Normal Astrocytes Research Articles

Chemosensitization of U-87 MG Glioblastoma Cells by Neobavaisoflavone towards Doxorubicin and Etoposide.

Glioblastoma (GB) is the most common type of glioma, which is distinguished by high mortality. Due to the rapid progression of the tumor and drug resistance, the treatment is often ineffective. The development of novel therapies in a big part concerns the application of anti-cancer agents already used in clinical practice, unfortunately often with limited effects. This could be overcome through the use of compounds that possess chemosensitizing properties. In our previous work, it has been shown that neobavaisoflavone (NBIF) enhances the in vitro activity of doxorubicin in GB cells. The aim of this study was a further investigation of the possible chemosensitizing effects of this isoflavone. The experimental panel involving image cytometry techniques, such as count assay, examination of mitochondrial membrane potential, Annexin V assay, and cell cycle analysis, was performed in human glioblastoma U-87 MG cells and normal human astrocytes (NHA) treated with NBIF, doxorubicin, etoposide, and their mixes with NBIF. NBIF in co-treatment with etoposide or doxorubicin caused an increase in the population of apoptotic cells and prompted alterations in the cell cycle. NBIF enhances the pro-apoptotic activity of etoposide and doxorubicin in U-87 MG cells, which could be a sign of the chemosensitizing properties of the isoflavone.

Apatinib Induces Ferroptosis of Glioma Cells through Modulation of the VEGFR2/Nrf2 Pathway.

Background Glioma is a common tumor that originated from the brain, and molecular targeted therapy is one of the important treatment modalities of glioma. Apatinib is a small-molecule tyrosine kinase inhibitor, which is widely used for the treatment of glioma. However, the underlying molecular mechanism has remained elusive. Recently, emerging evidence has proved the remarkable anticancer effects of ferroptosis. In this study, a new ferroptosis-related mechanism of apatinib inhibiting proliferation of glioma cells was investigated, which facilitated further study on inhibitory effects of apatinib on cancer cells. Methods Human glioma U251 and U87 cell lines and normal astrocytes were treated with apatinib. Ferroptosis, cell cycle, apoptosis, and proliferation were determined. A nude mouse xenograft model was constructed, and tumor growth rate was detected. Tumor tissues were collected to estimate ferroptosis levels and to identify the relevant pathways after treatment with apatinib. Results Treatment with apatinib could induce loss of cell viability of glioma cells, but not of normal astrocytes, through eliciting ferroptosis in vitro and in vivo. It was also revealed that apatinib triggered ferroptosis of glioma cells via inhibiting the activation of nuclear factor erythroid 2-related factor 2/vascular endothelial growth factor receptor 2 (Nrf2/VEFGR2) pathway. The overexpression of Nrf2 rescued the therapeutic effects of apatinib. Conclusion Our study proved that treatment with apatinib could restrain proliferation of glioma cells through induction of ferroptosis via inhibiting the activation of VEGFR2/Nrf2/Keap1 pathway. Overexpression of Nrf2 could counteract the induction of ferroptosis by apatinib.

Medullary astrocytes mediate irregular breathing patterns generation in chronic heart failure through purinergic P2X7 receptor signalling.

SummaryBackgroundBreathing disorders (BD) (apnoeas/hypopneas, periodic breathing) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte‐mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored.MethodsSelective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations.FindingsWe showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P"X/ P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7 P2X7r into astrocytes increases ATP bioavailability and abolished BD.InterpretationOur results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling.FundingThis study was funded by the National Research and Development Agency of Chile (ANID).

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation.

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.

N6-isopentenyladenosine induces cell death through necroptosis in human glioblastoma cells

Targeting necroptosis is considered a promising therapeutic strategy in cancer, including Glioblastoma Multiforme (GBM), one of the most lethal brain tumors. Necroptosis is a mechanism of programmed cell death overcoming the apoptosis resistance mechanism underlying GBM tumorigenesis and malignant progression. N6-isopentenyladenosine (iPA), adenosine modified with isoprenoid derivative, displays antitumor activity in different cancer models. In previous studies, we demonstrated that iPA interferes with EGFR signaling reducing glioma cell viability. Here, we show that iPA induces necroptosis in glioblastoma cell lines and in primary cells established from tumor explants, without affecting the viability of non-cancerous brain cell lines, (Normal Human Astrocyte). The activation of RIP1, RIP3, and MLKL and the upregulation of necrosome formation were increased upon iPA treatment while caspase-3, caspase-8, and PARP were not activated in GBM cells. Co-treatment with specific necroptosis inhibitor necrostatin-1 (Nec-1) or Necrosulfonamide (NSA) prevented cell death caused by iPA treatment while the general caspase inhibitor Z-VAD-fluoromethylketone (z-VAD-fmk) did not elicit any effect, suggesting that this molecule induces caspase-independent necroptosis. These results suggest that iPA treatment can be able to bypass the apoptosis resistance mechanism in glioblastoma thereby offering higher therapeutic efficacy.

Low-Concentrations of Fatty Acids Induce an Early Increase in IL-8 Levels in Normal Human Astrocytes

Fatty acids (FAs) have been shown to exhibit a pro-inflammatory response in various cell types, but astrocytes have been mostly overlooked. FAs, both saturated and unsaturated, have previously been shown to induce pro-inflammatory responses in astrocytes at high concentrations of hundreds of µg/mL. SSO (Sulfo-N-succinimidyl Oleate sodium), an inhibitor of FA translocase CD36, has been shown to prevent inflammation in the mouse brain by acting on local microglia and infiltrating monocytes. Our hypothesis was that SSO treatment would also impact astrocyte pro-inflammatory response to FA. In order to verify our assumption, we evaluated the expression of pro- and anti-inflammatory cytokines in normal human astrocyte cell culture pre-treated (or not) with SSO, and then exposed to low concentrations of both saturated (palmitic acid) and unsaturated (oleic acid) FAs. As a positive control for astrocyte inflammation, we used fibrillary amyloid. Neither Aβ 1–42 nor FAs induced CD36 protein expression in human astrocytes in cell culture At low concentrations, both types of FAs induced IL-8 protein secretion, and this effect was specifically inhibited by SSO pre-treatment. In conclusion, low concentrations of oleic acid are able to induce an early increase in IL-8 expression in normal human astrocytes, which is specifically downregulated by SSO.

The Pivotal Immunoregulatory Functions of Microglia and Macrophages in Glioma Pathogenesis and Therapy.

Gliomas are mixed solid tumors composed of both neoplastic and nonneoplastic cells. In glioma microenvironment, the most common nonneoplastic and infiltrating cells are macrophages and microglia. Microglia are the exact phagocytes of the central nervous system, whereas macrophages are myeloid immune cells that are depicted with ardent phagocytosis. Microglia are heterogeneously located in almost all nonoverlapping sections of the brain as well as the spinal cord, while macrophages are derived from circulating monocytes. Microglia and macrophages utilize a variety of receptors for the detection of molecules, particles, and cells that they engulf. Both microglia and peripheral macrophages interact directly with vessels both in the periphery of and within the tumor. In glioma milieu, normal human astrocytes, glioma cells, and microglia all exhibited the ability of phagocytosing glioma cells and precisely apoptotic tumor cells. Also, microglia and macrophages are robustly triggered by the glioma via the expression of chemoattractants such as monocyte chemoattractant protein, stromal-derived factor-1, and macrophage-colony stimulating factor. Glioma-associated microglia and/or macrophages positively correlated with glioma invasiveness, immunosuppression, and patients' poor outcome, making these cells a suitable target for immunotherapeutic schemes.

GRAPHENE OXIDE AFFECT THE EXPRESSION OF PROLIFERATION RELATED GENES AND microRNA IN NORMAL HUMAN ASTROCYTES

Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes. Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used. Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3′-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions. Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.

MicroRNA 3928 Suppresses Glioblastoma through Downregulation of Several Oncogenes and Upregulation of p53.

Glioblastoma (GBM) is the most frequent and lethal primary malignant brain tumor. Despite decades of research, therapeutic advances that significantly prolong life are non-existent. In recent years, microRNAs (miRNAs) have been a focus of study in the pathobiology of cancer because of their ability to simultaneously regulate multiple genes. The aim of this study was to determine the functional and mechanistic effects of miR-3928 in GBM both in vitro and in vivo. To the best of our knowledge, this is the first article investigating the role of miR-3928 in GBM. We measured endogenous miR-3928 expression levels in a panel of patient-derived GBM tissue samples and cell lines. We found that GBM tissue samples and cell lines express lower levels of miR-3928 than normal brain cortex and astrocytes, respectively. Therefore, we hypothesized that miR-3928 is a tumor suppressive microRNA. We verified this hypothesis by showing that exogenous expression of miR-3928 has a strong inhibitory effect on both cell growth and invasiveness of GBM cells. Stable ex vivo overexpression of miR-3928 in GBM cells led to a reduction in tumor size in nude mice xenografts. We identified many targets (MDM2, CD44, DDX3X, HMGA2, CCND1, BRAF, ATOH8, and BMI1) of miR-3928. Interestingly, inhibition of the oncogene MDM2 also led to an upregulation of wild-type p53 expression and phosphorylation. In conclusion, we find that miR-3928, through the downregulation of several oncogenes and upregulation and activation of wild-type p53, is a strong tumor suppressor in GBM. Furthermore, the fact that miR-3928 can target many important dysregulated proteins in GBM suggests it might be a “master” regulatory microRNA that could be therapeutically exploited.

A Comprehensive Analysis of the Glutathione Peroxidase 8 (GPX8) in Human Cancer.

ObjectiveNowadays, cancer is still a leading public health problem all over the world. Several studies have reported the GPX8 could be correlated with the poor prognostic of Gastric Cancer and Breast Cancer. However, the prognostic potential of GPX8 in pan-cancer remains unclear. In this work, we aimed to explore the prognostic and immunological role of GPX8 in human cancer and confirm the oncogenic value in GBM.MethodsThe data of TCGA, CPTAC and GEO databases were adopted for the survival analysis. Based on the RNAseq and Methylation450 data of TCGA, the R language and package “ggplot2” were used to analyze the DNA methylation at the region of the promoter of GPX8 in tumors. The genetic alteration of GPX8 from TCGA cancers was investigated in cBioPortal. The R package “GSVA” and “ssGSEA” were employed to evaluate the correlation of GPX8 expression with the immune infiltration. The KEGG website was used for pathway analysis. The STRING website and GEPIA were performed to predict GPX8-binding proteins. The R package “ggplot2” and “clusterprofile” were used to analyze and visualize the GO and KEGG analysis. A normal human astrocyte cell line and three GBM cell lines were cultured under suitable conditions. The shRNA was transferred to cells by Lipofectamine 3000. The qRT-PCR and WB were adopted to detect the expression of GPX8. The wound-healing assay and transwell assay were taken to analyze the invasive and metastatic abilities. The tumor tissues and paracancerous ones were collected from patients with GBM. WB assay was employed to analyze the expression of GPX8 protein.ResultsGPX8 was a valuable diagnostic biomarker in multiple cancers, including GBM/LGG (glioblastoma multiforme/Brain lower grade glioma), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma) and STAD (stomach adenocarcinoma). Moreover, we observed a correlation between the expression of GPX8 and the reduced DNA methylation at the promoter region in several tumors, such as GBM/LGG. Our results indicated a positive correlation between the GPX8 expression and immune infiltration. In addition, the enrichment analysis demonstrated that antioxidant activity was mainly involved in the functional mechanism of GPX8. In particular, we first confirmed the up-regulated of GPX8 in GBM cells and observed the suppression of migrative and invasive phenotypes by knockdown of GPX8. Furthermore, we confirmed the expression of GPX8 was higher in GBM tumor tissues than paracancerous ones.ConclusionOur study showed a correlation of GPX8 expression with clinical prognosis, DNA methylation and immune infiltrates. Furthermore, we first confirmed GPX8 was highly expressed in GBM cells and contributed to migration and invasion. These results provided a predictive biomarker and an inclusive understanding of the GPX8 expression in multiple tumors types, especially in GBM.

Cold Plasma Discharge Tube Enhances Antitumoral Efficacy of Temozolomide.

Glioblastoma (GBM) is a fatal human brain tumor with a low survival rate. Temozolomide (TMZ) has been widely used in GBM therapy with noticeable side effects. Cold plasma is an ionized gas that is generated near room temperature. Here, we demonstrated the enhancement therapeutic efficacy of TMZ via using a cold plasma source based on nonequilibrium plasma in a sealed glass tube, named a radial cold plasma discharge tube (PDT). The PDT affected glioblastoma cells' function just by its electromagnetic (EM) emission rather than any chemical factors in the plasma. The PDT selectively increased the cytotoxicity of TMZ on two typical glioblastoma cell lines, U87MG and A172, compared with normal astrocyte cell line hTERT/E6/E7 to some extent. Furthermore, on the basis of a patient-derived xenograft model, our preliminary in vivo studies demonstrated the drastically improved mean survival days of the tumor-barrier mice by more than 100% compared to control. The PDT is not only independent of continuous helium supply but is also capable of resisting the interference of environmental changes. Thus, the PDT was a stable and low-cost cold atmospheric plasma source. In short, this study is the first to demonstrate the promising application of PDTs in GBM therapy as a noninvasive and portable modality.

Astrocytes Protect Human Brain Microvascular Endothelial Cells from Hypoxia Injury by Regulating VEGF Expression.

Hypoxic-ischemic stroke has been associated with changes in neurovascular behavior, mediated, in part, by induction of the vascular endothelial growth factor (VEGF). The objective of this study was to investigate the effects of human astrocytes on the proliferation, apoptosis, and function of human microvascular endothelial cells (hBMEC) in vitro. Human microvascular endothelial cells (hBMEC) and human normal astrocytes (HA-1800) were used to establish in vitro cocultured cell models. The coculture model was used to simulate hypoxic-ischemic stroke, and it was found that astrocytes could promote hBMEC proliferation, inhibit apoptosis, reduce cell damage, and enhance antioxidant capacity by activating the VEGF signaling pathway. When VEGF is knocked out in astrocytes, the protective effect of astrocytes on hBMEC was partially lost. In conclusion, our study confirms the protective effect of hBMEC and laid a foundation for the study of hypoxic-ischemic stroke.

Tumor Treating Fields Suppression of Ciliogenesis Enhances Temozolomide Toxicity.

Tumor Treating Fields (TTFields) are low-intensity, alternating intermediate-frequency (200 kHz) electrical fields that extend survival of glioblastoma patients receiving maintenance temozolomide (TMZ) chemotherapy. How TTFields exert efficacy on cancer over normal cells or interact with TMZ is unclear. Primary cilia are microtubule-based organelles triggered by extracellular ligands, mechanical and electrical field stimulation and are capable of promoting cancer growth and TMZ chemoresistance. We found in both low- and high-grade patient glioma cell lines that TTFields ablated cilia within 24 h. Halting TTFields treatment led to recovered frequencies of elongated cilia. Cilia on normal primary astrocytes, neurons, and multiciliated/ependymal cells were less affected by TTFields. The TTFields-mediated loss of glioma cilia was partially rescued by chloroquine pretreatment, suggesting the effect is in part due to autophagy activation. We also observed death of ciliated cells during TTFields by live imaging. Notably, TMZ and TTFields have opposing effects on glioma ciliogenesis. TMZ-induced stimulation of ciliogenesis in both adherent cells and gliomaspheres was blocked by TTFields. Surprisingly, the inhibitory effects of TTFields and TMZ on tumor cell recurrence are linked to the relative timing of TMZ exposure to TTFields and ARL13B+ cilia. Finally, TTFields disrupted cilia in patient tumors treated ex vivo. Our findings suggest that the efficacy of TTFields may depend on the degree of tumor ciliogenesis and relative timing of TMZ treatment.

341 SOncomagnetics: A Potential New Noninvasive Magnetic Stimulation Technology to Treat Glioblastoma

INTRODUCTION: A new noninvasive oscillating magnetic field (OMF)-generating device called Oncomagnetic (OM) device, developed in our laboratory, disrupts mitochondrial electron transport to elevate intracellular reactive oxygen species (ROS) levels in patient derived glioblastoma (GBM) cells. METHODS: GBM cell death, ROS elevation and caspase 3 activation were studied by bright-field and fluorescence microscopy involving specific fluorescent probes. Device treatment of FOXN1-Nude mice with GBM xenografts was for 4 hours twice a day. Treatment of a 53-year-old male patient with recurrent GBM was done under a compassionate use protocol for 2–6 hours each weekday. Contrast-enhanced MRI scans assessed tumor size. RESULTS: OMF caused selective death of GBM cells, but not normal human cortical neurons and astrocytes. It altered mitochondrial oxygen consumption, increased mitochondrial and cellular ROS, and activated caspase 3. The overall survival of xenografted mice was prolonged by 6 days with treatment. A 26-day device treatment was well tolerated by the patient and decreased contrast-enhanced tumor volume by >30%. Patient’s autopsy after his death from closed head injury showed treatment effect, no tumor mass, and some evidence of active tumor. CONCLUSION: Noninvasive stimulation with the OM device is a potential powerful new treatment for GBM involving a novel mechanism of action.

CRB2 enhances malignancy of glioblastoma via activation of the NF-κB pathway

Glioblastoma (GBM) is one of the most lethal types of primary brain tumors in adults with a median survival of less than 15 months. Although comprehensive clinical treatment strategies including surgical resection followed by radiotherapy and chemotherapy are widely applied, the prognosis for GBM patients remains dismal. The Nuclear Factor-κB (NF-κB) signaling pathway is a complex network linking extracellular stimuli to cell survival and proliferation, and aberrant activation of NF-κB signaling has been implicated in the propagation of a wide range of cancers. However, the underlying mechanism of NF-κB activation still requires further investigation. Here, we report that crumbs homolog 2 (CRB2) is markedly up-regulated in human GBM relative to non-tumor tissues or normal astrocytes. Clinically, enriched CRB2 could be observed in high grade glioma with IDH IDH wild-type and 1p19q co-deletion and implied poor outcome in GBM. Consistent with this, malignant characteristics of GBM cells including proliferation, migration, invasion and tumorigenesis were significantly suppressed by lentivirus knock-down of CRB2. Furthermore, exogenous overexpression of CRB2 enhanced the malignant biological signatures of GBM cells as well as therapy resistance to temozolomide (TMZ). To further investigate the molecular mechanisms responsible, bioinformatics analysis was performed using 3 public databases, with the result that CRB2 was found to correlate closely with tumor necrosis factor α (TNFα)–NF–κB signaling. Mechanistically, elevated CRB2 increased the phosphorylation of IκB-kinase α (IKKα), thus activating NF-κB via reduction of Ikβ protein. Taken together, these data suggest that CRB2 might be a reliable prognostic biomarker and potential therapeutic target for GBM.

Integrated Metabolomics and Lipidomics Reveal High Accumulation of Glycerophospholipids in Human Astrocytes under the Lipotoxic Effect of Palmitic Acid and Tibolone Protection.

Lipotoxicity is a metabolic condition resulting from the accumulation of free fatty acids in non-adipose tissues which involves a series of pathological responses triggered after chronic exposure to high levels of fatty acids, severely detrimental to cellular homeostasis and viability. In brain, lipotoxicity affects both neurons and other cell types, notably astrocytes, leading to neurodegenerative processes, such as Alzheimer (AD) and Parkinson diseases (PD). In this study, we performed for the first time, a whole lipidomic characterization of Normal Human Astrocytes cultures exposed to toxic concentrations of palmitic acid and the protective compound tibolone, to establish and identify the set of potential metabolites that are modulated under these experimental treatments. The study covered 3843 features involved in the exo- and endo-metabolome extracts obtained from astrocytes with the mentioned treatments. Through multivariate statistical analysis such as PCA (principal component analysis), partial least squares (PLS-DA), clustering analysis, and machine learning enrichment analysis, it was possible to determine the specific metabolites that were affected by palmitic acid insult, such as phosphoethanolamines, phosphoserines phosphocholines and glycerophosphocholines, with their respective metabolic pathways impact. Moreover, our results suggest the importance of tibolone in the generation of neuroprotective metabolites by astrocytes and may be relevant to the development of neurodegenerative processes.

TMEM60 Promotes the Proliferation and Migration and Inhibits the Apoptosis of Glioma through Modulating AKT Signaling.

Glioma is a highly fatal malignancy with aggressive proliferation, migration, and invasion metastasis due to aberrant genetic regulation. This work aimed to determine the function of transmembrane protein 60 (TMEM60) during glioma development. The level of TMEM60 in glioma tissues and normal tissues and its correlation with glioma prognosis were checked in The Cancer Genome Atlas (TCGA) database. The levels of TMEM60 in glioma cell lines and normal astrocytes were determined by quantitative real-time PCR and western blotting assay. TMEM60 knockdown and overexpression were conducted, followed by detection of cell viability, migration, invasion, and apoptosis. CCK-8 and colony formation assay were adopted to detect cell viability proliferation. Transwell assay was performed to measure cell migration and invasion. Cell apoptosis was evaluated by flow cytometry. The alternation of key proteins in the PI3K/Akt signaling pathway was measured by western blotting. TMEM60 expression was significantly higher in glioma tissues than that in the healthy control and was correlated with poor overall survival of patients. The protein and mRNA levels of TMEM60 were both elevated in glioma cell lines in comparison with the normal cell lines. Elevated level of TMEM60 led to enhanced proliferation, migration, and invasion and suppressed cell apoptosis. TMEM60 promoted the activation of PI3K/Akt signaling. Our data suggested that TMEM60 plays an oncogenic role in glioma progression via activating the PI3K/Akt signaling pathway.

Matteucinol combined with temozolomide inhibits glioblastoma proliferation, invasion, and progression: an in vitro, in silico, and in vivo study.

Glioblastoma is the most prevalent and malignant brain tumor identified in adults. Surgical resection followed by radiotherapy and chemotherapy, mainly with temozolomide (TMZ), is the chosen treatment for this type of tumor. However, the average survival of patients is around 15 months. Novel approaches to glioblastoma treatment are greatly needed. Here, we aimed to investigate the anti-glioblastoma effect of the combination of matteucinol (Mat) (dihydroxyflavanone derived from Miconia chamissois Naudin) with the chemotherapeutic TMZ in vitro using tumor (U-251MG) and normal astrocyte (NHA) cell lines and in vivo using the chick embryo chorioallantoic membrane (CAM) assay. The combination was cytotoxic and selective for tumor cells (28 μg/mL Mat and 9.71 μg/mL TMZ). Additionally, the combination did not alter cell adhesion but caused morphological changes characteristic of apoptosis in vitro. Notably, the combination was also able to reduce tumor growth in the chick embryo model (CAM assay). The docking results showed that Mat was the best ligand to the cell death membrane receptor TNFR1 and to TNFR1/TMZ complex, suggesting that these two molecules may be working together increasing their potential. In conclusion, Mat-TMZ can be a good candidate for pharmacokinetic studies in view of clinical use for the treatment of glioblastoma.

Alterations in 3D chromatin organization contribute to tumorigenesis of EGFR-amplified glioblastoma

BackgroundEGFR amplification and/or mutation are found in more than half of the cases with glioblastoma. Yet, the role of chromatin interactions and its regulation of gene expression in EGFR-amplified glioblastoma remains unclear. MethodsIn this study, we explored alterations in 3D chromatin organization of EGFR-amplified glioblastoma and its subsequent impact by performing a comparative analysis of Hi-C, RNA-seq, and whole-genome sequencing (WGS) on EGFR-amplified glioblastoma-derived A172 and normal astrocytes (HA1800 cell line). ResultsA172 cells showed an elevated chromatin relaxation, and unexpected entanglement of chromosome regions. A genome-wide landscape of switched compartments and differentially expressed genes between HA1800 and A172 cell lines demonstrated that compartment activation reshaped chromatin accessibility and activated tumorigenesis-related genes. Topological associating domain (TAD) analysis revealed that altered TAD domains in A172 also contribute to oncogene activation and tumor repressor deactivation. Interestingly, glioblastoma-derived A172 cells showed a different chromatin loop contact propensity. Genes in tumorigenesis-associated signaling pathways were significantly enriched at the anchor loci of altered chromatin loops. Oncogene activation and tumor repressor deactivation were associated with chromatin loop alteration. Structure variations (SVs) had a dramatic impact on the chromatin conformation of EGFR-amplified glioblastoma-derived tumor cells. Moreover, our results revealed that 7p11.2 duplication activated EGFR expression in EGFR-amplified glioblastoma via neo-TAD formation and novel enhancer-promoter interaction emergence between LINC01446 and EGFR. ConclusionsThe disordered 3D genomic map and multi-omics data of EGFR-amplified glioblastoma provide a resource for future interrogation of the relationship between chromatin interactions and transcriptome in tumorigenesis.

KIF4A drives gliomas growth by transcriptional repression of Rac1/Cdc42 to induce cytoskeletal remodeling in glioma cells.

Glioma is one of the most prevalent cancers diseases in the worldwide. Kinesin superfamily protein 4 (KIF4), a KIF member classified in Kinesin 4 has been indicated as a mediator acted in tumorigenesis of human cancer. However, the mechanism of KIF4A on glioma is yet to be investigated. This study aimed to explore the potential function and mechanism of KIF4A in gliomas. We analyzed the KIF4A expression and the prognosis in gliomas patients using The Cancer Genome Atlas (TCGA) databases. KIF4A level in normal human astrocyte cell (NHA) and glioma cell lines were examined by Western blot. We studied the function of KIF4A on proliferation, migration, invasion, cell cycle in glioma cell lines using a series of in vitro and in vivo experiments. Chromatin Immunoprecipitation (ChIP) analysis was applied to searching potential KIF4A related downstream in glioma. We identified the significant up-regulated expression of KIF4A both in glioma tissues and cell. Glioma patients with elevated KIF4A expression have shorter survival. Down-regulation of KIF4A exerted inhibitory effect on cell proliferation, invasion and migration. We crucially identified that KIF4A drives gliomas growth by transcriptional repression of Rac1/Cdc42 to induce cytoskeletal remodeling in glioma cells. Knockdown of KIF4A decreased RohA, Rac1, Cdc42, Pak1 and Pak2 expression level. Our study provided a prospect that KIF4A functions as an oncogene in glioma.