Early VEGF inhibition attenuates blood-brain barrier disruption in ischemic rat brains by regulating the expression of MMPs.

Vascular endothelial growth factor (VEGF) inhibition has been demonstrated to be an effective strategy in preserving the integrity of the blood-brain barrier (BBB) in patients with acute ischemic stroke. Loss of the BBB is the key event associated with morbidity and mortality in these patients. However, the underlying mechanisms remain poorly understood. In the present study, the effects of VEGF inhibition and the possible mechanism that underlies acute cerebral ischemia in rats was investigated. Following the induction of transient middle cerebral artery occlusion for a 90­min period, either an anti­VEGF neutralizing antibody (RB­222; 5 or 10 µg), or IgG (control), was administered by intracerebroventricular injection at 1 h following reperfusion. Functional outcomes, BBB leakage, brain edema, microvessel numbers and the relative protein levels of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, occludin and collagen-IV were then determined using neurological assessments, Evans Blue staining, brain water content, CD31 staining and western blotting. Treatment with RB­222 at a dose of 5 and 10 µg significantly improved neurological functional outcomes and diminished infarct size, BBB leakage and brain edema compared with the MCAO and IgG groups at 24 h following reperfusion; 10 µg RB­222 was more effective than a 5 µg dose of the antibody. In addition, RB­222 reduced the number of immature microvessels, which subsequently attenuated BBB permeability. RB­222 significantly repressed VEGF expression as well as decreased MMP­2 and MMP­9 expression. However, it enhanced occludin and collagen­IV levels in the ischemic rat brain compared with the MCAO and IgG groups. Taken together, the results indicate that early inhibition of VEGF may have significant potential against cerebral ischemia, partly by regulating the expression of MMPs.

TRIM28 as an independent prognostic marker plays critical roles in glioma progression.

Tripartite motif (TRIM) proteins are involved in tumorigenesis. Here, we examined the expression, biological function, and clinical significance of tripartite motif containing 28 (TRIM28) in glioma, a locally aggressive brain tumor. First, TRIM28 expression was significantly higher in glioma (n = 138) than in non-glioma controls (n = 6). TRIM28 expression was positively correlated with tumor malignancy, and associated with poor overall survival (OS) and progression-free survival (PFS). Notably, TRIM28 expression was negatively correlated with p21 expression in patients with glioblastoma multiforme (GBM). A multivariate analysis that included relevant measures indicated that high TRIM28 expression is an independent prognostic factor for poor OS and PFS in GBM patients. In experiments with cultured glioma cells, down-regulating TRIM28 with shRNA increased p21 expression, and induced cell cycle arrest at the G1 phase. In a xenograft model, down-regulating TRIM28 suppressed tumor growth. These results indicate that over-expression of TRIM28 is associated with poor outcome in glioma patients.

Subgroup characteristics of insular low-grade glioma based on clinical and molecular analysis of 42 cases.

Although the classification of insular glioma has been established based on the anatomical location in order to facilitate personalized surgical resection, the diagnosis based on anatomical and functional characteristics becomes more complex when insular tumors extend into either the frontobasal brain region and/or the temporal lobe, as part of the limbic system. Moreover, prognosis of insular tumor resection is still controversial. Further analysis of subgroup characteristics of insular grade II gliomas based on clinical and molecular analysis is required to reliably determine patients' survival rates. In this retrospective study 20 purely insular grade II gliomas patients and 22 paralimbic grade II gliomas that involved frontal and/or temporal lobes were compared with regard to epidemiological and clinical characteristics. The molecular profiles including Isocitrate dehydrogenase 1 (IDH1), telomerase reverse transcriptase (TERT) promoter, and P53 mutations, 1p19q co-deletion were analyzed, and microRNA profiles were assessed by microarray and bioinformatics analysis. Purely insular grade II gliomas displayed a high frequency of IDH1 mutations with favorable outcome. IDH1 mutated paralimbic glioma shared many parameters with the purely insular glioma in respect to growth patterns, survival, and microRNA profile, but differed significantly from the IDH1 wild type paralimbic gliomas. Our findings suggest that IDH1 mutations can define subpopulations of insular gliomas with distinct disease entities regardless of tumor extension patterns. These findings could be useful to develop a customized treatment strategy for insular glioma patients.

miR-124 suppresses the migration and invasion of glioma cells in vitro via Capn4.

miR-124 and Capn4 are aberrantly expressed in glioblastoma multiforme (GBM) tissues. In the present study, we investigated miR-124 and Capn4 expression in GBM tissue specimens. The role of miR-124 and Capn4 in the migration and invasion of glioma cells in vitro was also examined. miR-124 and Capn4 expression in 20 GBM and 6 control brain specimens was examined using RT-qPCR and immuno-blotting. Data from The Cancer Genome Atlas were retrieved. Candidate mRNA target sites of miR-124 were predicted using TargetScan/microRNA and binding was examined using dual luciferase reporter assays. The U87 and U251 cells were transfected with scrambled microRNA, miR-124 mimics and/or pLenti-Capn4 prior to wound­healing and Transwell invasion assays. Proteins involved in the epithelial-mesenchymal transition were examined using immunoblotting. The results showed that miR-124 was significantly downregulated in GBM tissues. Immunoblotting showed a marked upregulation of Capn4 expression in GBM tissues. The Spearman's correlation analysis revealed a negative association between miR-124 expression and Capn4 protein levels. TargetScan/microRNA predicted the miR-124 binding site in the nucleotide 440-446 region within the Capn4 3'-UTR, which was confirmed by luciferase assays. Wound­healing and Transwell invasion assays demonstrated that Capn4 downregulation or miR-124 mimics suppressed the migration and invasion of glioma cells. Capn4 downregulation or miR-124 mimics reduced the level of phospho-FAK and MMP2, vimentin and N-cadherin in U87 cells. In conclusion, miR-124 was found to suppress the migration and invasion of glioma cells in vitro via Capn4.

TERT promoter mutated WHO grades II and III gliomas are located preferentially in the frontal lobe and avoid the midline.

The promoter region of telomerase reverse transcriptase (TERTp) and isocitrate dehydrogenase (IDH) have been regarded as biomarkers with distinct clinical and phenotypic features. Investigated the possible correlations between tumor location and genetic alterations would enhance our understanding of gliomagenesis and heterogeneity of glioma. We examined mutations of TERTp and IDH by direct sequencing and fluorescence in-situ hybridization in a cohort of 225 grades II and III diffuse gliomas. Correlation analysis between molecular markers and tumor locations was performed by Chi-square tests/Fisher's exact test and multivariate logistic regression analysis. We found gliomas in frontal lobe showed higher frequency of TERTp mutation (P=0.0337) and simultaneously mutations of IDH and TERTp (IDH (mut)-TERTp(mut)) (P=0.0281) than frequency of biomarkers mutation of tumors in no-Frontal lobes, while lower frequency of TERTp mutation (P<0.0001) and simultaneously wild type of IDH and TERTp (IDH (wt)-TERTp(wt)) (P<0.0001) in midline than no-midline lobes. Logistic regression analysis indicated that locations of tumors associated with TERTp mutation (OR=0.540, 95% CI 0.324-0.900, P=0.018) and status of combinations of IDH and TERTp (IDH (mut)-TERTp (mut) vs. IDH (wt)-TERTp (wt) OR=0.162, 95% CI 0.075-0.350, P<0.001). In conclusion, grades II and III gliomas harboring TERTp mutation were located preferentially in the frontal lobe and rarely in midline. Association of IDH-TERTp status and tumor location suggests their potential values in molecular classification of grades II and III gliomas.

TERT promoter mutations contribute to IDH mutations in predicting differential responses to adjuvant therapies in WHO grade II and III diffuse gliomas.

IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations (p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup.

MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab.

BACKGROUND: Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. METHODS: miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. RESULTS: In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the ß-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. CONCLUSIONS: miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.

Blockage of a miR-21/EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas.

Epidermal growth factor receptors (EGFR) expression is frequently amplified in human glioblastoma cells. Nimotuzumab, a monoclonal antibody (mAb) against EGFR, has been used globally in clinics as an anti-cancer agent. It is largely unknown whether the blockade of miR-21, a microRNA that is upregulated in glioma cells, could amplify the effects of nimotuzumab. Herein, we have demonstrated that miR-21 directly targets von Hippel-Lindau (VHL) and peroxisome-proliferator-activated receptor α (PPARα) and that miR-21 regulates EGFR/AKT signaling through VHL/ß-catenin and the PPARα/AP-1 axis. Further, the expression of miR-21 is regulated by EGFR via the activation of ß-catenin and AP-1. These data indicate that a feedback loop exists between miR-21 and EGFR. We also show that the combination of nimotuzumab and an inhibitor of miR-21 is superior to single-agent therapy. These results clarify a novel association between miR-21 and EGFR in the regulation of cancer cell progression.

Serological identification of URGCP as a potential biomarker for glioma.

AIM: Glioblastoma multiforme (GBM) is one of the most frequent human brain tumor and causes dismal outcome. To identify tumor-associated antigens in GBM patients may find potential diagnostic markers and immunotherapeutic targets. In this study, we identified a gene termed URGCP using the serological identification of antigens by recombinant A2B5 positive glioma cDNA library. The gene product of URGCP is immunogenic in GBM after tested in allogenic patients serum screening. METHODS AND RESULTS: GBM patients with an auto-antibody response against URGCP show longer survival than those without URGCP response. In additional, we show that URGCP was high expression in most GBM tissues and cell lines compared with normal brain tissues and majorly co-expressed with stem cell marker A2B5. CONCLUSION: We identified a potential new biomarker of GBM, URGCP. The findings indicate that URGCP is immunogenic in human GBM and suggest its potential use as diagnostic and immunotherapeutic for GBM patients.

Curcumin suppresses malignant glioma cells growth and induces apoptosis by inhibition of SHH/GLI1 signaling pathway in vitro and vivo.

AIMS: To study the role of curcumin on glioma cells via the SHH/GLI1 pathway in vitro and vivo. METHODS: The effects of curcumin on proliferation, migration, apoptosis, SHH/GLI1 signaling, and GLI1 target genes expression were evaluated in multiple glioma cell lines in vitro. A U87-implanted nude mice model was used to study the role of curcumin on tumor volume and the suppression efficacy of GLI1. RESULTS: Curcumin showed cytotoxic effects on glioma cell lines in vitro. Both mRNA and protein levels of SHH/GLI1 signaling (Shh, Smo, GLI1) were downregulated in a dose- and time-dependent manner. Several GLI1-dependent target genes (CyclinD1, Bcl-2, Foxm1) were also downregulated. Curcumin treatment prevented GLI1 translocating into the cell nucleus and reduced the concentration of its reporter. Curcumin suppressed cell proliferation, colony formation, migration, and induced apoptosis which was mediated partly through the mitochondrial pathway after an increase in the ratio of Bax to Bcl2. Intraperitoneal injection of curcumin in vivo reduced tumor volume, GLI1 expression, the number of positively stained cells, and prolonged the survival period compared with the control group. CONCLUSION: This study shows that curcumin holds a great promise for SHH/GLI1 targeted therapy against gliomas.

Mutation analysis of IDH1 in paired gliomas revealed IDH1 mutation was not associated with malignant progression but predicted longer survival.

Recurrence and progression to higher grade lesions are characteristic behaviors of gliomas. Though IDH1 mutation frequently occurs and is considered as an early event in gliomagenesis, little is known about its role in the recurrence and progression of gliomas. We therefore analysed IDH1 and IDH2 status at codon 132 of IDH1 and codon 172 of IDH2 by direct sequencing and anti-IDH1-R132H immunohistochemistry in 53 paired samples and their recurrences, including 29 low-grade gliomas, 16 anaplastic gliomas and 8 Glioblastomas. IDH1/IDH2 mutation was detected in 32 primary tumors, with 25 low-grade gliomas and 6 anaplastic gliomas harboring IDH1 mutation and 1 low-grade glioma harboring IDH2 mutation. All of the paired tumors showed consistent IDH1 and IDH2 status. Patients were analyzed according to IDH1 status and tumor-related factors. Malignant progression at recurrence was noted in 22 gliomas and was not associated with IDH1 mutation. Survival analysis revealed patients with IDH1 mutated gliomas had a significantly longer progression-free survival (PFS) and overall survival (OS). In conclusion, this study demonstrated a strong tendency of IDH1/IDH2 status being consistent during progression of glioma. IDH1 mutation was not a predictive marker for malignant progression and it was a potential prognostic marker for gliomas of Chinese patients.

MiR-139 inhibits Mcl-1 expression and potentiates TMZ-induced apoptosis in glioma.

AIMS: Mcl-1, an antiapoptotic member of the Bcl-2 family, is overexpressed in human glioblastoma, conferring a survival advantage to tumor cells. The mechanisms underlying its dysregulation have not been clarified. In this study, we explored the involvement of micro-RNAs that acted as endogenous sequence-specific suppressors of gene expression. METHODS AND RESULTS: Using computational and TCGA analysis, we identified miR-139 as being downregulated in glioblastoma in comparison with human brain tissue, as well as possessing a putative target site in Mcl-1 mRNA. Overexpression of miR-139 led to a clear decrease in Mcl-1 expression in gliomas. Reporter assays revealed direct post-transcriptional regulation involving miR-139 and the 3'-untranslated region of Mcl-1. Human glioma tissues with low expression of miR-139 displayed higher expression of Mcl-1 protein than those with high expression, suggesting that low miR-139 contributes to Mcl-1 overexpression. In addition, upregulation of miR-139 suppressed the proliferation and enhanced temozolomide (TMZ)-induced apoptosis. Finally, we observed that Mcl-1 knockdown resulted in similar effects compared with miR-139 transfection. CONCLUSION: Our results suggested that miR-139 negatively regulated Mcl-1 and induced apoptosis in cooperation with an anticancer drug TMZ in glioma.

Robertsonian translocations: an overview of a 30-year experience in a single tertiary medical center in Taiwan.

BACKGROUND: Advanced maternal age (AMA) is the most frequent indication for amniocentesis in predicting balanced reciprocal translocations, and abnormal ultrasound findings are indications in predicting unbalanced reciprocal translocations; however, to date, no studies have focused on Robertsonian translocations. METHODS: A retrospective review was conducted on 16,749 pregnant women who underwent midtrimester amniocentesis between January 1981 and December 2010. Robertsonian translocations were identified in 39 cases. RESULTS: The percentage of Robertsonian translocations in all amniocentesis cases was 0.23% (39/16,749); 31 were balanced and eight were unbalanced. De novo abnormality occurred in 17 cases, or in 43.6% of all Robertsonian translocations. The two major indications for amniocentesis with a diagnosis of Robertsonian translocations were AMA (41.0%, n = 16) and a parent with abnormal karyotypes (18.0%, n = 7). The highest percentage of Robertsonian translocations was found in parents with abnormal karyotypes (2.8%, 7/252), but neither of the indications were clearly superior for detecting de novo Robertsonian translocations. CONCLUSION: Although AMA is an indication for amniocentesis in approximately two-fifths of cases with Robertsonian translocations, the indication of parent with abnormal karyotypes was more likely to lead to the detection of non-de novo Robertsonian translocations, suggesting that parents with abnormal karyotypes need careful prenatal consultation.

Balanced and unbalanced reciprocal translocation: an overview of a 30-year experience in a single tertiary medical center in Taiwan.

BACKGROUND: Reciprocal translocation is the most common type of translocation; however, there are only a few studies that address the indications for reciprocal translocation in amniocentesis. Here we share our data, based on 30 years' experience in a single tertiary center, to investigate the rates and indications for amniocentesis in cases of reciprocal translocations. METHODS: A retrospective review of 16,749 pregnant women, who underwent midtrimester amniocentesis between January 1981 and December 2010, was conducted. Seventy-four cases of reciprocal translocation were identified. RESULTS: The percentage of reciprocal translocations in all amniocentesis cases was 0.44% (74/16,749); of these 74 cases, 56 were balanced and 18 unbalanced. De novo abnormality occurred in 23 cases, which constituted 31.1% of all reciprocal translocations. The three major indications for amniocentesis with a diagnosis of reciprocal translocation included advanced maternal age (AMA, 52.7%), a parent with an abnormal karyotype (17.6%), and abnormal biochemical markers in the maternal serum (12.2%). For individual types of reciprocal translocations (balanced and unbalanced), except for the presence of abnormal biochemical markers in maternal serum, both AMA and a parent with an abnormal karyotype were primary indications for amniocentesis. However, the highest percentage of reciprocal translocations in all amniocentesis cases was found in cases involving a parent with an abnormal karyotype (5.16%, 13/252). CONCLUSION: Patients with a parent who carries an abnormal karyotype should be encouraged to undergo amniocentesis in prenatal consultation, since the risk of a diagnosis of reciprocal translocation can be particularly high.

An overview of a 30-year experience with amniocentesis in a single tertiary medical center in Taiwan.

OBJECTIVE: Amniocentesis is a popular and effective prenatal diagnostic tool for chromosomal disorders. It is well-established that the risk of chromosomal abnormalities increases with maternal age; however, other related indications are seldom reported. Herein, we report our 30-year experience with amniocentesis from a single medical center, focusing on the indications and rates of abnormality. MATERIAL AND METHODS: A retrospective review of 16,749 pregnant women in the mid-trimester between January 1981 and December 2010 was conducted. The medical records were analyzed. RESULTS: The indications for amniocentesis were advanced maternal age (≥ 34 years old) (n=10,970, 65.5%), increasing-risk maternal triple-marker Down's screening test (≥ 1/270) (n=2090, 12.5%), history of abnormal offspring birth (n=792, 4.7%), abnormal ultrasound findings (n=484, 2.9%), parent with abnormal karyotype (n=252, 1.5%), family history of chromosomal abnormality (n=183, 1.1%), drug and radiation exposure (n=165), abnormal chorionic villus sampling (CVS) results (n=25), intrauterine fetal death (n=50), and other non-specific causes (n=1662, 9.9%). The rate of abnormality for each indication was 16% in the abnormal CVS group, 12% in the intrauterine fetal death group, 11.5% for parental chromosomal abnormality, 8.7% in the abnormal ultrasound finding group, 3.0% in the increasing-risk maternal triple-marker Down's screening test group, 2.5% in the advanced maternal age group, 1.5% for other non-specific causes, 1.4% for history of abnormal offspring birth, and 1.1% for family history of chromosomal abnormality. CONCLUSIONS: Both parents with abnormal karyotype and abnormal ultrasound findings are indications for which consideration of further amniocentesis is highly recommended.

Prenatal diagnosis of mosaic tetrasomy 10p associated with megacisterna magna, echogenic focus of left ventricle, umbilical cord cysts and distal arthrogryposis.

We present the first report of mosaic isochromosome 10p associated with multiple congenital anomalies including megacisterna magna, echogenic focus of the left ventricle, umbilical cord cysts, and distal arthrogryposis. The most obvious anomalies found on prenatal ultrasound were enlarged cisterna magna and lower limb flexion contractures which resembled clubfeet. Analyses of GTG-banded chromosomes of 42 cells harvested from 32 independent tissue culture colonies were examined. Thirty-five cells from 27 colonies had 46 chromosomes and appeared to be 46,XX, female karyotype. Seven cells from independent colonies had 47 chromosomes with abnormal karyotypes. The extra chromosome material was identified as isochromosome 10p without involvement of the heterochromatic region of the long arm [47,XX,+ i(10p)]. Mosaic tetrasomy 10p was confirmed using fluorescent in situ hybridization (FISH) of a 10p-specific probe to metaphase chromosomes of this patient.