Apoptosis In Glioblastoma Multiforme Cells Research Articles

In Vitro Experiment Present ROCK2 Inhibition Promotes the Therapeutic Effect of Bevacizumab in the Treatment of Glioblastoma Multiforme

Objective Gliomas are a general designation for neuroepithelial tumors derived from the glial cells of the central nervous system. According to the histopathological and immunohistochemical features, the World Health Organization classifies gliomas into four grades. Bevacizumab is a monoclonal antibody targeting vascular endothelial growth factor that has been approved for the treatment of glioblastoma multiforme (GBM) as a second-line therapy. However, its efficacy remains limited. Rho/Rho-associated kinase (ROCK) is a downstream molecule of small guanosine triphosphatases (GTPases) that regulates multiple cellular processes, including motility, migration, and proliferation. Thus, ROCK has been regarded as a therapeutic target for cardiovascular diseases, neurological diseases, immune diseases, and cancer, and ROCK inhibitors have high potential clinical value. Methods Viability rate of cells was detected using MTT assay, and apoptosis of cells was detected using FACS. Expression of target genes and proteins was detected using qPCR and western blotting analysis. Concentration of cytokines was detected using ELISA methods. Results Viability and migration of GBM cells were reduced after bevacizumab treatment and that these effects were enhanced by ROCK2 inhibition. We further found that ROCK2 inhibition promoting the effect of bevacizumab was mainly mediated by the RhoA/ROCK2 pathway, further inducing apoptosis in GBM cells. In addition, we found that angiogenesis and degradation of cellular matrix-related cytokines were reduced by ROCK2 inhibition. Conclusions ROCK2 inhibition contributes to the therapeutic effects of bevacizumab.

Hyperbaric Oxygen Therapy as a Novel Approach to Modulating Macrophage Polarization for the Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer with a poor prognosis despite current treatments. This is partially attributed to the immunosuppressive environment facilitated by tumor-associated macrophages, which predominantly underlie the tumor-promoting M2 phenotype. This study investigated the potential of hyperbaric oxygen (HBO) therapy, traditionally used to treat conditions such as decompression sickness, in modulating the macrophage phenotype toward the tumoricidal M1 state and disrupting the supportive tumor microenvironment. HBO has direct antiproliferative effects on tumor cells and reduces hypoxia, which may impair angiogenesis and tumor growth. This offers a novel approach to GBM treatment by targeting the role of the immune system within the tumor microenvironment. The effects of HBO on macrophage polarization and GBM cell viability and apoptosis were evaluated in this study. We detected that HBO promoted M1 macrophage cytokine expression while decreasing GBM cell viability and increasing apoptosis using GBM cell lines and THP-1-derived macrophage-conditioned media. These findings suggest that HBO therapy can shift macrophage polarization toward a tumoricidal M1 state. This can improve GBM cell survival and offers a potential therapeutic strategy. In conclusion, HBO can shift macrophages from a tumor-promoting M2 phenotype to a tumoricidal M1 phenotype in GBM. This can facilitate apoptosis and, in turn, improve treatment outcomes.

Bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently, bufotalin has been shown to have anti-cancer activity in a variety of cancers. This investigation aims to investigate the effect of bufotalin on GBM and elucidate its potential underlying mechanism. Our results show that bufotalin not only inhibits the proliferation and epithelial-mesenchymal transition (EMT) but also triggers apoptosis in GBM cells. The result of RNA-seq indicated that bufotalin could induce mitochondrial dysfunction. Moreover, our observations indicate that bufotalin induces an excessive accumulation of intracellular reactive oxygen species (ROS) in GBM cells, leading to mitochondrial dysfunction and the dephosphorylation of AKT. Moreover, bufotalin improved TMZ sensitivity of GBM cells in vitro and in vivo. In conclusion, bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

MAPK-activated protein kinase 2 is associated with poor prognosis of glioma patients and immune inhibition in glioma.

An effective therapeutic method to noticeably improve the prognosis of glioma patients has not been developed thus far. MAPK-activated protein kinase 2 (MAPKAPK2) is a serine/threonine kinase, which is involved in tumorigenesis, tumor growth, metastasis, and the inflammatory process. The clinical significance and molecular function of MAPKAPK2 in glioma remain unclear. MAPKAPK2 expression in human glioma tissues was detected by immunohistochemistry and analyzed from the transcriptome sequencing data in TCGA and CGGA. Prognostic nomogram was constructed to predict the survival risk of individual patients. GO and KEGG enrichment analyses were performed to analyze the function and pathways MAPKAPK2 involved. Single-cell RNA sequencing data was used to analyze the cell types in which MAPKAPK2 was enriched. Flow cytometry was used for cell cycle and apoptosis detection. The ability of cell proliferation and migration was analyzed by CCK8 and cell migration assay, respectively. Correlation analyses were performed to analyze the relationship of MAPKAPK2 with immune infiltration, immune regulators, chemokine, and chemokine receptors. MAPKAPK2 was not only aberrantly upregulated in glioma tissues but also correlated with poor clinical characteristics. Moreover, MAPKAPK2 was prevalent in isocitrate dehydrogenase (IDH) wild-type and 1p/19q non-codeletion glioma cohorts and predicted poor prognosis of glioma patients. MAPKAPK2 may be involved in cell proliferation, cell migration, DNA damage repair, and immune regulation in glioma. MAPKAPK2 was enriched in microglia/macrophages and malignant tumor cells. Further investigation into cellular function revealed that inhibiting MAPKAPK2 suppressed the proliferation and migration of glioblastoma multiforme (GBM) cells in vitro. The inhibition of MAPKAPK2 significantly induced the G1 cell cycle arrest and cell apoptosis of GBM cells. Consistent with the enriched function of MAPKAPK2 in immune regulation, MAPKAPK2 was correlated with immune cell infiltration in glioma tissues. Mechanistically, a series of immune regulators, immunomodulatory chemokine, and chemokine receptors were positively correlated with MAPKAPK2 expression. Our findings provide evidence of the clinical relevance of MAPKAPK2 in prognosis evaluation of glioma patients and highlight the underlying significance of MAPKAPK2 in glioma therapy.

The Combination of 5-FU and Resveratrol Can Suppress the Growth of Glioblastoma Cells Through Downregulation of TRPM2 and β-Catenin.

Glioblastoma multiforme (GBM) is the most common as well as the most fatal primary malignant tumor of the central nervous system (CNS), which still lacks a definitive cure. 5-FU is an anti-metabolite anti-cancer agent which has shown promising results for GBM treatment. Resveratrol (Res) is a phytochemical anti-oxidant that has also been effective in suppressing the progression of GBM. The combination of 5-FU and Res has been studied in a variety of cancers, but no study has assessed this combination in GBM. In this study, we investigated how 5-FU and Res, in combination and alone, may affect the growth and apoptosis of GBM cells and also the potential of TRPM2 and β-catenin as the mediator of their effects. U87 cells were cultured as the in vitro model. MTT assay was used for measuring cellular growth, and RT-qPCR was used to measure the level of caspase-3, TRPM2, and β-catenin; caspase-3 level served as the indicator of apoptotic rate. 5-FU and Res, in combination and alone, suppressed the growth while promoting the apoptosis of U87 cells; these effects were significantly greater when they were used in combination. RT-qPCR showed downregulation of TRPM-2 and β-catenin in response to this combination, which suggested that these two molecules may mediate the cited anti-oncogenic effects. In conclusion, our study confirmed the synergism between 5-FU and Res in suppressing the progression of GBM and suggested the putative axis of TRPM2/ β-catenin as the downstream mediator of this therapeutic regime. Future studies may be able to approve the eligibility of this therapeutic regime for GBM treatment and also the underlying mechanism.

Flavopiridol Suppresses Cell Proliferation and Migration and Induces Apoptotic Cell Death by Inhibiting Oncogenic FOXM1 Signaling in IDH Wild-Type and IDH-Mutant GBM Cells.

Glioblastoma multiforme (GBM) remains one of the most challenging solid cancers to treat due to its highly aggressive and drug-resistant nature. Flavopiridol is synthetic flavone that was recently approved by the FDA for the treatment of acute myeloid leukemia. Flavopiridol exhibits antiproliferative activity in several solid cancer cells and currently evaluated in clinical trials in several solid and hematological cancers. In this study, we investigated the molecular mechanisms underlying antiproliferative effects of flavopiridol in GBM cell lines with wild-type and mutant encoding isocitrate dehydrogenase 1 (IDH1). We found that flavopiridol inhibits proliferation, colony formation, and migration and induces apoptosis in IDH1 wild-type and IDH-mutant cells through inhibition of FOXM1 oncogenic signaling. Furthermore, flavopiridol treatment also inhibits of NF-KB, mediators unfolded protein response (UPR), including, GRP78, PERK and IRE1α, and DNA repair enzyme PARP, which have been shown to be potential therapeutic targets by downregulating FOXM1 in GBM cells. Our findings suggest for the first time that flavopiridol suppresses proliferation, survival, and migration and induces apoptosis in IDH1 wild-type and IDH1-mutant GBM cells by targeting FOXM1 oncogenic signaling which also regulates NF-KB, PARP, and UPR response in GBM cells. Flavopiridol may be a potential novel therapeutic strategy in the treatment of patients IDH1 wild-type and IDH1-mutant GBM.

Triethylammonium salt of a synthesized dicoumarol: Structural insight and human anti-glioblastoma activities

Glioblastoma multiforme (GBM) is the most common and primary brain tumor with poor prognosis. They are removed by following tedious and life threatening surgeries. GBM stem cells (GSCs) are the main source of tumor recurrence after surgery. Hence, drugs are designed to overcome the recurrent glioblastoma malignant cells. Currently used chemotherapies are not cost effective as well as bear resistance. New and effective chemotherapeutic compounds are developed to overcome the intrinsic and acquired resistance. Dicoumarol derivative 3,3′-[(4-methoxyphenyl)methanediyl]bis(4-hydroxy-2Hchromen-2-one) (HL) and its triethylammonium salt triethylammonium3-[(4-methoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-2-oxo-2H-chromen-4-olate (L) were synthesized and characterized using spectral and analytical techniques. The deprotonated compound L was further studied structurally using single crystal analysis. Cytotoxic studies against human glioblastoma cells A172 and LN229 were investigated both dose and time dependently and compared with the cytotoxicity of normal human astrocytes (NHA). The IC50 value of HL against A172 was found to be lying within the range 2.68–0.95 μM whereas against LN229 the range was found to be 9.55–0.85 μM. Similarly, the compound L revealed range of 1.9–0.271 μM against A172 and 1.2–0.27 μM against LN229. Cell cycle arrest was observed in GBM cells treated with L compared to the control group, which suggested that L may trigger apoptosis in GBM cells according to cytotoxicity and flow cytometry results. The antioxidant activity of synthesized compounds was also investigated using DPPH free radicals.

Chemical Activation and Mechanical Sensitization of Piezo1 Enhance TRAIL-Mediated Apoptosis in Glioblastoma Cells.

Glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, has a mean survival of less than 15 months after standard treatment. Treatment with the current standard of care, temozolomide (TMZ), may be ineffective if damaged tumor cells undergo DNA repair or acquire mutations that inactivate transcription factor p53. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in multiple tumor types, while evading healthy cells, through a transcription-independent mechanism. GBM is particularly resistant to TRAIL, but studies have found that the mechanoreceptor Piezo1 can be activated under static conditions via Yoda1 agonist to induce TRAIL sensitization in other cancer cell lines. This study examines the effects and the mechanism of chemical and mechanical activation of Piezo1, via Yoda1 and fluid shear stress (FSS) stimulation, on TRAIL-mediated apoptosis in GBM cells. Here, we demonstrate that Yoda1 + TRAIL and FSS + TRAIL combination therapies significantly increase apoptosis in two GBM cell lines relative to controls. Further, cells known to be resistant to TMZ were found to have higher levels of Piezo1 expression and were more susceptible to TRAIL sensitization by Piezo1 activation. The combinatory Yoda1 + TRAIL treatment significantly decreased cell viability in TMZ-resistant GBM cells when compared to treatment with both low and high doses of TMZ. The results of this study suggest the potential of a highly specific and minimally invasive approach to overcome TMZ resistance in GBM by sensitizing cancer cells to TRAIL treatment via chemical or mechanical activation of Piezo1.

Advanced Bioinformatics Analysis and Genetic Technologies for Targeting Autophagy in Glioblastoma Multiforme.

As the most malignant primary brain tumor in adults, a diagnosis of glioblastoma multiforme (GBM) continues to carry a poor prognosis. GBM is characterized by cytoprotective homeostatic processes such as the activation of autophagy, capability to confer therapeutic resistance, evasion of apoptosis, and survival strategy even in the hypoxic and nutrient-deprived tumor microenvironment. The current gold standard of therapy, which involves radiotherapy and concomitant and adjuvant chemotherapy with temozolomide (TMZ), has been a game-changer for patients with GBM, relatively improving both overall survival (OS) and progression-free survival (PFS); however, TMZ is now well-known to upregulate undesirable cytoprotective autophagy, limiting its therapeutic efficacy for induction of apoptosis in GBM cells. The identification of targets utilizing bioinformatics-driven approaches, advancement of modern molecular biology technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas9) or CRISPR-Cas9 genome editing, and usage of microRNA (miRNA)-mediated regulation of gene expression led to the selection of many novel targets for new therapeutic development and the creation of promising combination therapies. This review explores the current state of advanced bioinformatics analysis and genetic technologies and their utilization for synergistic combination with TMZ in the context of inhibition of autophagy for controlling the growth of GBM.

High concentration of γ‑H2AX correlates with a marker of apoptotic suppression and PI3K/Akt pathway upregulation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a very aggressive type of primary brain tumor in adults with a poor prognosis. DNA double-strand breaks are known to be associated with the development of numerous cancer types due to their ability to generate genomic instabilities. In GBM, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a common pathway that can be activated by exogenous and endogenous factors. Genomic instability may be an endogenous stimulating factor for activation of the PI3K/Akt pathway, which may inhibit the apoptosis of GBM cells. Spontaneous DNA double-strand breaks play an essential role in the survival of GBM cells, and apoptosis levels may reflect survival ability. However, no study has yet been conducted to analyse the association between spontaneous DNA double-strand breaks and apoptosis in patients with GBM prior to treatment. Therefore, the present study examined the concentrations of γ-histone 2AX (γ-H2AX), a sensitive marker of spontaneous DNA double-strand breaks, and cleaved caspase-3, a marker of apoptosis, in patients with GBM. The correlation of γ-H2AX with cleaved caspase-3, PI3K and Akt was also investigated. A total of 26 pre-treatment tumor tissue specimens from patient with GBM were analyzed to determine the concentrations of γ-H2AX, PI3K, Akt and cleaved caspase-3 using sandwich enzyme-linked immunosorbent assays. The results showed a moderate positive correlation between γ-H2AX and PI3K (r=0.52; P=0.007), a moderate positive correlation between γ-H2AX and Akt (r=0.4; P=0.041) and a strong negative correlation between γ-H2AX and cleaved caspase-3 (r=-0.61; P=0.0009). These analyses were also performed in seven tumor tissue specimens from patients with grade I glioma as controls, but no significant correlations were detected. The findings of the present study suggest that a high level of γ-H2AX may affect GBM cell apoptosis via the PI3K/Akt pathway.

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial-Dependent Apoptosis in GBM Cells, Which Is Regulated by Autophagy

Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3β-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma.

Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways. The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ. EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments. This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Radix Glycyrrhizae Preparata Induces Cell Cycle Arrest and Induced Caspase-Dependent Apoptosis in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive and devastating brain tumor characterized by poor prognosis and high rates of recurrence. Despite advances in multidisciplinary treatment, GBM constinues to have a poor overall survival. The Radix Glycyrrhizae Preparata (RGP) has been reported to possess anti-allergic, neuroprotective, antioxidative, and anti-inflammatory activities. However, it not clear what effect it may have on tumorigenesis in GBM. This study demonstrated that RGP reduced glioma cell viability and attenuated glioma cell locomotion in GBM8401 and U87MG cells. RGP treated cells had significant increase in the percentage of apoptotic cells and rise in the percentage of caspase-3 activity. In addition, the results of study’s cell cycle analysis also showed that RGP arrested glioma cells at G2/M phase and Cell failure pass the G2 checkpoint by RGP treatment in GBM8401 Cells. Based on the above results, it seems to imply that RGP activated DNA damage checkpoint system and cell cycle regulators and induce apoptosis in established GBM cells. In conclusion, RGP can inhibit proliferation, cell locomotion, cell cycle progression and induce apoptosis in GBM cells in vitro.

Isobavachalcone's Alleviation of Pyroptosis Contributes to Enhanced Apoptosis in Glioblastoma: Possible Involvement of NLRP3.

Glioblastoma multiforme (GBM) is the most malignant intracranial tumor with high mortality rates and invariably poor prognosis due to its limited clinical treatments. There is an urgent need to develop new therapeutic drugs for GBM treatment. As a natural prenylated chalcone compound, Isobavachalcone (IBC)'s favorable pharmacological activities have been widely revealed. However, potential inhibitory effects of IBC on GBM have not been explored. In the present study, we aimed to detect the effects of IBC on GBM and clarify its anti-GBM mechanisms for the first time. It was observed that IBC could inhibit GBM cell proliferation, migration, and invasion in vitro and prevent tumor growth without any significant drug toxicity in both subcutaneous and orthotopic GBM xenograft tumor models in vivo. Mechanistically, IBC may target NOD-like receptor family pyrin domain-containing 3 (NLRP3) transcription factor estrogen receptor α (ESR1 gene) by network pharmacology and molecular docking analysis. Experimentally, IBC alleviated NLRP3 inflammasome-related pyroptosis and inflammation, arrested cell cycle at G1 phase, and induced mitochondria-dependent apoptosis in GBM cells. IBC's inhibition on NLRP3 could be rescued by the NLRP3 antagonist CY-09 both in vitro and in vivo. These results indicate that IBC is a potential therapeutic drug against GBM and provide a new insight into GBM treatment.

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.

Isolinderalactone inhibits glioblastoma cell supernatant-induced angiogenesis.

Glioblastoma multiforme (GBM) is the most frequently occurring malignant brain tumor in adults and is characterized by a high degree of vascularization. Glioblastoma cells communicate with their microenvironment and stimulate blood vessel formation to support tumor progression. It has previously been reported that isolinderalactone induces apoptosis in GBM cells and suppresses the growth of glioblastoma xenograft tumors in vivo. Furthermore, isolinderalactone has been shown to inhibit the hypoxia-driven upregulation of vascular endothelial growth factor (VEGF) in U-87 GBM cells and strongly reduce VEGF-triggered angiogenesis in vitro and in vivo. In the present study, the direct angiogenic effect of GBM and the effect of isolinderalactone on tumor angiogenesis were investigated. Culture supernatants were obtained from U-87 cells under normoxic or hypoxic conditions to provide normoxic conditioned medium (NCM) and hypoxic conditioned medium (HCM) respectively. The NCM and HCM were each used to treat to human brain microvascular endothelial cells (HBMECs), and their effects were observed using wounding migration and tube formation assays. HCM increased the migration and capillary-like tube formation of HBMECs when compared with NCM, and treatment with isolinderalactone suppressed the HCM-driven angiogenesis in vitro. Additionally, isolinderalactone decreased HCM-triggered angiogenic sprouting in HBMECs in a 3D microfluidic device after the application of an HCM-containing interstitial fluid flow. Furthermore, isolinderalactone strongly reduced HCM-triggered angiogenesis in an in vivo Matrigel plug assay in mice. These findings provide evidence of angiogenesis inhibition by isolinderalactone, and demonstrate the anti-angiogenic effect of isolinderalactone against the direct angiogenic effect of GBM tumor cells.

Morusin Enhances Temozolomide Efficiency in GBM by Inducing Cytoplasmic Vacuolization and Endoplasmic Reticulum Stress.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with high risks of recurrence and mortality. Chemoradiotherapy resistance has been considered a major factor contributing to the extremely poor prognosis of GBM patients. Therefore, there is an urgent need to develop highly effective therapeutic agents. Here, we demonstrate the anti-tumor effect of morusin, a typical prenylated flavonoid, in GBM through in vivo and in vitro models. Morusin showed selective cytotoxicity toward GBM cell lines without harming normal human astrocytes when the concentration was less than 20 µM. Morusin treatment significantly induced apoptosis of GBM cells, accompanied by the activation of endoplasmic reticulum (ER) stress, and the appearance of cytoplasmic vacuolation and autophagosomes in cells. Then, we found the ER stress activation and cytotoxicity of morusin were rescued by ER stress inhibitor 4-PBA. Furthermore, morusin arrested cell cycle at the G1 phase and inhibited cell proliferation of GBM cells through the Akt–mTOR–p70S6K pathway. Dysregulation of ERs and cell cycle in morusin exposed GBM cells were confirmed by RNA-seq analysis. Finally, we demonstrated the combination of morusin and TMZ remarkably enhanced ER stress and displayed a synergistic effect in GBM cells, and suppressed tumor progression in an orthotopic xenograft model. In conclusion, these findings reveal the toxicity of morusin to GBM cells and its ability to enhance drug sensitivity to TMZ, suggesting the potential application value of morusin in the development of therapeutic strategies for human GBM.

LncRNA H19 promotes glioblastoma multiforme development by activating autophagy by sponging miR-491-5p

ABSTRACT Glioblastoma multiforme (GBM) is a malignant cancer with severely poor survival, and the cells continue to thrive during hypoxia and toxic stress through autophagy. To validate the oncogenic role of long noncoding RNA H19 in GBM progression and examine whether autophagy and/or miR-491-5p participate in the process. The expression of H19 and autophagy-related genes in GBM and healthy control tissues was assessed via quantitative polymerase chain reaction. In addition, cell viability, proliferation, apoptosis and autophagy were respectively determined via cell counting kit-8 assay, clone formation assay, flow cytometry, western blotting and green fluorescent protein–microtubule-associated protein 1 light chain 3 alpha fluorescence analysis in vitro. Furthermore, a rescue assay was performed using rapamycin or miR-491-5p antagomir to examine the role of autophagy or miR-491-5p in H19-mediated regulation of proliferation and apoptosis. RNA pull-down and dual-luciferase reporter assays were employed to analyze the interaction between H19 and miR-491-5p. Additionally, tumor growth in a xenograft-bearing mouse model and autophagy in tumor mass were analyzed in vivo. The expression H19 was increased in GBM and was positively correlated with LC3 or Beclin-1. Silencing H19 inhibited growth and promoted apoptosis in GBM cells both in vitro and in vivo, and miR-491-5p was identified as one of the important mediators. H19 regulated the autophagy signaling pathway at least partly via miR-491-5p. Increased H19 expression in GBM exerts oncogenic effects by sponging miR-491-5p and enhancing autophagy. Therefore, H19 may be explored as a target for GBM therapy.

CircPOLR2A Promotes Proliferation and Impedes Apoptosis of Glioblastoma Multiforme Cells by Up-regulating POU3F2 to Facilitate SOX9 Transcription

Glioblastoma multiforme (GBM) is the most common cancer in nervous system around the world. Little advancement has been achieved in promoting prognosis of GBM patients. Circular RNAs (circRNAs) are suggested as crucial effectors in modulating GBM development. Hsa_circRNA_092437 (circPOLR2A), an up-regulated circRNA in GBM cells, has not been studied yet. In this study, RT-qPCR and western blot assays were applied to detect RNA and protein levels. Cell proliferation and apoptosis were analyzed via functional assays. Subcellular fractionation assay was carried out to determine circPOLR2A distribution in cells. Bioinformatics analysis and mechanism assays were done for detecting relationships among different factors. Rescue assays were performed to confirm validity of circPOLR2A/SOX9 axis. According to experimental results, circPOLR2A was up-regulated in GBM cells and promoted GBM cell proliferation while inhibiting GBM cell apoptosis. CircPOLR2A mainly existed in cell cytoplasm and sponged miR-2113 to positively regulate POU3F2 expression. POU3F2 activated the transcription of SOX9 through interacting with SOX9 promoter (1–500). Rescue assays validated that circPOLR2A influenced GBM cell proliferation and apoptosis via SOX9. To conclude, circPOLR2A enhanced the transcription of SOX9 through miR-2113/POU3F2 axis, thus exacerbating GBM cells growth.

TMBIM1 promotes proliferation and attenuates apoptosis in glioblastoma cells by targeting the p38 MAPK signalling pathway

Glioblastoma multiforme (GBM) is the most common and most fatal primary malignant brain tumour in adults. The average survival time of patients after diagnosis is only 12–15 months. And its characteristics of excessive proliferation and apoptosis evasion play a crucial role in the poor prognosis of patients. Therefore, it is worth investigating the molecular mechanism of GBM to find an effective therapeutic target to overcome the dilemma. In the current study, Transmembrane BAX inhibitor motif containing 1 (TMBIM1) was highly expressed in GBM tissues and high TMBIM1 expression in GBM cell lines (U87 and U251) could promote cell proliferation and inhibit cell cycle arrest. In addition, TMBIM1 could significantly attenuate GBM cell apoptosis and decrease the sensitivity of GBM cells to temozolomide (TMZ). In terms of the molecular mechanism, we revealed that TMBIM1 interferes with the p38/MAPK pathway by inhibiting p38 phosphorylation to promote cell proliferation and attenuate cell apoptosis. In vivo experiments showed that the survival time of mice in TMBIM1 knockdown group was significantly prolonged. Our discovery provided an important basis for future intensive molecular mechanism research in GBM and presented a potential target for the treatment of GBM.