ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

BACKGROUND: The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot. RESULTS: The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment. CONCLUSION: Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells

​Occult extracranial malignancy after complete remission of pineal mixed germ cell tumors: a rare case report and literature review

Abstract Background ​Extracranial metastasis can occur in intracranial germ cell tumors (GCTs), but it is very rare. Recurrence or metastasis of non-germinomatous germ cell tumors (NGGCTs) is often accompanied by elevated tumor markers. ​Occult extracranial metastases or recurrences with negative markers are often difficult to detect in time, resulting in a very poor prognosis. Case presentation A 12-year-old boy was admitted to our institution with dizziness, headache, vomiting, and sleepiness. Magnetic resonance imaging (MRI) showed a pineal mass, accompanied by a significant increase in serum alpha-fetoprotein (AFP). The patient subsequently underwent total removal of the tumor. Pathology revealed that the tumor was a mixed GCT, consisting of mature teratoma, germinoma, and yolk sac tumor. Intracranial GCT achieved complete remission after intensive adjuvant chemotherapy and radiotherapy. Regular follow-up MRI revealed no recurrence of the intracranial tumor and continued monitoring of tumor markers revealed no abnormalities. ​Eight months later, the patient was readmitted due to progressive abdominal pain. Imaging and physical examination revealed abdominal occupation and lymphatic mass in the neck. He received salvage chemotherapy, anti-PD-1 immunotherapy, and palliative chemotherapy, but still developed multiple organ dysfunction syndromes (MODS) due to tumor progression and eventually died after one month. Conclusions ​This profound case suggests that intracranial NGGCTs may develop occult extracranial malignancy, which can be very severe at the time of clinical symptoms and has an extremely poor prognosis. Therefore, in addition to tumor marker monitoring, regular follow-up with extracranial imaging may be warranted to detect extracranial tumors as early as possible, although perhaps not as frequently as with neuroimaging

Downregulation of Death-associated Protein Kinase 3 and Caspase-3 Correlate to the Progression and Poor Prognosis of Gliomas

Aim: To investigate the role of death-associated protein kinase 3 (DAPK3) and activated caspase-3 in glioma condition. Methods: Immunohistochemical staining for DAPK3 and activated caspase-3 was performed on 136 paraffin-embedded glioma samples and 15 normal brain tissues, and the relationship between their expression levels and clinico-pathological features of glioma was statistically analyzed. Univariate and multivariate analyses were used to evaluate their prognostic value and patients′ survival. Results: The expression of both DAPK3 and activated caspase-3 was found suppressed in glioma tissues (P = 0.001 and P = 0.043). Further, DAPK3 and activated caspase-3 expression were markedly correlated with World Health Organization (WHO) Grading (I-II vs. III-IV) of the glioma condition (P = 0.002 and P < 0.001). A significantly positive correlation was observed between DAPK3 and activated caspase-3 expression (Spearman′s correlation coefficient = 0.706; P < 0.001). Univariate analysis revealed that both DAPK3 and activated caspase-3 were significantly associated with the overall survival of glioma patients (P < 0.001 and P < 0.001). In addition, multivariate analysis demonstrated that only DAPK3 and activated caspase-3 protein levels, but not WHO grading, significantly correlated with patients′ survival (P = 0.008 and P = 0.042). Conclusion: Downregulation of DAPK3 and activated caspase-3 is strongly associated with the clinical progression and poor prognosis of glioma, suggesting their use as a reliable clinical predictor

Prediction and Analysis of Key Genes in Glioblastoma Based on Bioinformatics

Understanding the mechanisms of glioblastoma at the molecular and structural level is not only interesting for basic science but also valuable for biotechnological application, such as the clinical treatment. In the present study, bioinformatics analysis was performed to reveal and identify the key genes of glioblastoma multiforme (GBM). The results obtained in the present study signified the importance of some genes, such as COL3A1, FN1, and MMP9, for glioblastoma. Based on the selected genes, a prediction model was built, which achieved 94.4% prediction accuracy. These findings might provide more insights into the genetic basis of glioblastoma

Determining an Optimal Cutoff of Serum β-Human Chorionic Gonadotropin for Assisting the Diagnosis of Intracranial Germinomas.

BACKGROUND:Beta (β)-human chorionic gonadotropin (β-HCG) is used to confirm the diagnosis and plan treatment of intracranial germinomas. However, the cutoff values of serum β-HCG in diagnosis of intracranial germinomas reported in the literature are inconsistent. To establish an appropriate cutoff value of serum β-HCG for diagnosis of intracranial germinomas, we retrospectively reviewed the records of intracranial tumor patients who received serum β-HCG and α-fetoprotein (AFP) tests for diagnostic purposes at our hospital from 2005 to 2014. METHODS:A total of 93 intracranial germinomas and 289 intracranial non-germ cell tumors were included in this study. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of 3 cutoffs (0.1, 0.4, and 0.5 mIU/mL) for diagnosing intracranial germinomas. The serum β-HCG level of intracranial germinoma patients was further analyzed to investigate the effect of metastasis status and tumor location on serum β-HCG level. RESULTS:The area under the ROC curve was 0.81 (P < .001), suggesting β-HCG is an effective marker. Of the 3 cutoff values, 0.1 mIU/mL possessed a highest sensitivity (66.67%) and good specificity (91%). Although there was no β-HCG level difference between metastatic and non-metastatic intracranial germinoma patients, the diagnostic rate of metastatic neurohypophyseal germinomas was significantly higher than that of its non-metastatic counterpart (P < .05), implying that the location of the germinoma might need to be considered when β-HCG is used as a marker to predict metastasis. CONCLUSIONS:Determining an optimal cutoff of serum β-HCG is helpful for assisting the diagnosis of intracranial germinoma

Diagnosis of intracranial germinoma by pre-treatment serum β-human chorionic gonadotropin (β-HCG) level.

<p>Area under the receiver operating characteristic curve = 0.81 (<i>P</i> < 0.001).</p

Protein expression of ZEB2 between glioma and normal brain tissues.

<p>Protein expression of ZEB2 between glioma and normal brain tissues.</p

ZEB2 downregulation induces apoptosis by the activation of Caspase-3 in glioma cells.

<p><b>A.</b> Apoptosis in U251 and U87 cells was measured by Annexin V-FITC/propidium iodide (PI) staining following siZEB2 or control treatment. Early apoptotic cell populations were significantly increased (P<0.01) after siZEB2 transfection. <b>B.</b> Western blot analysis for antiapoptotic PARP and effector Caspase-3, Caspase-6 and Caspase-9. Decreased PARP and increased caspase-3,-6,-9, and Cleaved PARP expression were observed in U251 and U87 cells after ZEB2 downregulation at 72 hr post transfection. β-actin was used as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean ± SD for three independent experiments. *<i>P</i><0.05, statistically significant difference.</p

ZEB2 downregulation promotes expression of E-cadherin but suppresses EMT progression in glioma cells.

<p>ZEB2 reduction enhanced expression of E-cadherin and expression level changes of N-cadherin, Snail, β-Catenin and Vimentin in U251 and U87 cells at 72 hr post-transfection. β-actin was used as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean ± SD for three independent experiments. *<i>P</i><0.05, statistically significant difference.</p

Effect of siRNA interference on ZEB2 expression in human glioma cell lines U251 and U87.

<p>Different treatments included negative control (NC) and siZEB2-transfected groups (siZEB2). <b>A.</b> RT-PCR shows transcriptional levels of the ZEB2 gene 48 hr post transfection and ARF was used as a loading control. The arbitrary units were plotted using mean ± SD of at least three individual repetitions. <b>B.</b> Western blot showing protein expression levels in NC and siZEB2 treatments. At 72 hr post transfection, cells were harvested and whole cell lysates prepared using RIPA buffer. The representative image of three different repetitions is shown. β-actin served as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented as mean ± SD for three independent experiments. <b>C.</b> Immunofluorescence study using blinded analysis showing the expression of ZEB2 in NC and siZEB2-treated U251 and U87 cells at 48 hr post-transfection (Original magnification 1000×). *<i>P</i><0.05, statistically significant difference.</p