GBM-derived Cells Research Articles

Pharmacological Modulation of the Cytosolic Oscillator Affects Glioblastoma Cell Biology

The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12–15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1 ε/δ (CK1ε/δ) with specific inhibitors (CHIR99021 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99021-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition compared to control cells. The combined treatment of CHIR99021 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects GBM cell biology.

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Hypoxia-inducible factor 1alpha and vascular endothelial growth factor in Glioblastoma Multiforme: a systematic review going beyond pathologic implications.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor characterized by extensive heterogeneity and vascular proliferation. Hypoxic conditions in the tissue microenvironment are considered a pivotal player leading tumor progression. Specifically, hypoxia is known to activate inducible factors, such as hypoxia-inducible factor 1alpha (HIF-1α), which in turn can stimulate tumor neo-angiogenesis through activation of various downward mediators, such as the vascular endothelial growth factor (VEGF). Here, we aimed to explore the role of HIF-1α/VEGF immunophenotypes alone and in combination with other prognostic markers or clinical and image analysis data, as potential biomarkers of GBM prognosis and treatment efficacy. We performed a systematic review (Medline/Embase, and Pubmed database search was completed by 16th of April 2024 by two independent teams; PRISMA 2020). We evaluated methods of immunoassays, cell viability, or animal or patient survival methods of the retrieved studies to assess unbiased data. We used inclusion criteria, such as the evaluation of GBM prognosis based on HIF-1α/VEGF expression, other biomarkers or clinical and imaging manifestations in GBM related to HIF-1α/VEGF expression, application of immunoassays for protein expression, and evaluation of the effectiveness of GBM therapeutic strategies based on HIF-1α/VEGF expression. We used exclusion criteria, such as data not reporting both HIF-1α and VEGF or prognosis. We included 50 studies investigating in total 1319 GBM human specimens, 18 different cell lines or GBM-derived stem cells, and 6 different animal models, to identify the association of HIF-1α/VEGF immunophenotypes, and with other prognostic factors, clinical and macroscopic data in GBM prognosis and therapeutic approaches. We found that increased HIF-1α/VEGF expression in GBM correlates with oncogenic factors, such as miR-210-3p, Oct4, AKT, COX-2, PDGF-C, PLDO3, M2 polarization, or ALK, leading to unfavorable survival. Reduced HIF-1α/VEGF expression correlates with FIH-1, ADNP, or STAT1 upregulation, as well as with clinical manifestations, like epileptogenicity, and a favorable prognosis of GBM. Based on our data, HIF-1α or VEGF immunophenotypes may be a useful tool to clarify MRI-PET imaging data distinguishing between GBM tumor progression and pseudoprogression. Finally, HIF-1α/VEGF immunophenotypes can reflect GBM treatment efficacy, including combined first-line treatment with histone deacetylase inhibitors, thimerosal, or an active metabolite of irinotecan, as well as STAT3 inhibitors alone, and resulting in a favorable tumor prognosis and patient survival. These data were supported by a combination of variable methods used to evaluate HIF-1α/VEGF immunophenotypes. Data limitations may include the use of less sensitive detection methods in some cases. Overall, our data support HIF-1α/VEGF's role as biomarkers of GBM prognosis and treatment efficacy.

Exploring Novel Therapeutic Opportunities for Glioblastoma Using Patient-Derived Cell Cultures

Glioblastomas (GBM) are the most common, primary brain tumors in adults. Despite advances in neurosurgery and radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNA-seq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), and the expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking intertumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at the mRNA/protein levels suggested increased epithelial-to-mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures, the strongest accumulation of the apoptotic markers caspase 7 and PARP were found in WG4 cells with methylated MGMT, suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, and thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity, and that identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.

Regulatory networks driving expression of genes critical for glioblastoma are controlled by the transcription factor c-Jun and the pre-existing epigenetic modifications

BackgroundGlioblastoma (GBM, WHO grade IV) is an aggressive, primary brain tumor. Despite extensive tumor resection followed by radio- and chemotherapy, life expectancy of GBM patients did not improve over decades. Several studies reported transcription deregulation in GBMs, but regulatory mechanisms driving overexpression of GBM-specific genes remain largely unknown. Transcription in open chromatin regions is directed by transcription factors (TFs) that bind to specific motifs, recruit co-activators/repressors and the transcriptional machinery. Identification of GBM-related TFs-gene regulatory networks may reveal new and targetable mechanisms of gliomagenesis.ResultsWe predicted TFs-regulated networks in GBMs in silico and intersected them with putative TF binding sites identified in the accessible chromatin in human glioma cells and GBM patient samples. The Cancer Genome Atlas and Glioma Atlas datasets (DNA methylation, H3K27 acetylation, transcriptomic profiles) were explored to elucidate TFs-gene regulatory networks and effects of the epigenetic background. In contrast to the majority of tumors, c-Jun expression was higher in GBMs than in normal brain and c-Jun binding sites were found in multiple genes overexpressed in GBMs, including VIM, FOSL2 or UPP1. Binding of c-Jun to the VIM gene promoter was stronger in GBM-derived cells than in cells derived from benign glioma as evidenced by gel shift and supershift assays. Regulatory regions of the majority of c-Jun targets have distinct DNA methylation patterns in GBMs as compared to benign gliomas, suggesting the contribution of DNA methylation to the c-Jun-dependent gene expression.ConclusionsGBM-specific TFs-gene networks identified in GBMs differ from regulatory pathways attributed to benign brain tumors and imply a decisive role of c-Jun in controlling genes that drive glioma growth and invasion as well as a modulatory role of DNA methylation.

Eltanexor Effectively Reduces Viability of Glioblastoma and Glioblastoma Stem-Like Cells at Nano-Molar Concentrations and Sensitizes to Radiotherapy and Temozolomide.

Current standard adjuvant therapy of glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to therapy resistance. Thus, novel therapeutic approaches are highly demanded. We tested the therapeutic efficacy of the second-generation XPO1 inhibitor Eltanexor using assays for cell viability and apoptosis in GBM cell lines and GBM stem-like cells. For most GBM-derived cells, IC50 concentrations for Eltanexor were below 100 nM. In correlation with reduced cell viability, apoptosis rates were significantly increased. GBM stem-like cells presented a combinatorial effect of Eltanexor with TMZ on cell viability. Furthermore, pretreatment of GBM cell lines with Eltanexor significantly enhanced radiosensitivity in vitro. To explore the mechanism of apoptosis induction by Eltanexor, TP53-dependent genes were analyzed at the mRNA and protein level. Eltanexor caused induction of TP53-related genes, TP53i3, PUMA, CDKN1A, and PML on both mRNA and protein level. Immunofluorescence of GBM cell lines treated with Eltanexor revealed a strong accumulation of CDKN1A, and, to a lesser extent, of p53 and Tp53i3 in cell nuclei as a plausible mechanism for Eltanexor-induced apoptosis. From these data, we conclude that monotherapy with Eltanexor effectively induces apoptosis in GBM cells and can be combined with current adjuvant therapies to provide a more effective therapy of GBM.

Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD+ Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase.

Simple SummaryGlioblastoma is the most prevalent and lethal brain tumor type, often treated with the DNA alkylating agent temozolomide (TMZ). The cytotoxic DNA lesion O6-methylguanine only accounts for about 9% of the DNA lesions induced by TMZ. The other DNA lesions (>80%) are quickly repaired by the base excision repair (BER) pathway. However, resistance to cytotoxicity of the O6-methylguanine lesion is common in cancer due to defects in the mismatch repair pathway or overexpression of the MGMT repair protein. Therefore, the aim of this study was to find approaches to inhibit the BER pathway to overcome TMZ resistance. We found that combining TMZ with an NAD+ precursor (dihydronicotinamide riboside) and a PARG inhibitor strongly inhibited BER and overcame TMZ resistance. This combination treatment regimen provides a novel approach to consider for glioblastoma.Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway. These resistance mechanisms are either somatic or arise in response to treatment, highlighting the need to uncover treatments to overcome resistance. We found that administration of the NAD+ precursor dihydronicotinamide riboside (NRH) to raise cellular NAD+ levels combined with PARG inhibition (PARGi) triggers hyperaccumulation of poly(ADP-ribose) (PAR), resulting from both DNA damage-induced and replication-stress-induced PARP1 activation. Here, we show that the NRH/PARGi combination enhances the cytotoxicity of TMZ. Specifically, NRH rapidly increases NAD+ levels in both TMZ-sensitive and TMZ-resistant GBM-derived cells and enhances the accumulation of PAR following TMZ treatment. Furthermore, NRH promotes hyperaccumulation of PAR in the presence of TMZ and PARGi. This combination strongly suppresses the cell growth of GBM cells depleted of MSH6 or cells expressing MGMT, suggesting that this regimen may improve the efficacy of TMZ to overcome treatment resistance in GBM.

Glioblastoma Embryonic-like Stem Cells Exhibit Immune-Evasive Phenotype.

Simple SummaryMost glioblastoma (GBM) patients relapse after an initial response to treatment. These aggressive traits are often associated with the presence of glioma stem cells (GSCs) within the tumor bulk, which are thought to participate in GBM therapy resistance. Given GBM cellular heterogeneity, we hypothesized that GSCs might also display cellular hierarchies associated with different degrees of stemness. Based on single-cell RNAseq data from GBM patients, we identified a subpopulation of GSCs, named core-GSCs (c-GSCs), with a similar profile to embryonic stem cells and downregulation of immune-associated pathways. In addition, we developed an in vitro induced c-GSC (ic-GSC) model resembling their tumor counterpart. The characterization of immune-privileged c-GSCs provides a valuable resource to study immune evasion mechanisms in GBM and to identify potential unexplored targets to improve immunotherapy treatments.Background: Glioma stem cells (GSCs) have self-renewal and tumor-initiating capacities involved in drug resistance and immune evasion mechanisms in glioblastoma (GBM). Methods: Core-GSCs (c-GSCs) were identified by selecting cells co-expressing high levels of embryonic stem cell (ESC) markers from a single-cell RNA-seq patient-derived GBM dataset (n = 28). Induced c-GSCs (ic-GSCs) were generated by reprogramming GBM-derived cells (GBM-DCs) using induced pluripotent stem cell (iPSC) technology. The characterization of ic-GSCs and GBM-DCs was conducted by immunostaining, transcriptomic, and DNA methylation (DNAm) analysis. Results: We identified a GSC population (4.22% ± 0.59) exhibiting concurrent high expression of ESC markers and downregulation of immune-associated pathways, named c-GSCs. In vitro ic-GSCs presented high expression of ESC markers and downregulation of antigen presentation HLA proteins. Transcriptomic analysis revealed a strong agreement of enriched biological pathways between tumor c-GSCs and in vitro ic-GSCs (κ = 0.71). Integration of our epigenomic profiling with 833 functional ENCODE epigenetic maps identifies increased DNA methylation on HLA genes’ regulatory regions associated with polycomb repressive marks in a stem-like phenotype. Conclusions: This study unravels glioblastoma immune-evasive mechanisms involving a c-GSC population. In addition, it provides a cellular model with paired gene expression, and DNA methylation maps to explore potential therapeutic complements for GBM immunotherapy.

Molecular pathways in glioblastoma-derived stem cells to identify effective drug agents: A bioinformatics study.

ABSTRACTBackground and Aim:Glioblastoma multiform (GBM) is considered as one of the malignant brain tumors that affect a wide range of people every year. Cancer stem cells, as essential factors, are resistant to chemotherapy drugs and complicate treatments. Therefore, finding critical molecular pathways in GBM-derived stem cells, and selecting the appropriate drug agents can prove more effective treatment approaches for GBM.Method:In this study, using RNA-Seq data, we performed continuous bioinformatics analyses and examined the up-and down-regulated genes from GBM-derived stem cells samples. Afterward, we separated the signaling pathways using the KEGG database and measured the protein interactions with the STRING database. Then, using the Drug matrix database, we nominated drugs that could affect these genes.Results:The first 20 pathways on tumorigenesis and 41 up-regulated and 73 down-regulated genes were selected. These genes were most active in the pathways involved in cell division, metabolism, cytoskeleton, cell adhesion molecules, and extracellular space. We then examined the candidate genes and the approach of the drugs that target these genes. Chlorambucil, cyclosporine A, doxorubicin, and etoposide were selected as the drug agents.Conclusion:Using integrated bioinformatics analyses, it was found that prominent genes in the cell cycle and cytoskeletal pathways are more expressed in cancer stem cells and that Chlorambucil, cyclosporine A, doxorubicin, and etoposide can be effective compounds to attenuate these cells.

Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1.

BackgroundGlioblastoma (GBM), the most common primary brain tumor, has a median survival of 15–16 months. Immunotherapy is promising but GBM-mediated immunosuppression remains a barrier. GBMs express the interferon-gamma (IFN-γ)-responsive immunosuppressive molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1). Extracellular vesicles (EVs) have also been implicated in GBM-mediated immunosuppression, in part through PD-L1. We therefore sought to determine if GBM IFN-γ exposure increased GBM EV-mediated immunosuppression and mechanisms underlying this.MethodsHuman GBM-derived cells were cultured in the presence/absence of IFN-γ. EVs were harvested. PD-L1, IDO1, and EV-associated protein expression was assessed. GBM EVs (+/−IFN-γ) were cultured with healthy donor monocytes. Immunosuppressive myeloid-derived suppressor cell (MDSC) and nonclassical monocyte (NCM) frequency was determined. Impact of GBM (+/−IFN-γ) EV-treated monocytes on CD3/CD28-mediated T cell proliferation was assessed. The impact of PD-L1 and IDO1 knockdown in GBM EVs in this system was evaluated.ResultsIFN-γ exposure increased PD-L1 and IDO1 expression in GBM cells and EVs without altering EV size or frequency. IFN-γ-exposed GBM EVs induced more MDSC and NCM differentiation in monocytes and these monocytes caused more T cell inhibition than IFN-γ-naive GBM EVs. PD-L1 and/or IDO1 knockdown in GBM cells abrogated the immunosuppressive effects of IFN-γ-exposed GBM EVs on monocytes.ConclusionsIFN-γ exposure such as might occur during an antitumor immune response results in superinduction of GBM EVs’ baseline immunosuppressive effects on monocytes. These effects are mediated by increased PD-L1 and IDO1 expression in GBM EVs. These data highlight mechanisms of GBM EV-mediated immunosuppression and identify therapeutic targets (PD-L1, IDO1) to reverse these effects.

Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells.

Simple SummaryHuman glioblastoma (GBM) cells are particularly resistant to nuclear fragmentation upon cytotoxic insult. To date, nuclear disassembly is the biological point-of-no-return of the apoptotic process. The injured cell that does not go beyond this point goes into a failed apoptosis and has potential for recovery. Nuclear disassembly is governed by caspases and, ultimately, by DFF40/CAD endonuclease. GBM cells express low levels of DFF40/CAD protein. Our aim was to assess whether this endonuclease could be activated to facilitate nuclear fragmentation in GBM cells. We revealed that GBM cells can activate their nuclear pool of DFF40/CAD in a caspase-dependent manner when treated with gossypol. Gossypol enabled DFF40/CAD assembly into high-order structures, facilitating nuclear dismantling. The identification of such compounds, pushing cells toward the point-of-no-return of apoptosis, will provide new tools to hamper the recovery of injured cells, slowing down tumor progression.Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

Crosstalk between 17β-Estradiol and TGF-β Signaling Modulates Glioblastoma Progression.

Epithelial–mesenchymal transition (EMT) is an essential mechanism contributing to glioblastoma multiforme (GBM) progression, the most common and malignant brain tumor. EMT is induced by signaling pathways that crosstalk and regulate an intricate regulatory network of transcription factors. It has been shown that downstream components of 17β-estradiol (E2) and transforming growth factor β (TGF-β) signaling pathways crosstalk in estrogen-sensitive tumors. However, little is known about the interaction between the E2 and TGF-β signaling components in brain tumors. We have investigated the relationship between E2 and TGF-β signaling pathways and their effects on EMT induction in human GBM-derived cells. Here, we showed that E2 and TGF-β negatively regulated the expression of estrogen receptor α (ER-α) and Smad2/3. TGF-β induced Smad2 phosphorylation and its subsequent nuclear translocation, which E2 inhibited. Both TGF-β and E2 induced cellular processes related to EMT, such as morphological changes, actin filament reorganization, and mesenchymal markers (N-cadherin and vimentin) expression. Interestingly, we found that the co-treatment of E2 and TGF-β blocked EMT activation. Our results suggest that E2 and TGF-β signaling pathways interact through ER-α and Smad2/3 mediators in cells derived from human GBM and inhibit EMT activation induced by both factors alone.

Glioblastoma stem cell (GSC)-derived PD-L1-containing exosomes activates AMPK/ULK1 pathway mediated autophagy to increase temozolomide-resistance in glioblastoma

Temozolomide (TMZ)-resistance hampers the therapeutic efficacy of this drug for glioblastoma (GBM) treatment in clinic, and emerging evidences suggested that exosomes from GBM-derived stem cells (GSCs) contributed to this process, but the detailed mechanisms are still largely unknown. In the present study, we reported that GSCs derived programmed death-ligand 1 (PD-L1) containing exosomes activated AMPK/ULK1 pathway mediated protective autophagy enhanced TMZ-resistance in GBM in vitro and in vivo. Specifically, we noticed that continuous low-dose TMZ stimulation promoted GSCs generation and PD-L1 containing exosomes (PD-L1-ex) secretion in GBM cells, and that PD-L1-ex inhibited cell apoptosis and promoted cell autophagy to increased TMZ-resistance in GBM cells, which were reversed by co-treating cells with the autophagy inhibitor 3-methyladenine (3-MA). Consistently, upregulation of PD-L1 also increased TMZ-resistance in TS-GBM cells, and silencing of PD-L1 sensitized TR-GBM cells to TMZ. In addition, PD-L1-ex activated AMPK/ULK1 pathway to induce autophagy in TMZ treated GBM cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) promoted cell apoptosis in GBM cells co-treated with PD-L1-ex and high-dose TMZ. Finally, we evidenced that PD-L1-ex promoted tumor growth and Ki67 protein expressions to increase TMZ-resistance in GBM in vivo. Collectively, we concluded that GSCs-derived PD-L1-ex activated AMPK1/ULK1 signaling cascade mediated autophagy to increase TMZ-resistance in GBM, and this study provided potential strategies to improve the therapeutic efficacy of TMZ in GBM.

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

Suppression of the USP10/CCND1 axis induces glioblastoma cell apoptosis.

Recent studies show that the expression of CCND1, a key factor in cell cycle control, is increased following the progress and deteriotation of glioma and predicts poor outcomes. On the other hand, dysregulated deubiquitinase USP10 also predicts poor prognosis for patients with glioblastoma (GBM). In the present study, we investigated the interplay between CCND1 protein and USP10 in GBM cells. We showed that the expression of CCND1 was significantly higher in both GBM tissues and GBM-derived stem cells. USP10 interacted with CCND1 and prevented its K48- but not K63-linked polyubiquitination in GBM U251 and HS683 cells, which led to increased CCND1 stability. Consistent with the action of USP10 on CCND1, knockdown of USP10 by single-guided RNA downregulated CCND1 and caused GBM cell cycle arrest at the G1 phase and induced GBM cell apoptosis. To implement this finding in the treatment of GBMs, we screened a natural product library and found that acevaltrate (AVT), an active component derived from the herbal plant Valeriana jatamansi Jones was strikingly potent to induce GBM cell apoptosis, which was confirmed by the Annexin V staining and activation of the apoptotic signals. Furthermore, we revealed that AVT concentration-dependently suppressed USP10-mediated deubiquitination on CCND1 therefore inducing CCND1 protein degradation. Collectively, the present study demonstrates that the USP10/CCND1 axis could be a promising therapeutic target for patients with GBMs.

Estradiol Induces Epithelial to Mesenchymal Transition of Human Glioblastoma Cells

The mesenchymal phenotype of glioblastoma multiforme (GBM), the most frequent and malignant brain tumor, is associated with the worst prognosis. The epithelial–mesenchymal transition (EMT) is a cell plasticity mechanism involved in GBM malignancy. In this study, we determined 17β-estradiol (E2)-induced EMT by changes in cell morphology, expression of EMT markers, and cell migration and invasion assays in human GBM-derived cell lines. E2 (10 nM) modified the shape and size of GBM cells due to a reorganization of actin filaments. We evaluated EMT markers expression by RT-qPCR, Western blot, and immunofluorescence.We found that E2 upregulated the expression of the mesenchymal markers, vimentin, and N-cadherin. Scratch and transwell assays showed that E2 increased migration and invasion of GBM cells. The estrogen receptor-α (ER-α)-selective agonist 4,4’,4’’-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 10 nM) affected similarly to E2 in terms of the expression of EMT markers and cell migration, and the treatment with the ER-α antagonist methyl-piperidino-pyrazole (MPP, 1 μM) blocked E2 and PPT effects. ER-β-selective agonist diarylpropionitrile (DNP, 10 nM) and antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazole[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 1 μM) showed no effects on EMT marker expression. These data suggest that E2 induces EMT activation through ER-α in human GBM-derived cells.

A new tyrosine kinase inhibitor K905-0266 inhibits proliferation and sphere formation of glioblastoma cancer cells

Glioblastoma (GBM) is the most prevalent malignant tumour of the central nervous system and carries a poor prognosis; average survival time after diagnosis is 14 months. Because of its unfavourable prognosis, novel therapies are needed. The aim of this study was to assess whether inhibition of GBM and GBM-derived cancer stem cells (CSCs) by a new tyrosine kinase inhibitor (TKI), K905-0266, is possible. To do this, we generated GBM (D54 and U87MG) cells expressing luciferase and characterised the inhibitory effects of the TKI with bioluminescent imaging (BLI) and western blot (WB). The effect of the TKI was then evaluated in CSCs. BLI showed significant inhibition of D54 and U87MG cells by TKI treatment. WB showed that the TKI decreased pERK and Bcl-2 level and increased cleaved caspase-3 level. Sphere formation was significantly reduced by the TKI in CSCs. Our results showed that a new TKI, K905-0266, effectively inhibited GBM and CSCs, making this a candidate for GBM therapy.

β2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the β2-adrenergic receptor (β2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective β2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a β2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the β2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of β2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

Ferritin in glioblastoma

Elevated levels of serum ferritin (SF) are observed in several types of cancer; however, little is known on the association between ferritin and glioma, the most frequent type of human primary brain tumour. Here we report that GBM patients show significantly increased pre-surgical SF levels (i.e. ferritinaemia) within the SF reference range and a marked ferritin immunoreactivity of resected tumour tissue. Our findings account for an indirect association between ferritin synthesis in glioma-tissue and altered SF levels, which limits the clinical value of SF as a tumour marker in glioma. Importantly, we show for the first time that GBM-derived glioma cells release ferritin in vitro, which exerts an apoptosis-stimulating activity. Albeit the pathophysiologic context of apoptosis induction by a tumour-derived ferritin remains to be defined, our findings account for a distinct growth-regulatory role of these ferritin species in tumour biology.

Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival

BackgroundResistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ.MethodsCell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan–Meier method.ResultsPre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood–brain barrier, diminished proteasome activity and significantly prolonged animal survival.ConclusionBTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.