A prospective multicenter assessor blinded pilot study using confocal laser endomicroscopy for intraoperative brain tumor diagnosis.

In this multi-center, assessor-blinded pilot study, the diagnostic efficacy of cCeLL-Ex vivo, a second-generation confocal laser endomicroscopy (CLE), was compared against the gold standard frozen section analysis for intraoperative brain tumor diagnosis. The study was conducted across three tertiary medical institutions in the Republic of Korea. Biopsy samples from newly diagnosed brain tumor patients were categorized based on location and divided for permanent section analysis, frozen section analysis, and cCeLL-Ex vivo imaging. Of the 74 samples from 55 patients, the majority were from the tumor core (74.3%). cCeLL-Ex vivo exhibited a relatively higher diagnostic accuracy (89.2%) than frozen section analysis (86.5%), with both methods showing a sensitivity of 92.2%. cCeLL-Ex vivo also demonstrated higher specificity (70% vs. 50%), positive predictive value (PPV) (95.2% vs. 92.2%), and negative predictive value (NPV) (58.3% vs. 50%). Furthermore, the time from sample preparation to diagnosis was notably shorter with cCeLL-Ex vivo (13 min 17 s) compared to frozen section analysis (28 min 28 s) (p-value < 0.005). These findings underscore cCeLL-Ex vivo's potential as a supplementary tool for intraoperative brain tumor diagnosis, with future studies anticipated to further validate its clinical utility.

Repurposing Penfluridol in Combination with Temozolomide for the Treatment of Glioblastoma.

Despite the presence of aggressive treatment strategies, glioblastoma remains intractable, warranting a novel therapeutic modality. An oral antipsychotic agent, penflurido (PFD), used for schizophrenia treatment, has shown an antitumor effect on various types of cancer cells. As glioma sphere-forming cells (GSCs) are known to mediate drug resistance in glioblastoma, and considering that antipsychotics can easily penetrate the blood-brain barrier, we investigated the antitumor effect of PFD on patient-derived GSCs. Using five GSCs, we found that PFD exerts an antiproliferative effect in a time- and dose-dependent manner. At IC50, spheroid size and second-generation spheroid formation were significantly suppressed. Stemness factors, SOX2 and OCT4, were decreased. PFD treatment reduced cancer cell migration and invasion by reducing the Integrin α6 and uPAR levels and suppression of the expression of epithelial-to-mesenchymal transition (EMT) factors, vimentin and Zeb1. GLI1 was found to be involved in PFD-induced EMT inhibition. Furthermore, combinatorial treatment of PFD with temozolomide (TMZ) significantly suppressed tumor growth and prolonged survival in vivo. Immunostaining revealed decreased expression of GLI1, SOX2, and vimentin in the PFD treatment group but not in the TMZ-only treatment group. Therefore, PFD can be effectively repurposed for the treatment of glioblastoma by combining it with TMZ.

Role of vincristine in the inhibition of angiogenesis in glioblastoma.

OBJECTIVE: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α). METHODS: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O2 (hypoxia). RESULTS: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G2-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC50), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels. CONCLUSIONS: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.

Reactive oxygen species production has a critical role in hypoxia-induced Stat3 activation and angiogenesis in human glioblastoma.

Glioblastoma is the most aggressive primary brain tumor with hypoxia-associated morphologic features including pseudopalisading necrosis and endothelial hyperplasia. It has been known that hypoxia can activate signal transducer and activator of transcription 3 (Stat3) and subsequently induce angiogenesis. However, the molecular mechanism underlying hypoxia-induced Stat3 activation has not been defined. In this study, we explored the possible implication of reactive oxygen species (ROS) in hypoxia-driven Stat3 activation in human glioblastoma. We found that hypoxic stress increased ROS production as well as Stat3 activation and that ROS inhibitors (diphenyleneiodonium, rotenone and myxothiazol) and an antioxidant (N-acetyl-L-cysteine) blocked Stat3 activation under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is involved in hypoxia-induced ROS production. Nox4 expression was found to be increased at both mRNA and protein levels in hypoxic glioblastoma cells. In addition, siRNA-mediated knockdown of Nox4 expression abolished hypoxia induced Stat3 activation and vascular endothelial growth factor expression, which is associated with tumor cells' ability to trigger tube formation of endothelial cells in vitro. Our findings indicate that elevated ROS production plays a crucial role for Stat3 activation and angiogenesis in hypoxic glioblastoma cells.

Romo1 is associated with ROS production and cellular growth in human gliomas.

Romo1 is a mitochondrial protein whose elevated expression is commonly observed in various types of human cancers. However, the expression status of Romo1 and its implication in the pathogenesis of human glioblastoma (GBM) remain largely undefined. To understand the role of Romo1 in the progression of GBM, we explored its expression in a series of GBM tissues and cell lines and determined its effect on ROS production, cell proliferation, and tumor growth. Romo1 was frequently overexpressed at the mRNA level in both primary tumors and cell lines and its elevation was more commonly observed in high grade tumors versus low grade tumors. Romo1 expression was associated with ROS production and its knockdown led to a marked reduction of in vitro cellular growth and anchorage-independent growth of GBM. Consistently, Romo1 depletion induced a G2/M arrest of the cell cycle that was accompanied with accumulation of phospho-cdc2. Furthermore, a mouse xenograft assay revealed that Romo1 depletion significantly decreased tumor formation and growth. Therefore, our data demonstrate that Romo1 upregulation is a common event in human GBMs and contributes to the malignant tumor progression, suggesting that Romo1 could be a new therapeutic target for human GBM.

Expression of astrocyte elevated gene-1 (AEG-1) in human meningiomas and its roles in cell proliferation and survival.

Astrocyte elevated gene-1 (AEG-1) has recently been proposed to be involved in tumor development, invasion, and metastasis in several human cancers. However, the functional importance of AEG-1 expression in human meningioma has not been determined. We investigate the level of AEG-1 expression by quantitative reverse transcription PCR, immunohistochemistry analysis, and western blotting in various human meningioma tissues and cells. To determine the suppressive effect of AEG-1 on meningioma progression, we inhibited AEG-1 expression using small interfering RNA and examined cell proliferation, apoptosis, colony formation and tumorigenicity in a mouse xenograft model. AEG-1 expression was frequently elevated at both mRNA and protein levels in meningioma tumor tissues and in meningioma-derived cells as well. This elevation was more commonly observed in high-grade tumors than in benign ones. The knockdown of AEG-1 led to a decrease in overall cell proliferation, as well as anchorage-independent growth of malignant meningioma. In addition, apoptotic cell death occurred in AEG-1 depleted meningioma cells through p-Akt and Bcl-2 suppression. Furthermore, a mouse xenograft meningioma model showed that inhibition of AEG-1 expression significantly decreased tumor growth. Altogether, these data show that the elevation of AEG-1 contributes to the malignant progression of meningiomas, suggesting that AEG-1 could be a novel therapeutic target against human meningiomas.

O6-methylguanine DNA methyltransferase status determined by promoter methylation and immunohistochemistry in gliosarcoma and their clinical implications.

O(6)-methylguanine-DNA methyltransferase (MGMT) is known as a DNA repair protein, and loss of function in MGMT is related to an increase in survival in patients with malignant gliomas treated with alkylating agents. In the present study, we determined the status of MGMT using methylation-specific polymerase chain reaction (PCR) and immunohistochemistry on paraffin-embedded specimens in 12 human gliosarcomas, and these results were then related to overall survival (OS) and response to alkylating agents. The MGMT promoter was methylated in six patients. Immunostaining of MGMT was positive in 58.3% of patients. MGMT methylation status was correlated with immunostaining results in five patients (41.7%). The median OS and progression-free survival (PFS) of the whole population were 13.4 months [95% confidence interval (CI), 12.3-14.5 months] and 8.3 months (95% CI, 7.4-9.2 months), respectively. In patients with methylated MGMT promoter, median OS was 15.0 months, compared with 11.3 months in the unmethylated group. Median PFS of gliosarcoma patients was 10.3 months for the methylated group, whereas it was 7.3 months for the unmethylated group. On multivariate analysis, patients with methylated MGMT promoter had better prognosis than patients with unmethylated MGMT promoter with respect to OS and PFS (P = 0.045 and 0.034, respectively). However, there was no statistical significance between MGMT protein expression and survival. The results show that a significant fraction of gliosarcomas have MGMT promoter methylation and protein expression, and suggest that patient survival is associated with MGMT methylation status.

Activated STAT3 regulates hypoxia-induced angiogenesis and cell migration in human glioblastoma.

BACKGROUND: Glioblastoma is the most common primary brain tumor, with typical histopathologic findings, pseudopalisading necrosis, and microvascular proliferation, all of which are associated with a poor prognosis. Hypoxia is known to affect these morphological features, but the underlying molecular mechanism has been poorly understood. OBJECTIVE: To determine the role of signal transducer and activator of transcription 3 (STAT3) in the malignant progression of glioblastoma under hypoxic conditions. METHODS: We studied STAT3 activation by hypoxic stress and its effect on hypoxia-induced angiogenesis and cell migration using U87, A172, T98, and U373 human glioblastoma cell lines. RESULTS: All four glioblastoma cells analyzed expressed detectable levels of STAT3 phosphorylation. Hypoxic stress markedly increased phosphorylated STAT3 level in a time-dependent fashion, and activated STAT3 was translocated into the nucleus. Hypoxic conditions led to a 30-50% increase in angiogenesis and cell migration, but these effects were significantly attenuated by small interfering ribonucleic acid-mediated knockdown of STAT3. Furthermore, STAT3 activation was associated with an elevated expression of hypoxic inducible factor-1, vascular endothelial growth factor, matrix metalloproteinase 2, and TWIST messenger ribonucleic acid and protein, which may play a critical role in hypoxia-induced angiogenesis and migration. CONCLUSION: STAT3 plays an important role in glioblastoma angiogenesis and migration triggered by hypoxia. Therefore, STAT3 might be a target for control of pseudopalisading necrosis and angiogenesis in glioblastoma.

Influence of interleukin-6 on the development of peritumoral brain edema in meningiomas.

OBJECT: Peritumoral brain edema (PTBE) is associated with perioperative neurological deficits in patients with meningiomas. However, the pathogenesis of meningioma-associated edema remains unclear. In the present study, the authors investigated the expression of interleukin-6 (IL-6) and its relationship with PTBE in resected meningiomas. METHODS: Thirty-six benign meningiomas obtained in 36 patients were studied retrospectively. Edema volume was assessed on MR images, and an edema index (EI) was calculated. Interleukin-6 mRNA and protein expression were examined by real-time reverse transcriptase polymerase chain reaction and immunohistochemical staining. RESULTS: Peritumoral brain edema was found in 16 patients (44%). Neither age, sex, histological subtype, nor tumor location were related to PTBE. The level of IL-6 mRNA was 7.72 times greater in the edema group (EI > 0.2) than in the nonedema group (EI < 0.2; p = 0.011). On immunohistochemical analysis, IL-6 protein was found localized in the cytoplasm of the tumor cells, and was detected in 12 (75%) of 16 cases of edematous meningiomas, but in only 6 (30%) of 20 nonedematous cases. There was a significant correlation between the severity of PTBE and IL-6 expression (p = 0.004). CONCLUSIONS: The authors' results in this study indicate that IL-6 expression may contribute to the development of brain edema associated with meningiomas.