of caveolae structure proteins caveolin-1 and caveolin-2 in BMECs was increased after VEGF injection, peaked at 45 min, and then decreased to the untreated level. The time peak of expression level of caveolin-1 and caveolin-2 was identical with the peak time of permeability of the BTB and the density of pinocytotic vesicles. All of these results strongly indicated that VEGF increased permeability of the BTB caused by enhancement of the density of pinocytotic vesicles, and the molecular mechanism might be associated with upregulated expression of caveolin-1 and caveolin-2.

Objective: To investigate the influence of adherent culture on the acquisition of CD133+ cells in glioblastomas and the expressions of acetaldehyde dehydrogenase 1 (ALDH1) in the undifferentiated and differentiated cells., Methods: Adherent culture was performed with immunomagnetic bead technique in 7 glioblastoma samples to gain CD133+ cells. The cell differentiation was induced via serum medium culture. Immunocytochemistry staining was used to detect the expressions of ALDH1 and Sox2 in the undifferentiated and differentiated cells., Results: CD133+ cells were obtained in 6 samples and typical tumor spheres were observed. Positive expressions of ALDH1 and Sox2 were detected in the undifferentiated cells and co-localization was found. No expressions of those two markers in the differentiated cells were observed., Conclusion: CD133+ cells can be efficiently obtained via adherent culture method. ALDH1 expression appears only in the undifferentiated cells, which can be a new marker for glioma stem cells.

This study was performed to determine whether low frequency ultrasound (LFU) irradiation, Papaverine (PA) infusion and combination LFU irradiation with PA infusion opened the blood-tumor barrier (BTB) by affecting tight junctions (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin and caludin-5. In a rat brain glioma model, we found that the mRNA and protein expression levels of ZO-1, occludin and claudin-5 were decreased by LFU irradiation and PA infusion. LFU-induced and PA-induced decrease of ZO-1, occludin and claudin-5 was further decreased after combining LFU irradiation with PA infusion. Immunohistochemistry assay showed that the decreased expression of ZO-1, occludin and claudin-5 was the most obvious in the tumor capillaries. Meanwhile, Evans blue assay showed that the permeability of BTB was increased, and transmission electron microscopy (TEM) indicated that TJ was opened. This led to the conclusion that LFU irradiation and PA infusion together can open the BTB by paracellular pathway. Significantly down-regulated expression levels of ZO-1, occludin and claudin-5 might be one of the molecular mechanisms of combining LFU and PA enhancing the permeability of BTB.

Objective: To investigate the influence of trauma on the tumorigenicity of rat glial tumor stem cells (C6-side population cell, C6-SP) in vivo., Methods: Rat glial tumor stem cells C6-SP were isolated by flowcytometry. The biological behavior of C6-SP cells were observed by means of MTT experiment, single cell cloning, and cell cycle study with FCM CD133 expression was measured by immunofluorescence and FCM.. The tumorigenicity of C6-SP cells in vivo was evaluated by in situ tumor growth after intracranial implantation. The rat model was established by intracranial implantation of C6-SP cells. 10 days later, the rats in experimental groups were subjected to orthotopic or ectopic trauma. 24 days later, brain specimen was retrieved, gross tumor volume was measured, and Ki67 was evaluated by immunochemistry. The migration of stem cell was studied by the method to observe the relocation of C6-SP cells., Results: Clustrus tumor growth was seen when C6-SP cell was cultured in serum-free medium. The doubling time of C6-SP cell was shorter than ordinary C6 cell. Single stem cell cloning efficiency of C6-SP cell was 77% whereas that of ordinary C6 cell was 16.4%. Among cloned C6-SP cell and ordinary C6 cell, 49.7% +/- 5.2% and 35.2 +/- 4.3% were at G0/G1 phase respectively. CD133 expression of C6-SP cells was positively shown by immunofluorescence. Tumorigenesis was 100% 2 weeks after in situ implantation of C6-SP cells. Gross tumor volume and Ki67 expression of orthotopic trauma group were larger and higher than those of ectopic trauma group or blank control group (P < 0.05) whereas difference was insignificant in the later two groups (P > 0.05). Red stained cells relocation was seen in both traumatized groups and absent in controls., Conclusion: C6-SP cells are the tumor stem cells (TSCs) for rat glioma. Trauma at the lesional site could increase tumorigenicity of the C6-SP cells. Moreover, trauma could induce migration of C6-SP cells in brain.

Intra-arterial administration of papaverine has been revealed to cause an increase in the blood-brain tumor barrier (BTB) permeability. The exact mechanism of papaverine opening the BTB in chemotherapy of malignant cerebral tumors, however, has not been well described. We used a rat brain glioma (C6) model for studying how papaverine modulates the permeability of BTB by monitoring the activities of the tight junction (TJ)-associated protein occludin, claudin-5 and cytoskeletal protein filamentous actin (F-actin) and whether protein kinase A (PKA) and heat shock protein 70 (HSP70) were involved in the regulation of this biological process. The levels of occludin, claudin-5 and F-actin protein in the tumor tissues were down-regulated by papaverine via immunohistochemistry, immunofluorescence assays and Western blot, corresponding to the time-dependent change of the BTB permeability. The most obvious attenuation of occludin, claudin-5 and F-actin protein was observed at 1h after papaverine perfusion, companied by a significant decrease in expression levels of PKA protein. The expression level of HSP70 in the tumor tissues was also progressively increased after papaverine perfusion and reached the maximum at 3h. The results demonstrate that the reversible openning of BTB mediated by papaverine may be associated with the functional combination between PKA and HSP70. That is, BTB opening may be attributable to the down-regulation of occludin, claudin-5 and F-actin, and cAMP/PKA signaling pathway might be involved in this process. HSP70 is likely responsible for the BTB closing, which helping the repairment of injured TJ protein and the rebuilding of the BTB., (2010 Elsevier Inc. All rights reserved.)

Bradykinin (BK) increases the permeability of the blood-tumor barrier (BTB) selectively through the transcellular pathway; however, the role of the caveolae structural proteins caveolin-1 and caveolin-2 in this process has not been precisely elucidated. Thus, this study was performed to examine whether caveolin-1 and caveolin-2 are involved in the regulation of this biological process. In the rat brain glioma (C6) model, western blot, immunohistochemistry, and immunofluorescence assays were used to detect the expression levels and locations of caveolin-1 and caveolin-2. The results showed that caveolin-1 and caveolin-2 levels increased 5 min after BK infusion, peaked at 15 min, and then decreased. Meanwhile, Evans blue (EB) assay showed that the permeability of the BTB increased significantly after BK infusion. In our previous study we demonstrated that the quantity of pinocytotic vesicles in the endothelial cells was dramatically augmented 15 min after BK infusion. The time point at which changes of caveolin-1 and caveolin-2 reached their peak was the same as that at which EB and the quantity of pinocytotic vesicles reached their peaks. This led to the conclusion that the BK-mediated BTB permeability increase resulting from augmentation of the quantity of pinocytotic vesicles (transcellular pathway) is associated with the significantly up-regulated expression of caveolin-1 and caveolin-2. This study thus contributes further to elucidating the molecular mechanism of opening of the BTB by BK and provides a theoretical basis for clinical application of BK.

This study was performed to determine whether endothelial-monocyte-activating polypeptide (EMAP) II increases the permeability of the blood-tumor barrier (BTB) in the rat model of C6 glioma, and whether EMAP II opens the BTB by affecting tight junction (TJ) associated proteins zonula occluden-1 (ZO-1), occludin and claudin-5. The rats were divided into eight groups randomly: control group, EMAPII 0h group, EMAPII 0.5h group, EMAPII 1h group, EMAPII 2h group, EMAPII 3h group, EMAPII 6h group and EMAPII 12h group. The BTB permeability was assessed by Evans blue extravasation. The mRNA and protein expressions of ZO-1, occludin, and claudin-5 were determined by reverse transcriptase-polymerase chain reaction, western blot, and immunohistochemistry assays. The BTB permeability significantly increased after EMAP II injection in different doses (40ng/kg, 80ng/kg and 160ng/kg). The BTB permeability started to increase from 0.5h, reached a peak at 1h, and finally returned to the level of EMAP II 0h group after EMAP II injection at dose of 80ng/kg. The mRNA and protein expression levels of ZO-1, occludin and claudin-5 were significantly decreased after EMAP II injection. This study demonstrates for the first time that EMAP II increases the permeability of BTB selectively, and the possible mechanism is associated with the down-regulation of ZO-1, occludin and claudin-5., (Copyright 2010 Elsevier B.V. All rights reserved.)

The research was conducted to study the characteristics of the noninvasive, reversible, targeted opening of the blood-brain barrier (BBB) by use of low-frequency ultrasound (LFU) irradiation and the selective opening of the blood-tumor barrier (BTB) by intracarotid infusion of bradykinin (BK) in small-dose, with the objective of exploring maximum opening of the BTB by combining LFU irradiation with BK infusion. Thus, it provides new therapeutic strategies for targeted transport of macromolecular or granular drugs to the brain. By using the rat C6 glioma model it was shown that extravasation of Evans blue (EB) through the BTB was significantly increased by combining LFU irradiation (frequency = 1.0 MHz, power = 12 mW, duration = 20 s) with intracarotid small-dose BK infusion, compared with utilizing the two methods separately. By transmission electron microscopy (TEM) we observed that this combination significantly increased the number of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB. An even more significant increase was observed by using RT-PCR, western blot, immunohistochemistry, and immunofluorescence to detect mRNA and changes of expression of the caveolae structure proteins caveolin-1 and caveolin-2 of BMECs. In summary, this research concludes that LFU irradiation and small-dose BK together selectively enhance the permeability of the BTB and increase the number of pinocytic vesicles of BMECs to a maximum. Significant up-regulation of the level of expression of caveolae structure proteins caveolin-1 and caveolin-2 might be the molecular mechanism of the co-enhanced endocytotic transport by BMECs. Thus, this research provides new therapeutic strategies for targeted transport of macromolecular drugs and the design of drugs.

Objective: To investigate the expression of aquaporin-4 (AQP4) in the brains of patients with glioblastoma and its association with brain edema., Methods: Immunofluorescence cytochemistry and western blot tests were performed to detect the expression of AQP4 in the brain tumors and the adjacent tissues in 30 patients with glioblastoma. The association between AQP4 and the extent of brain edema was analysed., Results: The AQP4 immunoreactive cells were mainly astrocytes in the brains, which were extensively distributed in the intracytoplasm. Higher expressions of AQP4 were found in the brain tumors and adjacent tissues in the patients with glioblastoma than in the normal controls (P<0.05). More AQP4 were distributed in the tumor adjacent tissues than in the tumors (P<0.05). The AQP4 was positively correlated with the extent of brain edema., Conclusion: AQP4 overexpress in the brain tumors and adjacent tissues, which is associated with the extent of brain edema. Cytotoxic and vasogenic edemas may coexist in the cerebral edema induced by glioblastoma.