Glioblastoma Multiforme Cell Lines Research Articles

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin β1/FAK signaling pathway

BackgroundTemozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PurposeThis study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. MethodsThe differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. ResultsDEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway. ConclusionWe first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

Exploring the Therapeutic Effects of Radix Astragalus on Glioblastoma Multiforme Cell Culture

Objective: Glioblastomas, recognized for their aggressive and invasive nature, present a daunting challenge for effective treatment. At present, available therapies are not entirely efficacious in combating this primary brain tumor. In light of this, the present study aims to explore novel therapeutic strategies by investigating the antitumorigenic potential of Radix astragalus against Glioblastoma Multiforme (GBM) cell-lines in vitro. Methods: To accomplish this, the study employed a comprehensive research methodology that involved evaluating GBM cell viability and proliferation through MTT and LDH assays. Additionally, the study analyzed the oxidative burden of the GBM cell-line by measuring total antioxidant capacity (TAC), total oxidant status (TOS) and malondialdehyde (MDA) values, in order to further investigate the effects of Radix astragalus extracts. These advanced techniques allowed us to gain an in-depth understanding of the intricate molecular interactions between GBM cells and the extracts, paving the way for the development of novel therapies that can effectively combat this highly malignant disease. Results: The findings of this study demonstrate the remarkable antitumorigenic activity of Radix astragalus, as it significantly reduced cell viability in the GBM cell-line. Moreover, the extract inhibited the oxidative stress of GBM cells, which is a crucial factor that contributes to the development and progression of GBMs. Furthermore, the antioxidant assays revealed increased antioxidant activity in Radix astragalus-treated GBM cells compared to normal GBM cells. These findings offer a promising solution for treating GBMs, which are notoriously challenging to treat. Conclusion: The remarkable antitumorigenic activity of Radix astragalus identified in this study presents a novel avenue for cancer treatment research, with potential applications in the development of efficacious treatments against malignant brain tumors. Further exploration of the potential of Radix astragalus is warranted to pave the way for innovative therapies that can effectively combat GBMs.

STAG2 mutations regulate 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme

Inactivating mutations of genes encoding the cohesin complex are common in a wide range of human cancers. STAG2 is the most commonly mutated subunit. Here we report the impact of stable correction of endogenous, naturally occurring STAG2 mutations on gene expression, 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme (GBM). In two GBM cell lines, correction of their STAG2 mutations significantly altered the expression of ∼10% of all expressed genes. Virtually all the most highly regulated genes were negatively regulated by STAG2 (i.e., expressed higher in STAG2-mutant cells), and one of them - HEPH - was regulated by STAG2 in uncultured GBM tumors as well. While STAG2 correction had little effect on large scale features of 3D genome organization (A/B compartments, TADs), STAG2 correction did alter thousands of individual chromatin loops, some of which controlled the expression of adjacent genes. Loops specific to STAG2-mutant cells, which were regulated by STAG1-containing cohesin complexes, were very large, supporting prior findings that STAG1-containing cohesin complexes have greater loop extrusion processivity than STAG2-containing cohesin complexes and suggesting that long loops may be a general feature of STAG2-mutant cancers. Finally, STAG2 mutation activated Polycomb activity leading to increased H3K27me3 marks, identifying Polycomb signaling a potential target for therapeutic intervention in STAG2-mutant GBM tumors. Together, these findings illuminate the landscape of STAG2-regulated genes, A/B compartments, chromatin loops, and pathways in GBM, providing important clues into the largely still unknown mechanism of STAG2 tumor suppression.

Autophagy-related CMTM6 promotes glioblastoma progression by activating Wnt/β-catenin pathway and acts as an onco-immunological biomarker.

Glioblastoma multiforme (GBM) is identified as one of the most prevalent and malignant brain tumors, characterized by poor treatment outcomes and a limited prognosis. CMTM6, a membrane protein, has been found to upregulate the expression of programmed cell death 1 ligand 1 protein (PD-L1) and acts as an immune checkpoint inhibitor by inhibiting the programmed death 1 protein/PD-L1 signaling pathway. Recent research has demonstrated a high expression of CMTM6 in GBM, suggesting its potential role in influencing the pathogenesis and progression of GBM, as well as its association with immune cell infiltration in the tumor microenvironment. However, the underlying mechanism of CMTM6 in GBM requires further investigation. Data from cancer patients in The Cancer Genome Atlas, Gene Expression Omnibus and Chinese Glioma Genome Atlas cohorts were consolidated for the current study. Through multi-omics analysis, the study systematically examined the expression profile of CMTM6, epigenetic modifications, prognostic significance, biological functions, potential mechanisms of action and alterations in the immune microenvironment. Additionally, the study investigated CMTM6 expression in GBM cell lines and normal cells using reverse transcription PCR and western blot analysis. The impact of CMTM6 on GBM cell proliferation, migration and invasion was evaluated using a combination of cell counting kit-8 assay, clone formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, wound healing assay and Transwell assay. In order to explore the mechanism of CMTM6, the Wnt/β-catenin signaling pathway and autophagy-related genes were further verified through western blot analysis. CMTM6 is highly expressed in multiple tumors, particularly GBM. CMTM6 has been shown to be a valuable diagnostic and prognostic biomarker by various bioinformatics approaches. Additionally, CMTM6 plays a pivotal role in the pathogenesis of cancer, specifically GBM, by modulating various biological processes such as DNA methyltransferase expression, RNA modification, copy number variation, genomic heterogeneity, tumor stemness and DNA methylation. The findings of the experiment indicate a significant correlation between elevated CMTM6 expression and the proliferation, invasion, migration and autophagy of GBM cells, with potential key roles mediated through the Wnt/β-catenin signaling pathway. Furthermore, CMTM6 is implicated in modulating tumor immune cell infiltration and is closely linked to the expression of various immune checkpoint inhibitors and immune modulators, particularly within the context of GBM. High levels of CMTM6 expression also enhance the responsiveness of GBM patients to radiotherapy and chemotherapy, thereby offering valuable insights for guiding treatment strategies for GBM. Autophagy-related CMTM6 is highly expressed in various types of cancer, especially GBM, and it can regulate GBM progression through the Wnt/β-catenin signaling pathway and is capable of being used as an underlying target for the diagnosis, treatment selection and prognosis of patients with GBM.

Investigation of Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG)

Natural products are appealing as anticancer agents because of their low toxicity profiles. Cinnamon is a naturally occurring component with a variety of pharmacological properties, including anti-oxidant, anti-microbial, and anti-cancer properties for cancer cell apoptosis. Therefore, the current studies were performed to investigate Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG) since the proteins are directly involved in cell survival and function.Hot and cold extraction for Cinnamon performed from Cinnamon verum followed by phytochemicals tests. The Glioblastoma Multiforme Cell Lines U87 was subjected to cell viability by MTT Assay with Addition of cinnamon extract. The protein content was determined by spectrometric assay and its expression was investigated by SDS PAGE.The aqueous Cinnamon extract found to be positive for Alkaloids, Tannins, Saponins, Flavonoids, Terpenoids (only for Hot extract), Phenols and Reducing sugars (only for Cold extract). The cell viability at cinnamon extract concentration of 0.051 mg/100μl is shown to be highest and cell death at maximum cinnamon extract concentration of 1.65 mg/100μl is highest. Further these phytochemicals together showed less protein content on cultured U-87 cells and displayed lower protein expression in gel band pattern when compared to their control. The Cinnamon extract phytochemicals showed significant anticancer properties for reduced U87 cell viability with IC50 0.729. Thus positive phytochemicals result has shown to play an important role in inhibiting cancer properties.by showing less protein content and its expression for cinnamon phytochemicals treated U87 cell lines.

Urease and human glioblastoma (U87) cells growth inhibitory iridoid glycoside acetates from Nyctanthes arbor-tristis Linn.

The present study was undertaken on the chemical constituents of ethanol extract of aerial parts of Nyctanthes arbor-tristis Linn., and their determination of growth inhibitory activity against glioblastoma multiforme cell line (U87) and urease inhibitory activity. Six constituents were isolated including two new arbortristoside F tetraacetate (1) and arbortristoside G heptaacetate (2) and four known arborside A tetraacetate (3), arborside C pentaacetate (4), 6,7-di-O-acetyl-6β-hydroxyloganin hexaacetate (5) and nyctanthoside heptaacetate (6) iridoid glycoside acetates. Their structures were elucidated using spectroscopic methods, including 1D and 2D NMR and mass analyses. Compounds 3 and 6 showed significant growth inhibition of U87 cell line (76.41 and 87.62% inhibition) respectively while 2, 4 and 5 showed moderate inhibition. 3 and 6 showed notable urease inhibition (IC50 = 17.2 ± 0.4 and 17.2 ± 0.7 µM) respectively, and IC50 of 2, 4 and 5 were 23.8 − 56.3 µM. Compound 1 was inactive.

Nuclear autoantigenic sperm protein facilitates glioblastoma progression and radioresistance by regulating the ANXA2/STAT3 axis.

Although radiotherapy is a core treatment modality for various human cancers, including glioblastoma multiforme (GBM), its clinical effects are often limited by radioresistance. The specific molecular mechanisms underlying radioresistance are largely unknown, and the reduction of radioresistance is an unresolved challenge in GBM research. We analyzed and verified the expression of nuclear autoantigenic sperm protein (NASP) in gliomas and its relationship with patient prognosis. We also explored the function of NASP in GBM cell lines. We performed further mechanistic experiments to investigate the mechanisms by which NASP facilitates GBM progression and radioresistance. An intracranial mouse model was used to verify the effectiveness of combination therapy. NASP was highly expressed in gliomas, and its expression was negatively correlated with the prognosis of glioma. Functionally, NASP facilitated GBM cell proliferation, migration, invasion, and radioresistance. Mechanistically, NASP interacted directly with annexin A2 (ANXA2) and promoted its nuclear localization, which may have been mediated by phospho-annexin A2 (Tyr23). The NASP/ANXA2 axis was involved in DNA damage repair after radiotherapy, which explains the radioresistance of GBM cells that highly express NASP. NASP overexpression significantly activated the signal transducer and activator of transcription 3 (STAT3) signaling pathway. The combination of WP1066 (a STAT3 pathway inhibitor) and radiotherapy significantly inhibited GBM growth invitro and invivo. Our findings indicate that NASP may serve as a potential biomarker of GBM radioresistance and has important implications for improving clinical radiotherapy.

FOXP3 promote the progression of glioblastoma via inhibiting ferroptosis mediated by linc00857/miR-1290/GPX4 axis

The oncogenic properties of members belonging to the forkhead box (FOX) family have been extensively documented in different types of cancers. In this study, our objective was to investigate the impact of FOXP3 on glioblastoma multiforme (GBM) cells. By conducting a screen using a small hairpin RNA (shRNA) library, we discovered a significant association between FOXP3 and ferroptosis in GBM cells. Furthermore, we observed elevated levels of FOXP3 in both GBM tissues and cell lines, which correlated with a poorer prognosis. FOXP3 was found to promote the proliferation of GBM cells by inhibiting cell ferroptosis in vitro and in vivo. Mechanistically, FOXP3 not only directly upregulated the transcription of GPX4, but also attenuated the degradation of GPX4 mRNA through the linc00857/miR-1290 axis, thereby suppressing ferroptosis and promoting proliferation. Additionally, the FOXP3 inhibitor epirubicin exhibited the ability to impede proliferation and induce ferroptosis in GBM cells both in vitro and in vivo. In summary, our study provided evidences that FOXP3 facilitates the progression of glioblastoma by inhibiting ferroptosis via the linc00857/miR-1290/GPX4 axis, highlighting FOXP3 as a potential therapeutic target for GBM.

Abstract 6355: Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC)

Abstract Background: Glioblastoma Multiforme (GBM), primarily arising in the brain's frontal and temporal lobes, is a devastating malignancy originating from astrocytes. Despite treatment advancements, the prognosis remains bleak, with a median survival of 15-18 months and a 10% five-year survival rate.Our study explored a novel targeted approach to combat this challenge. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types but highly overexpressed on the majority of Soft-Tissue- and Bone Sarcomas. It internalizes fragments of collagen and transfers them to the lysosome for degradation. Methods & Results: First, we analyzed formalin-fixed paraffin-embedded (FFPE) tumors from GBM patients through immunohistochemistry (IHC) staining. Interestingly, a majority of the samples showed strong but varying uPARAP expression. In parallel, we detected and quantified uPARAP receptor expression in GBM cell lines by flow cytometry, underscoring its potential as a therapeutic target.Next, we generated an antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), a potent antimitotic agent. Confocal microscopy showed rapid internalization of the ADC in several GBM cell lines in vitro. Cellular cytotoxicity was demonstrated in Incucyte and Alamar Blue in vitro assays. Finally, anti-tumor activity of the uPARAP-ADC was studied in a GBM patient-derived xenograft (PDX) model in vivo, where it resulted in significant tumor regression with a tumor growth inhibition (TGI) of 92 and 97% at 3 and 6 mg/kg, respectively. Conclusions: Our findings show that uPARAP is overexpressed on GBM tumors and that a uPARAP-targeted ADC has anti-tumor activity in a PDX model of GBM. This research provides initial evidence that uPARAP-ADCs could become a novel treatment option for this highly aggressive disease. Citation Format: Ida Gregersen, Cecillie R. Løkke, Jette B. Lange, Pernille Barkholt, Christophé Côme, Tina Broberg, Marianne M. Petersen, Carmel M. Lynch, Dominik Mumberg, Niels Behrendt, Lars H. Engelholm. Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6355.

Abstract 5365: Characterizing signaling in macrophage-GBM cell coculture

Abstract Macrophages are a type of immune cell that play a crucial role in the tumor microenvironment. In tumors, macrophages can be polarized towards a pro-inflammatory phenotype, referred to as M1, or towards an anti-inflammatory and tumor-promoting phenotype, referred to as M2. M2 macrophages promote tumor growth by secreting cytokines that increase tumor proliferation, angiogenesis, and dampen the immune response from other immune cells such as T cells and dendritic cells. The balance between M1 and M2 macrophages in the tumor microenvironment is critical in determining the outcome of cancer progression and response to therapy. By understanding the pathways that lead to macrophage polarization, we can develop therapeutics that inhibit this process and support an anti-tumor macrophage response. Co-culture is a powerful model to study interactions between different cell types. We created co-cultures with macrophages and glioblastoma multiforme (GBM) cells to understand how these cell-cell interactions can lead to altered cell phenotype. Several studies have looked at the interaction of two populations using a transwell system where one cell population is placed in a well with a permeable membrane and the other population is placed below, allowing for the exchange of soluble factors. However, this does not account for an important mode of cell-cell interaction, which is physical contact. Therefore, we tested how macrophages derived from primary monocytes polarized in the context of conditioned media (supernatant), cell lysate, and direct co-culture. We used U87-MG as a model GBM cell line. We assayed the M2 phenotype by using flow cytometry with CD163 as a marker. Our findings show that direct co-culture is crucial for promoting macrophage M2 polarization. Using our in vitro model for tumor-induced macrophage polarization, we conducted phosphoproteomic analysis to characterize signals associated with this phenotypic change. To study how co-culture induced differential signaling in tumor populations and macrophages, we devised a method to fix cells to preserve cell signaling, followed by separation using flow cytometry and LC-MS/MS analysis of individual cell populations. We created co-cultures of U87-MG tumor cells and macrophage-differentiated THP-1 cells, a monocyte cell line. We then tested whether our method could reliably separate the different populations, whether we could detect altered signals due to co-culture, and showcase how separating cell populations yields information that could not be gathered from bulk analysis of co-cultures. Our data shows that the analysis was highly reproducible and that the signaling from cells separated from co-culture was highly similar to signaling in cells that were grown individually. However, some phosphorylation residues were uniquely upregulated in the co-culture condition, pointing to signaling that may occur in response to tumor interaction. Citation Format: Alicia D. D'Souza, Rachit Mukkamala, Ryuhjin Ahn, Forest M. White. Characterizing signaling in macrophage-GBM cell coculture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5365.

Exposure to Low-Frequency Radiation Changes the Expression of Nestin, VEGF, BCRP and Apoptosis Markers During Glioma Treatment Strategy: An In Vitro Study

Background: Exposure to physical contamination during chemotherapy, including non-ionizing electromagnetic fields, raises concerns about the widespread sources of exposure to this type of radiation. Glioblastoma multiforme (GBM) is an aggressive central nervous system tumor that is hard to treat due to resistance to drugs such as temozolomide (TMZ). Objective: Electromagnetic fields (EMF) and haloperidol (HLP) may have anticancer effects. In this study, we investigated the effects of TMZ, HLP, and EMF on GBM cell lines and analyzed the association between non-ionizing radiation and the risk of change in drug performance. Methods: Cell viability and reactive oxygen species (ROS) generation were measured by MTT and NBT assay, respectively. Then, the expression levels of breast cancer-resistant protein (BCRP), Bax, Bcl2, Nestin, vascular endothelial growth factor (VEGF) genes, and P53, Bax, and Bcl2 Proteins were evaluated by real-time PCR and western blot. Results: Co-treatment of GBM cells by HLP and TMZ enhanced apoptosis in T-98G and A172 cells by increasing the expression of P53 and Bax and decreasing Bcl-2. Interestingly, exposure of GBM cells to EMF decreased apoptosis in the TMZ+HLP group. Conclusion: In conclusion, EMF reduced the synergistic effect of TMZ and HLP. This hypothesis that patients who are treated for brain tumors and suffer from depression should not be exposed to EMF is proposed in the present study. There appears to be an urgent need to reconsider exposure limits for low-frequency magnetic fields, based on experimental and epidemiological research, the relationship between exposure to non-ionizing radiation and adverse human health effects.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

NOVA1 acts as an oncogenic RNA-binding protein to regulate cholesterol homeostasis in human glioblastoma cells

NOVA1 is a neuronal RNA-binding protein identified as the target antigen of a rare autoimmune disorder associated with cancer and neurological symptoms, termed paraneoplastic opsoclonus-myoclonus ataxia. Despite the strong association between NOVA1 and cancer, it has been unclear how NOVA1 function might contribute to cancer biology. In this study, we find that NOVA1 acts as an oncogenic factor in a GBM (glioblastoma multiforme) cell line established from a patient. Interestingly, NOVA1 and Argonaute (AGO) CLIP identified common 3' untranslated region (UTR) targets, which were down-regulated in NOVA1 knockdown GBM cells, indicating a transcriptome-wide intersection of NOVA1 and AGO-microRNA (miRNA) targets regulation. NOVA1 binding to 3'UTR targets stabilized transcripts including those encoding cholesterol homeostasis related proteins. Selective inhibition of NOVA1-RNA interactions with antisense oligonucleotides disrupted GBM cancer cell fitness. The precision of our GBM CLIP studies point to both mechanism and precise RNA sequence sites to selectively inhibit oncogenic NOVA1-RNA interactions. Taken together, we find that NOVA1 is commonly overexpressed in GBM, where it can antagonize AGO2-miRNA actions and consequently up-regulates cholesterol synthesis, promoting cell viability.

Transduction Efficiency of Zika Virus E Protein Pseudotyped HIV-1gfp and Its Oncolytic Activity Tested in Primary Glioblastoma Cell Cultures.

The development of new tools against glioblastoma multiforme (GBM), the most aggressive and common cancer originating in the brain, remains of utmost importance. Lentiviral vectors (LVs) are among the tools of future concepts, and pseudotyping offers the possibility of tailoring LVs to efficiently transduce and inactivate GBM tumor cells. Zika virus (ZIKV) has a specificity for GBM cells, leaving healthy brain cells unharmed, which makes it a prime candidate for the development of LVs with a ZIKV coat. Here, primary GBM cell cultures were transduced with different LVs encased with ZIKV envelope variants. LVs were generated by using the pNLgfpAM plasmid, which produces the lentiviral, HIV-1-based, core particle with GFP (green fluorescent protein) as a reporter (HIVgfp). Using five different GBM primary cell cultures and three laboratory-adapted GBM cell lines, we showed that ZIKV/HIVgfp achieved a 4-6 times higher transduction efficiency compared to the commonly used VSV/HIVgfp. Transduced GBM cell cultures were monitored over a period of 9 days to identify GFP+ cells to study the oncolytic effect due to ZIKV/HIVgfp entry. Tests of GBM tumor specificity by transduction of GBM tumor and normal brain cells showed a high specificity for GBM cells.

Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines.

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer, with a poor prognosis. GBM cells, which develop in the environment of neural tissue, often exploit neurotransmitters and their receptors to promote their own growth and invasion. Nicotinic acetylcholine receptors (nAChRs), which play a crucial role in central nervous system signal transmission, are widely represented in the brain, and GBM cells express several subtypes of nAChRs that are suggested to transmit signals from neurons, promoting tumor invasion and growth. Analysis of published GBM transcriptomes revealed spatial heterogeneity in nAChR subtype expression, and functional nAChRs of α1*, α7, and α9 subtypes are demonstrated in our work on several patient-derived GBM microsphere cultures and on the U87MG GBM cell line using subtype-selective neurotoxins and fluorescent calcium mobilization assay. The U87MG cell line shows reactions to nicotinic agonists similar to those of GBM patient-derived culture. Selective α1*, α7, and α9 nAChR neurotoxins stimulated cell growth in the presence of nicotinic agonists. Several cultivating conditions with varying growth factor content have been proposed and tested. The use of selective neurotoxins confirmed that cell cultures obtained from patients are representative GBM models, but the use of media containing fetal bovine serum can lead to alterations in nAChR expression and functioning.

Furanocoumarins as Enhancers of Antitumor Potential of Sorafenib and LY294002 toward Human Glioma Cells In Vitro.

Furanocoumarins are naturally occurring compounds in the plant world, characterized by low molecular weight, simple chemical structure, and high solubility in most organic solvents. Additionally, they have a broad spectrum of activity, and their properties depend on the location and type of attached substituents. Therefore, the aim of our study was to investigate the anticancer activity of furanocoumarins (imperatorin, isoimperatorin, bergapten, and xanthotoxin) in relation to human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cell lines. The tested compounds were used for the first time in combination with LY294002 (PI3K inhibitor) and sorafenib (Raf inhibitor). Apoptosis, autophagy, and necrosis were identified microscopically after straining with Hoechst 33342, acridine orange, and propidium iodide, respectively. The levels of caspase 3 and Beclin 1 were estimated by immunoblotting and for the blocking of Raf and PI3K kinases, the transfection with specific siRNA was used. The scratch test was used to assess the migration potential of glioma cells. Our studies showed that the anticancer activity of furanocoumarins strictly depended on the presence, type, and location of substituents. The obtained results suggest that achieving higher pro-apoptotic activity is determined by the presence of an isoprenyl moiety at the C8 position of the coumarin skeleton. In both anaplastic astrocytoma and glioblastoma, imperatorin was the most effective in induction apoptosis. Furthermore, the usage of imperatorin, alone and in combination with sorafenib or LY294002, decreased the migratory potential of MOGGCCM and T98G cells.

HSV1 microRNAs in glioblastoma development: an in silico study

Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor. Recent findings highlighted the significance of viral microRNAs (miRs) in regulating post-transcriptional mRNA expression in various human conditions. Although HSV1 encodes viral miRs and affects the central nervous system, no study investigated the roles of HSV1-encoding miRs in GBM development. This study applied in silico approaches to investigate whether HSV1-encoding miRs are involved in GBM development and, if so, how they regulate tumor-suppressive/oncogenes expression in GBM. This study leveraged bioinformatics approaches to identify the potential effect of HSV1 miRs in GBM development. The GSE158284, GSE153679, and GSE182109 datasets were analyzed to identify differentially expressed genes in GBM tissues and cell lines using the limma package in the R software. The GSE182109 dataset was analyzed to determine gene expression at the single-cell levels using the Seurat package in the R software. The TCGA-GTEX, GDSC, CTRP, immunogenetic, and enrichment analyses were performed to study the impact of identified viral HSV1 miRs targets in GBM development. hsv1-miR-H6-3p is upregulated in GBM and can be responsible for EPB41L1 and SH3PXD2A downregulation in GBM tissues. Also, hsv1-miR-H1-5p is upregulated in GBM and can decrease the expression of MELK, FZD2, NOVA1, TMEM97, PTPRZ1, and PDGFC in GBM development. The single-cell RNA sequencing analyses have demonstrated that MELK, FZD2, NOVA1, TMEM97, PTPRZ1, and PDGFC are expressed in astrocytes residing in the GBM microenvironment. This study provides novel insights into the potential roles of HSV1 miRs in GBM pathogenesis and offers a reference for further studies on the significance of HSV1 miRs in GBM development.

A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO.

N 6-Methyladenosine (m6A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m6A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, TD19, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. TD19 irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m6A-containing RNA. Moreover, TD19 displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.

Investigating the effect of hydroalcoholic extract of Prosopis farcta fruit on the toxicity induced by temozolomide in glioblastoma cell line in vitro

Introduction: Glioblastoma multiforme (GBM) is one of the most common human brain tumors. The use of temozolomide drug can be mentioned among the treatment methods. Researchers are trying to use methods to overcome resistance to temozolomide. Regarding the anticancer effects of Prosopis farcta in traditional Iranian medicine, the present study aimed to investigate the effect of the fruit hydroalcoholic extract of Prosopis farcta on the toxicity induced by temozolomide in the GBM cell line. Method: The T98G cell line was treated with plant extract and temozolomide. The cell survival rate was evaluated by MTT assay. The type of interaction between herbal extract and temozolomide was evaluated using the MTT test and Compusyn software. In addition, the amount of nitric oxide production in cancer cells was measured. Furthermore, the expression of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene was evaluated by Real-time PCR. Results: Hydroalcoholic extract of the plant and temozolomide decreased cell viability in a concentration- and time-dependent manner. The combination of extract and temozolomide had a synergistic effect in reducing cell survival. The hydroalcoholic extract and/or temozolomide caused a significant decrease in nitric oxide production and secretion (P<0.05). Considering the expression of MGMT, the hydroalcoholic extract and temozolomide significantly decreased and increased the expression of this gene, respectively (P<0.05). Simultaneous treatment caused a significant decrease in MGMT expression compared to the group treated with temozolomide (P<0.05). Conclusion: The combination of Prosopis farcta fruit extract and temozolomide had synergistic antitumor potential in GBM cells. This extract could reduce cellular resistance to temozolomide through the downregulation of MGMT expression.

Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma

BackgroundGlioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM.MethodsThe synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts.ResultsCombined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity.ConclusionOur findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials.396yfhm1sdcH9_KhVkr-McVideo