Change in microRNAs Expression Profile of Glioblastoma Multiforme (GBM) Cell Lines Following Treatment By Ascorbic Acid

Whereas several clinical trials are ongoing or have been completed examining the benefits of Ascorbic acid-based therapy of cancer patients, its efficacy at GBM treatment has not been sufficiently investigated. In the present study, the influence of L-Ascorbic acid (Vitamin C, VC) on two GBM cell lines (U87 and U251) was evaluated in terms of cytotoxicity, induction of cell cycle arrest, reactive oxygen species (ROS) production, and alteration in the level of GBM related microRNAs. The half maximal inhibitory concentration (IC50) of VC was obtained by Crystal Violet assay as 2 mM and 1.8 mM for U87 cell line following 24 and 48 h treatment, respectively. These values were obtained in a similar way for U251 cell line as 3.2 mM and 2.9 mM. Propidium iodide (PI) staining of the cells revealed that ascorbic acid caused cell cycle arrest in G2/M phase in both of studied GBM cell lines. Moreover, RT-qPCR results indicated that VC-treatment of GBM cell lines causes downregulation of Bcl-2 alongside increase in BAK-1 and BAX expressions. Flowcytometry-based DCFH assay confirmed drastic increase in reactive oxygen species (ROS) within U87 and U251 cells following VC-treatment. Eventually, study on microRNAs expression profile implied significant increase in four tumor suppressor miRNAs including miR-7, miR-34a, miR-128, and miR-182 in both of U87 and U251 cell lines after treatment with ascorbic acid. Besides, the expression levels of three onco-miRs (i.e., miR-10b in both of cell lines, miR-222 in U87 and miR-93 in U251) were significantly diminished.

Pathological Features of Echovirus-11-Associated Brain Damage in Mice Based on RNA-Seq Analysis

Echovirus 11 (E11) is a neurotropic virus that occasionally causes fatal neurological diseases in infected children. However, the molecular mechanism underlying the disease and pathological spectrum of E11 infection remains unclear. Therefore, we modelled E11 infection in 2-day-old type I interferon receptor knockout (IFNAR−/−) mice, which are susceptible to enteroviruses, with E11, and identified symptoms consistent with the clinical signs observed in human cases. All organs of infected suckling mice were found to show viral replication and pathological changes; the muscle tissue showed the highest viral replication, whereas the brain and muscle tissues showed the most obvious pathological changes. Brain tissues showed oedema and a large number of dead nerve cells; RNA-Seq analysis of the brain and hindlimb muscle tissues revealed differentially expressed genes to be abundantly enriched in immune response-related pathways, with changes in the Guanylate-binding protein (GBP) and MHC class genes, causing aseptic meningitis-related symptoms. Furthermore, human glioma U251 cell was identified as sensitive target cells for E11 infection. Overall, these results provide new insights into the pathogenesis and progress of aseptic meningitis caused by E11.

Bufalin induces mitochondrial dysfunction and promotes apoptosis of glioma cells by regulating Annexin A2 and DRP1 protein expression

Background Glioma is a common primary central nervous system tumour, and therapeutic drugs that can effectively improve the survival rate of patients in the clinic are lacking. Bufalin is effective in treating various tumours, but the mechanism by which it promotes the apoptosis of glioma cells is unclear. The aim of this study was to investigate the drug targets of bufalin in glioma cells and to clarify the apoptotic mechanism. Methods Cell viability and proliferation were evaluated by CCK-8 and colony formation assays. Then, the cell cycle and apoptosis, intracellular ion homeostasis, oxidative stress levels and mitochondrial damage were assessed after bufalin treatment. DARTS-PAGE technology was employed and LC–MS/MS was performed to explore the drug targets of bufalin in U251 cells. Molecular docking and western blotting were performed to identify potential targets. siRNA targeting Annexin A2 and the DRP1 protein inhibitor Mdivi-1 were used to confirm the targets of bufalin. Results Bufalin upregulated the expression of cytochrome C, cleaved caspase 3, p-Chk1 and p-p53 proteins to induce U251 cell apoptosis and cycle arrest in the S phase. Bufalin also induced oxidative stress in U251 cells, destroyed intracellular ion homeostasis, and caused mitochondrial damage. The expression of mitochondrial division-/fusion-related proteins in U251 cells was abnormal, the Annexin A2 and DRP1 proteins were translocated from the cytoplasm to mitochondria, and the MFN2 protein was released from mitochondria into the cytoplasm after bufalin treatment, disrupting the mitochondrial division/fusion balance in U251 cells. Conclusions Our research indicated that bufalin can cause Annexin A2 and DRP1 oligomerization on the surface of mitochondria and disrupt the mitochondrial division/fusion balance to induce U251 cell apoptosis. Graphic Abstract

Expressions and Significances of CTSL, The Target of COVID‐19 on GBM

CTSL is one of the SARS-entry-associated CoV-2's proteases and plays a key role in the virus's entry into the cell and subsequent infection. We investigated the association between the expression level of CTSL and overall survival in TCGA and CGGA databases, in order to better understand the possible route and risks of new coronavirus infection for patients with GBM. Meanwhile, the relationship between CTSL and immune infiltration levels was analyzed by means of the TIMER database. The impact of CTSL inhibitors on GBM biological activity was tested. The findings revealed that GBM tissues had higher CTSL expression levels than that of normal brain tissues, which was associated with a significantly lower survival rate in GBM patients. Meanwhile, CTSL was very negatively correlated with purity, B cell and CD8+ T cell in GBM. CTSL inhibitor significantly reduced U251 cell growth and invasion in vitro and induced mitochondrial apoptosis. According to the findings of this study, CTSL acts as an independent prognostic factor and can be considered as promising therapeutic target for GBM.

Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines

Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation ( P = 0.003 ) and lower Ki-67 protein expression ( P = 0.011 ). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.

Cannabinoid WIN 55,212-2 Inhibits Human Glioma Cell Growth by Triggering ROS-Mediated Signal Pathways

Glioblastoma is a highly invasive primary malignant tumor of the central nervous system. Cannabinoid analogue WIN 55,212-2 (WIN) exhibited a novel anticancer effect against human tumors. However, the anticancer potential and underlying mechanism of WIN against human glioma remain unclear. Herein, the anticancer efficiency and mechanism of WIN in U251 human glioma cells were investigated. The results showed that WIN dose-dependently inhibited U251 cell proliferation, migration, and invasion in vitro. WIN treatment also effectively suppressed U251 tumor spheroids growth ex vivo. Further studies found that WIN induced significant apoptosis as convinced by the caspase-3 activation and release of cytochrome C. Mechanism investigation revealed that WIN triggered ROS-mediated DNA damage and caused dysfunction of VEGF-AKT/FAK signal axis. However, ROS inhibition effectively attenuated WIN-induced DNA damage and dysfunction of VEGF-AKT/FAK signal axis and eventually improved U251 cell proliferation, migration, and invasion. Taken together, our findings validated that WIN had the potential to inhibit U251 cell proliferation, migration, and invasion and induce apoptosis by triggering ROS-dependent DNA damage and dysfunction of VEGF-AKT/FAK signal axis.

4-Acetylantrocamol LT3 Inhibits Glioblastoma Cell Growth and Downregulates DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase

Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.

AdipoR2 inhibits human glioblastoma cell growth through the AMPK/mTOR pathway

Background: AdipoR2, which belongs to the seven-transmembrane-domain receptor family, has been shown to play an important role in the development of human tumours, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the antiproliferative effects of AdipoR2 in U251 cells (a human glioma cell line) using colony formation and WST-8 growth assays.Methods: The U251 cell line was cultured in vitro. Western blotting was used to detect the expression of relevant proteins. Quantitative RT-PCR was used to detect AdipoR1 and AdipoR2 expression. Flow cytometry was used to detect cell cycle assay results. The gene expression profiles of glioma samples from the CGGA database were analysed by MATLAB and GSEA software.Results: We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA suggested that AdipoR2 is a cell cycle-associated gene. The results of the flow cytometry analysis indicated that AdipoR2 induced G0/G1 cell cycle arrest in U251 cells. Furthermore, we identified the AMPK/Mtor signalling axis to be involved in AdipoR2-induced cell cycle arrest.Conclusions: Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggest that AdipoR2 may be a promising therapeutic target in GBM patients.