TMZ-resistant Glioblastoma Multiforme Cells 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.

Polymer-Drug Conjugates Codeliver a Temozolomide Intermediate and Nitric Oxide for Enhanced Chemotherapy against Glioblastoma Multiforme.

Polymer-drug conjugates (PDCs) provide possibilities for the development of multiresponsive drug delivery and release platforms utilized in cancer therapy. The delivery of Temozolomide (TMZ, a DNA methylation agent) by PDCs has been developed to improve TMZ stability under physiological conditions for the treatment of glioblastoma multiforme (GBM); however, with inefficient chemotherapeutic efficacy. In this work, we synthesized an amphiphilic triblock copolymer (P1-SNO) with four pendant functionalities, including (1) a TMZ intermediate (named MTIC) as a prodrug moiety, (2) a disulfide bond as a redox-responsive trigger to cage MTIC, (3) S-nitrosothiol as a light/heat-responsive donor of nitric oxide (NO), and (4) a poly(ethylene glycol) chain to enable self-assembly in aqueous media. P1-SNO was demonstrated to liberate MTIC in the presence of reduced glutathione and release gaseous NO upon exposure to light or heat. The in vitro results revealed a synergistic effect of released MTIC and NO on both TMZ-sensitive and TMZ-resistant GBM cells. The environment-responsive PDC system for codelivery of MTIC and NO is promising to overcome the efficacy issue in TMZ-based cancer therapy.

Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

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.

The Effect of Hydroquinidine on Proliferation and Apoptosis of TMZ-sensitive and -resistant GBM Cells.

Glioblastoma multiforme (GBM) is a lethal form of central nervous system cancer with a lack of efficient therapy options. Aggressiveness and invasiveness of the GBM result in poor prognosis and low overall survival. Therefore, the necessity to develop new anti-carcinogenic agents in GBM treatment is still a priority for researchers. Ion channels are one of the primary regulators of physiological homeostasis with additional critical roles in many essential biological processes related to cancer, such as invasion and metastasis. A multi-channel blocker, hydroquinidine (HQ), is currently in use to treat short-QT and Brugada arrhythmia syndromes. The objective of the study was to examine the alterations in survival, clonogenicity, migration, tumorigenicity, proliferation, apoptosis, and gene expression profile of temozolomide (TMZ)-sensitive and TMZ-resistant GBM cells upon HQ treatment. The possible anti-neoplastic activity of HQ on GBM cells was investigated by several widely applied cell culture methods. The IC50 values were determined using the MTT assay. Upon HQ treatment, the clonogenicity and migration capacity of cells were evaluated via colony-formation and wound healing assay, respectively. For antiproliferative and apoptotic effects, EdU and CFSE, and Annexin-V labeling were applied. Tumorigenicity level was depicted by employing soft agar assay. The expression level of multiple genes functioning in the cell cycle and apoptosis- related processes was checked utilizing qPCR. A significant anti-carcinogenic effect of HQ on TMZ-sensitive and -resistant GBM cells characterized by the increased apoptosis and decreased proliferation rate was revealed due to the altered gene expression profile related to cell cycle and cell death. In this study, the anti-carcinogenic effect of HQ has been demonstrated for the first time. Our data suggest the possible utilization of HQ to suppress the growth of GBM cells. Further studies on GBM-bearing animal models are required to assess its therapeutic potential in GBM treatment.

NADPH Oxidase Subunit CYBB Confers Chemotherapy and Ferroptosis Resistance in Mesenchymal Glioblastoma via Nrf2/SOD2 Modulation.

Glioblastoma multiforme (GBM) is a highly heterogeneous disease with a mesenchymal subtype tending to exhibit more aggressive and multitherapy-resistant features. Glioblastoma stem-cells derived from mesenchymal cells are reliant on iron supply, accumulated with high reactive oxygen species (ROS), and susceptible to ferroptosis. Temozolomide (TMZ) treatment is the mainstay drug for GBM despite the rapid development of resistance in mesenchymal GBM. The main interconnection between mesenchymal features, TMZ resistance, and ferroptosis are poorly understood. Herein, we demonstrated that a subunit of NADPH oxidase, CYBB, orchestrated mesenchymal shift and promoted TMZ resistance by modulating the anti-ferroptosis circuitry Nrf2/SOD2 axis. Public transcriptomic data re-analysis found that CYBB and SOD2 were highly upregulated in the mesenchymal subtype of GBM. Accordingly, our GBM cohort confirmed a high expression of CYBB in the GBM tumor and was associated with mesenchymal features and poor clinical outcome. An in vitro study demonstrated that TMZ-resistant GBM cells displayed mesenchymal and stemness features while remaining resilient to erastin-mediated ferroptosis by activating the CYBB/Nrf2/SOD2 axis. The CYBB maintained a high ROS state to sustain the mesenchymal phenotype, TMZ resistance, and reduced erastin sensitivity. Mechanistically, CYBB interacted with Nrf2 and consequently regulated SOD2 transcription. Compensatory antioxidant SOD2 essentially protected against the deleterious effect of high ROS while attenuating ferroptosis in TMZ-resistant cells. An animal study highlighted the protective role of SOD2 to mitigate erastin-triggered ferroptosis and tolerate oxidative stress burden in mice harboring TMZ-resistant GBM cell xenografts. Therefore, CYBB captured ferroptosis resilience in mesenchymal GBM. The downstream compensatory activity of CYBB via the Nrf2/SOD2 axis is exploitable through erastin-induced ferroptosis to overcome TMZ resistance.

The PI3K Inhibitor XH30 Enhances Response to Temozolomide in Drug-Resistant Glioblastoma via the Noncanonical Hedgehog Signaling Pathway.

Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system. Temozolomide (TMZ)–based adjuvant treatment has improved overall survival, but clinical outcomes remain poor; TMZ resistance is one of the main reasons for this. Here, we report a new phosphatidylinositide 3-kinase inhibitor, XH30; this study aimed to assess the antitumor activity of this compound against TMZ-resistant GBM. XH30 inhibited cell proliferation in TMZ-resistant GBM cells (U251/TMZ and T98G) and induced cell cycle arrest in the G1 phase. In an orthotopic mouse model, XH30 suppressed TMZ-resistant tumor growth. XH30 was also shown to enhance TMZ cytotoxicity both in vitro and in vivo. Mechanistically, the synergistic effect of XH30 may be attributed to its repression of the key transcription factor GLI1 via the noncanonical hedgehog signaling pathway. XH30 reversed sonic hedgehog–triggered GLI1 activation and decreased GLI1 activation by insulin-like growth factor 1 via the noncanonical hedgehog signaling pathway. These results indicate that XH30 may represent a novel therapeutic option for TMZ-resistant GBM.

Visible Light and Glutathione Dually Responsive Delivery of a Polymer-Conjugated Temozolomide Intermediate for Glioblastoma Chemotherapy.

Temozolomide (TMZ) is a prodrug of 5-(3-methyltriazene-1-yl)imidazole-4-carboxamide (MTIC, short-lived) and used as a first-line therapy drug for glioblastoma multiforme (GBM). However, little progress has been made in regulating the kinetics of TMZ to MTIC degradation to improve the therapeutic effect, particularly in the case of TMZ-resistant GBM. In this work, we introduced a strategy to cage MTIC by N-acylation of the triazene moiety to boost the MTIC stability, designed a diblock copolymer-based MTIC prodrug installed with a disulfide linkage, and achieved self-assembled polymer micelles without the concern of MTIC leakage under physiological conditions. Polymer micelles could be induced to disassemble by stimuli factors such as glutathione (GSH) and visible light irradiation through thiol/sulfide exchange and homolytic sulfide scission mechanisms, which contributed to MTIC release in GSH-dependent and GSH-independent pathways. The in vitro results demonstrated that microenvironment-responsive polymeric micelles benefited the suppression of both TMZ-sensitive and TMZ-resistant GBM cells. The chemistry of polymer-MTIC prodrug provided a new option for TMZ-based glioma treatment.

Oxyphyllanene B overcomes temozolomide resistance in glioblastoma: Structure–activity relationship and mitochondria-associated ER membrane dysfunction

The identification of novel therapeutic candidates from natural products for the development of chemoresistant glioblastoma multiforme (GBM) treatment has been a highly significant and effective strategy. Sesquiterpenes are a class of naturally occurring 15-carbon isoprenoid compounds, and several types of sesquiterpenes have the ability to induce growth inhibition and apoptosis in a variety of cancer cell lines. In the present study, 56 sesquiterpenes of five types, namely, eudesmane-type (I) (1-24), eremophilane-type (II) (25-32), cadinane-type (III) (33-41), guaiane-type (IV) (42-49), and oplopanone-type (V) (50-56), were screened for their antiglioma activity, structure-activity relationship analysis (SAR), and underlying mechanism based on patient-derived recurrent GBM strains, patient-derived GBM cell sphere, GBM organoid (GBO) models, and temozolomide (TMZ)-resistant GBM cell lines. We found that compound 12 (oxyphyllanene B, OLB) showed the most potent antiglioma activity, and we confirmed that OLB could induce apoptosis in a time- and dose-dependent manner in TMZ-resistant GBM cells and GBOs. SAR announced that the presence of an α, β-unsaturated carbonyl moiety was likely to enhance cytotoxic activities. Mechanistic studies demonstrated that OLB induced abnormal changes in ER and mitochondria-associated membrane (MAM) networks, which triggered ER stress, mitochondrial dysfunction, and apoptosis. Furthermore, our findings suggested that OLB-triggered PACS2 activation might form a committed step to disrupt ER-mitochondria communication and showed for the first time that the expression levels of PACS2 might positively correlate with the progression and chemotherapy resistance of glioma. Our results indicated that OLB might be a promising candidate for treating TMZ-resistant GBM cells by activating PACS2, which triggered a crucial event to promote the disruption of ER-mitochondria communication and overcome chemotherapy resistance of GBM.

Chemoattractants driven and microglia based biomimetic nanoparticle treating TMZ-resistant glioblastoma multiforme

Currently, clinical treatment for temozolomide (TMZ)-resistant glioblastoma multiforme (GBM) is still a difficult problem. The aim of this paper is to set up a new GBM-targeted drug delivery system to treat TMZ-resistant GBM. Zoledronate (ZOL) not only induces apoptosis of TMZ-resistant GBM cells by down-regulation of farnesyl pyrophosphate synthetase (FPPS) but also increases the proportion of M1-type GBM associated macrophages (GAM). Based on chemoattractants secreted by GBM cells, a ZOL loaded nanoparticle coated with microglia cell membrane (ZOL@CNPs) was prepared to deliver ZOL to central nervous system to treat TMZ-resistant GBM. ZOL@CNPs was actively recruited to TMZ-resistant GBM region by CX3CL1/CX3CR1 and CSF-1/CSF-1R signal axis, and the release of ZOL from ZOL@CNPs was triggered by glutathione in GBM cells. ZOL@CNPs inhibited the growth of TMZ-resistant GBM through inducing apoptosis and inhibiting the migration and invasion of TMZ-resistant GBM cells. Besides, the immunosuppressive and hypoxic microenvironment, playing an important role in the growth of TMZ-resistant GBM, was significantly improved by ZOL@CNPs through increasing the proportion of M1-type GAM and blocking the expression of HIF-1α. ZOL@CNPs has a great potential application in the treatment for TMZ-resistant GBM.

NBM-BMX, an HDAC8 Inhibitor, Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the β-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition.

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.

Abstract 2984: Developing a new combinational therapy to combat temozolomide resistance in glioblastoma multiforme

Abstract Background: Glioblastoma multiforme (GBM) is the most lethal central nervous system malignancy, and recurrence is seen in nearly all cases. Temozolomide (TMZ), a DNA alkylating agent, shows initial promise in eradicating primary GBM, but has little effect on recurrent GBM. Our previous work identified the Phosphatidylinositol 3-kinase Catalytic Subunit Beta (PIK3CB) of the PI3K pathway to be selectively important for the survival and progression of GBM (1). Other previous research also shows that a gap-junction protein, Connexin 43 (Cx43), promotes TMZ resistance in GBM by activating PI3K signaling and that this resistance can be reversed by treatment with a peptide compound named αCT1 (2). More recently, it was discovered that G-protein coupled receptors (GPCRs), specifically the Gβγ subunit, are upstream activators of Cx43/PI3K/AKT signaling in TMZ-resistant GBM cells. We believe that selectively targeting and blocking the activators of these signaling pathways will help to restore and enhance sensitivity to TMZ, yielding successful eradication of GBM cells. We therefore propose to test the effect of a quadruple combination of TMZ, αCT1, TGX-221, and gallein, a selective inhibitor of the Gβγ subunit of GPCRs, on GBM tumor growth. Hypothesis: Gβγ subunit inhibition will substantially increase GBM cytotoxicity in combination with αCT1, TGX-221 (a PIK3CB inhibitor), and TMZ. Methods: MTS cell viability assay was used to determine optimum gallein dosage both alone and in combinational therapies in experimental cell lines with variable Cx43 expression rates and TMZ sensitivities. This assay was also used to determine cell viability after treatment with an optimized quadruple combination therapy in these same lines. Current work is focused on quantifying Cx43/PIK3CB/AKT signaling inhibition via Western blot, as well as applying the optimized quadruple combination treatment in GBM stem cells, three-dimensional GBM tumor models, primary GBM patient samples, and healthy astrocytes. Results: Significant inhibitory effects have been shown with an optimized quadruple combination therapy compared to TMZ alone in TMZ-resistant experimental cell lines. Conclusions: While results in experimental lines have been favorable, additional work is still needed to elucidate the efficacy of the optimized quadruple combination therapy, which will include testing primary GBM patient samples, GBM stem cells, healthy astrocytes and three-dimensional tumor models.

STAT3 inhibition induced temozolomide-resistant glioblastoma apoptosis via triggering mitochondrial STAT3 translocation and respiratory chain dysfunction

Recent evidence has demonstrated that the signal transducer and activator of transcription 3 (STAT3) gene are abnormally active in glioblastoma multiforme (GBM), and this change is crucial for the tumor survival and chemotherapy-resistant. Certain preclinical pharmacology studies have focused on STAT3 phosphorylation and homodimerization, and have developed a class of salicylic acid-based inhibitors, which blocks the nuclear translocation-dependent canonical STAT3 signaling. In the present study, we demonstrated that the salicylic acid-based compound SH-4-54 was quite toxic to temozolomide (TMZ)-resistant GBM cells and could trigger apoptosis in these cells via enhancing mitochondrial translocation–dependent non-canonical STAT3 pathway. We demonstrated that incubation of TMZ-resistant GBM cells with SH-4-54 led to mitochondrial STAT3 (mitoSTAT3) activation and respiratory dysfunction reflected by disrupted (or suppressed) activities of oxidative phosphorylation complexes and oxygen consumption rate. Mechanistically, we proved that SH-4-54 could increase mitoSTAT3 transmembrane import via GRIM-19 and reinforce the association between mitoSTAT3 and mitochondrial transcription factor A (TFAM), indicating that SH-4-54 could facilitate the binding of mitoSTAT3 to mitochondria DNA (mtDNA) and negatively regulate mitochondrial-encoded genes, thus leading to the abnormal oxidation respiratory. Lastly, using GRIM-19 knockout cell line and subcutaneous xenotransplanted tumor model, we elaborately showed the enrichment of SH-4-54 in mitochondria by LC-MS/MS analysis. In conclusion, our data demonstrate thatthe salicylic acid-based compound SH-4-54 is quite effective in killing TMZ-resistant GBM cells and this cytotoxicity is attributed to mitoSTAT3 activation.

Downregulation of SNRPG induces cell cycle arrest and sensitizes human glioblastoma cells to temozolomide by targeting Myc through a p53-dependent signaling pathway.

Objective: Temozolomide (TMZ) is commonly used for glioblastoma multiforme (GBM) chemotherapy. However, drug resistance limits its therapeutic effect in GBM treatment. RNA-binding proteins (RBPs) have vital roles in posttranscriptional events. While disturbance of RBP-RNA network activity is potentially associated with cancer development, the precise mechanisms are not fully known. The SNRPG gene, encoding small nuclear ribonucleoprotein polypeptide G, was recently found to be related to cancer incidence, but its exact function has yet to be elucidated.Methods: SNRPG knockdown was achieved via short hairpin RNAs. Gene expression profiling and Western blot analyses were used to identify potential glioma cell growth signaling pathways affected by SNRPG. Xenograft tumors were examined to determine the carcinogenic effects of SNRPG on glioma tissues.Results: The SNRPG-mediated inhibitory effect on glioma cells might be due to the targeted prevention of Myc and p53. In addition, the effects of SNRPG loss on p53 levels and cell cycle progression were found to be Myc-dependent. Furthermore, SNRPG was increased in TMZ-resistant GBM cells, and downregulation of SNRPG potentially sensitized resistant cells to TMZ, suggesting that SNRPG deficiency decreases the chemoresistance of GBM cells to TMZ via the p53 signaling pathway. Our data confirmed that SNRPG suppression sensitizes GBM cells to TMZ by targeting Myc via the p53 signaling cascade.Conclusions: These results indicated that SNRPG is a probable molecular target of GBM and suggested that suppressing SNRPG in resistant GBM cells might be a substantially beneficial method for overcoming essential drug resistance.

Cycling Quiescence in Temozolomide Resistant Glioblastoma Cells Is Partly Explained by microRNA-93 and -193-Mediated Decrease of Cyclin D.

Glioblastoma multiforme (GBM) is a fatal malignancy of the central nervous system, commonly associated with chemoresistance. The alkylating agent Temozolomide (TMZ) is the front-line chemotherapeutic agent and has undergone intense studies on resistance. These studies reported on mismatch repair gene upregulation, ABC-targeted drug efflux, and cell cycle alterations. The mechanism by which TMZ induces cell cycle arrest has not been well-established. TMZ-resistant GBM cells have been linked to microRNA (miRNA) and exosomes. A cell cycle miRNA array identified distinct miRNAs only in exosomes from TMZ-resistant GBM cell lines and primary spheres. We narrowed the miRs to miR-93 and -193 and showed in computational analyses that they could target Cyclin D1. Since Cyclin D1 is a major regulator of cell cycle progression, we performed cause-effect studies and showed a blunting effects of miR-93 and -193 in Cyclin D1 expression. These two miRs also decreased cell cycling quiescence and induced resistance to TMZ. Taken together, our data provide a mechanism by which GBM cells can exhibit TMZ-induced resistance through miRNA targeting of Cyclin D1. The data provide a number of therapeutic approaches to reverse chemoresistance at the miRNA, exosomal and cell cycle points.

Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma.

Glioblastoma Multiforme (GBM) is still an incurable disease. The front-line Temozolomide (TMZ)-based therapy suffers from poor efficacy, underlining the need of new therapies.Preclinically, Aldoxorubicin (Aldox), a novel prodrug of Doxorubicin (Dox), has been successfully tested against GBM, encouraging the study of its association with other agents.For the first time, we evaluated the effectiveness of Aldox combined to TMZ in preclinical models of GBM.Our in vitro results demonstrated that the anti–glioma effect of Aldox was more marked than TMZ and their combination increased the killing effect of the anthracycline in TMZ-resistant GBM cells. Moreover, unlike Dox, Aldox was able to accumulate in P-glycoprotein (P-gp)-overexpressed cells due to a negative regulation of the P-gp function.We also compared efficacy and safety of weekly administrations of Aldox (16 mg/kg), with or without TMZ (0.9 mg/kg, daily injections), in the U87 xenograft mouse model.Aldox therapy induced a moderate tumor volume inhibition (TVI) and an increased survival rate (+12.5% vs vehicle). On the other hand, when combined to TMZ, Aldox caused a significant TVI (P=0.0175 vs vehicle) and delayed the mortality during the experimental period, although TVI and endpoint survival percentage (+37.5% vs vehicle) were not significantly different from TMZ alone.Our preliminary data showed that Aldox exerts anti–glioma effects in vitro and in vivo. It also enhances its antitumor activity when combined with TMZ, resulting in a superior efficacy compared to the single agents, without adverse side effects.

Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma

Chemoresistance blunts the effect of Temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Whether exosomal transfer of miRNAs derived from TMZ-resistant GBM cells could confer TMZ resistance remains to be determined. qPCR was used to determine miR-151a expression in two TMZ-resistant GBM cell lines. The direct targets of miR-151a were identified by microarray assays, bioinformatics and further RNA chromatin immunoprecipitation (RNA-ChIP) assay. We characterized exosomes from TMZ-resistant cell lines, serum and cerebrospinal fluid (CSF) and determined the effect of exosomes from TMZ-resistant cells on recipient GBM cells. miR-151a loss drove the acquisition of TMZ resistance. Restored miR-151a expression sensitized TMZ-resistant GBM cells via inhibiting XRCC4-mediated DNA repair. TMZ-resistant GBM cells conferred TMZ chemoresistance to recipient TMZ-sensitive cells in an exosomal miR-151a loss-dependent manner. Restoration of exosomal miR-151a from donor TMZ-resistant cells abolished the chemoresistance dissemination that was directed by donor TMZ-resistant cells. CSF-derived exosomes contained miRNA signatures reflective of the underlying chemoresistant status of GBMs in terms of miR-151a expression levels. Exosomal miR-151a is not only essentially a less-invasive ‘liquid biopsy’ that might predict chemotherapy response, but also represents a promising therapeutic target for therapy-refractory GBMs.

P.038 Investigating the role of long non-coding RNAs in glioblastoma multiforme

Background: Malignant gliomas are the most common and deadly brain tumors. Mean survival rate for a patient diagnosed with a glioblastoma multiforme (GBM) remains slightly over one year. Standard of care consists of treatment with temozolomide (TMZ) and radiotherapy. Recent work has highlighted functions of long non-coding RNAs (lncRNAs) in GBM progression and TMZ response even though the information regarding these newly discovered molecules is sparse. The overarching objective of this project was thus to assess the expression of select lncRNAs in GBM tumor samples and in models of TMZ resistance. Methods: A qRT-PCR-based approach was undertaken to measure six lncRNAs in 19 primary GBM samples, four GBM cell lines and in-house developed TMZ-resistant GBM cells. Results: Elevated levels of Hotair and H19 were observed in primary GBM tumors while decreased expression of MEG3 was recorded in the same samples. Interestingly, levels of PANDA increased 3.4-fold in GBM cells resistant to TMZ when compared with their sensitive counterparts. Conclusions: Overall, this work provides evidence of lncRNA deregulation in GBM tumors and reveals a previously unexplored lncRNA potentially involved in TMZ resistance. Modulation of lncRNA targets via RNAi-mediated approaches is envisioned to clarify their function and to strengthen their position as therapeutic options in GBMs.

LncRNA NEAT1 is involved in temozolomide resistance by regulating MGMT in glioblastoma multiforme

Introduction: As one of the most aggressive and lethal tumors, glioblastoma multiforme (GBM) is commonly treated by surgical resection combined with radiotherapy and chemotherapy. Temozolomide (TMZ) is the preferred chemotherapy medicine against GBM. However, recurrent GBM patients exhibit TMZ resistance. As reported in previous studies, NEAT1 is over-expressed in glioma cells; thus, we explored the relationship between NEAT1 and TMZ resistance in GBM. Materials and Methods: The expression levels of NEAT1 and O -6-methylguanine-DNA methyltransferase (MGMT) in GBM tissues and cells were determined by quantitative real-time PCR. Si-RNA and the over-expression vector were transfected into GBM cells to modulate the level of related molecules. Western blot analysis was used to investigate the protein expression of MGMT. The cell viability and IC-50 were analyzed by CCK-8, and apoptosis was detected by flow cytometry assay. Result: The expression level of NEAT1 in the TMZ-sensitive GBM tissues and cells was lower than that in the TMZ-resistant GBM tissues and cells. Furthermore, due to the down-regulation of NEAT1 in TMZ-resistant GBM cells transfected with Si-RNA, the viability and IC-50 value of the GBM cell lines were decreased, and the knockdown of NEAT1 significantly enhanced TMZ-induced cell apoptosis in GBM cells. We also determined that the mRNA and protein level of MGMT were up-regulated in TMZ-resistant GBM cells. Interference with MGMT expression led to a decrease in viability and IC-50 value in the GBM cells, and the knockdown of NEAT1 suppressed the transcription and translation levels of MGMT. However, the over-expression of MGMT enhanced TMZ resistance in NEAT1-silenced U87 and U251 cells. Conclusion: NEAT1 participates in the TMZ resistance of GBM cells by regulating MGMT.

Quinacrine enhances temozolomide cytotoxicity in temozolomide-sensitive and -resistant glioblastoma cells

Background: The alkylating agent temozolomide (TMZ) is widely used in glioblastoma multiforme (GBM) therapy. Unfortunately, TMZ-resistance frequently occurs in recurrent GBM and is the major cause of treatment failure. The anti-malarial drug quinacrine (QC) harbors antitumor and chemosensitivity properties, but its interactions with TMZ in GBM remain unclear. This study aimed to investigate whether QC would sensitize TMZ in TMZ-sensitive and TMZ-resistant GBM cells as well as the underlying mechanisms. Materials and Methods: The cytotoxicity of QC and TMZ in TMZ-sensitive and TMZ-resistant GBM cells was evaluated using in vitro cell viability assay and colony formation assay. Cellular apoptosis and protein expression levels were determined using TUNEL assay and immunoblotting, respectively. Results: QC substantially enhanced TMZ cytotoxicity in both TMZ-sensitive and TMZ-resistant cells. Such cytotoxic effect was accompanied by changes in the expression levels of LC3II, p62 and cleaved caspase 3, and increased cellular apoptosis. The results suggested that QC could sensitize GBM cells to TMZ at least partially through apoptosis induction, in which autophagy inhibition might be involved. Conclusion: The antimalarial drug QC may hold promise as a potentiation of TMZ treatment in GBM, especially in cases of TMZ-resistance.