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PIMREG expression strongly correlates with cellular proliferation in both malignant and normal cells. Throughout embryo development, PIMREG expression is prominent in the central nervous system. Recent studies have described elevated PIMREG expression in different types of tumors, which correlates with patient survival and tumor aggressiveness. Given the emerging significance of PIMREG in carcinogenesis and its putative role in the context of the nervous system, we investigated the expression and function of PIMREG in gliomas, the most common primary brain tumors. We performed an extensive analysis of PIMREG expression in tumors samples from glioma patients. We then assessed the effects of PIMREG silencing and overexpression on the sensitivity of glioblastoma cell lines treated with genotoxic agents commonly used for treating patients and assessed for treatment response, proliferation and migration. Our analysis shows that glioblastoma exhibits the highest levels of PIMREG expression among all cancers analyzed and that elevated PIMREG expression is a biomarker for glioma progression and patient outcome. Moreover, PIMREG is induced by genotoxic agents, and its silencing renders glioblastoma cells sensitive to temozolomide treatment and affects ATR- and ATM-dependent signaling. Our data demonstrate that PIMREG is involved in DNA damage response and temozolomide resistance of glioblastoma cells and further supports a role for PIMREG in tumorigenesis. • PIMREG expression is a biomarker for glioma progression and patient outcome. • GBM exhibits the highest levels of PIMREG expression among all cancers analyzed. • PIMREG is upregulated upon cell exposure to genotoxic agents and DNA damage. • PIMREG is involved in DNA damage response (DDR) in GBM cells. • PIMREG silencing sensitizes GBM cells to TMZ treatment. [ABSTRACT FROM AUTHOR]

Tumor suppressor p53 are frequently mutated in glioblastomas (GBMs) and appears to contribute, in part, to resistance to temozolomide and therapeutic drugs. WW domain-containing oxidoreductase WWOX (FOR or WOX1) is a proapoptotic protein and is considered as a tumor suppressor. Loss of WWOX gene expression is frequently seen in malignant cancer cells due to promoter hypermethylation, genetic alterations, and translational blockade. Intriguingly, ectopic expression of wild type WWOX preferentially induces apoptosis in human glioblastoma cells harboring mutant p53. WWOX is known to physically bind and stabilize wild type p53. Here, we provide an overview for the updated knowledge in p53 and WWOX, and postulate a potential scenarios that wild type and mutant p53, or isoforms, modulate the apoptotic function of WWOX. We propose that triggering WWOX activation by therapeutic drugs under p53 functional deficiency is needed to overcome TMZ resistance and induce GBM cell death.

Temozolomide (TMZ) has remained the chemotherapy of choice in patients with glioblastoma multiforme (GBM) primarily due to the lack of more effective drugs. Tumors, however, quickly develop resistance to this line of treatment creating a critical need for alternative approaches and strategies to resensitize the cells. Withaferin A (WA), a steroidal lactone derived from several genera of the Solanaceae plant family has previously demonstrated potent anti-cancer activity in multiple tumor models. Here, we examine the effects of WA against TMZ-resistant GBM cells as a monotherapy and in combination with TMZ. WA prevented GBM cell proliferation by dose-dependent G2/M cell cycle arrest and cell death through both intrinsic and extrinsic apoptotic pathways. This effect correlated with depletion of principle proteins of the Akt/mTOR and MAPK survival and proliferation pathways with diminished phosphorylation of Akt, mTOR, and p70 S6K but compensatory activation of ERK1/2. Depletion of tyrosine kinase cell surface receptors c-Met, EGFR, and Her2 was also observed. WA demonstrated induction of N-acetyl-L-cysteine-repressible oxidative stress as measured directly and through a subsequent heat shock response with HSP32 and HSP70 upregulation and decreased HSF1. Finally, pretreatment of TMZ-resistant GBM cells with WA was associated with O6-methylguanine-DNA methyltransferase (MGMT) depletion which potentiated TMZ-mediated MGMT degradation. Combination treatment with both WA and TMZ resulted in resensitization of MGMT-mediated TMZ-resistance but not resistance through mismatch repair mutations. These studies suggest great clinical potential for the utilization of WA in TMZ-resistant GBM as both a monotherapy and a resensitizer in combination with the standard chemotherapeutic agent TMZ. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most frequently observed and aggressive type of high-grade malignant glioma. Temozolomide (TMZ) is the primary agent for GBM treatment. However, TMZ resistance remains a major challenge. In this study, we report that MDK is overexpressed in GBM, which leads to enhanced proliferation, apoptosis inhibition, increased invasion and TMZ resistance in GBM cells. It was also determined that MDK could significantly improve the stem-like properties of GBM cells. Mechanistically, MDK enhanced p-JNK through Notch1 and subsequently increased the expression of stemness markers, such as CD133 and Nanog, thereby promoting TMZ resistance. Finally, xenograft experiments and clinical sample analysis also demonstrated that MDK knockdown could significantly inhibit tumor growth in vivo , and the expression of MDK was positively correlated with Notch1, p-JNK and CD133. This study revealed that MDK induces TMZ resistance by improving the stem-like properties of GBM by upregulating the Notch1/p-JNK signaling pathway, which provides a possible target for therapeutic intervention of GBM, especially in TMZ-resistant GBM with high MDK expression., Competing Interests: None., (AJCR Copyright © 2022.)

Background: Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes., Methods: Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study., Results: Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico . In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients., Conclusions: In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Glioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear.Purpose: This study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells.Methods: CCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting.Results: Here we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway.Conclusion: We first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT+ GBM patients. [ABSTRACT FROM AUTHOR]

Simple Summary: This study reports a potential new drug—Cerebraca wafer—that is designed to deliver its active pharmaceutical ingredient, (Z)-n-butylidenephthalide (BP), directly into the surgical cavity created when a brain tumor is resected. The therapeutic mechanism of Cerebraca wafer was shown to involve the following: (1) an IC50 of BP against tumor stem cells four times lower than that of bis-chloroethylnitrosourea (BCNU); (2) a synergistic effect between BP and temozolomide (TMZ), as demonstrated by a reduction in O6-methylguanine-DNA-methyltransferase (MGMT) expression level; (3) BP inhibition of programmed cell death-ligand 1 (PD-L1) protein levels, thereby activating T-cell cytotoxicity and increasing interferon-gamma (IFN-γ) secretion. The implantation of Cerebraca wafer is safe, no drug-related adverse events (AEs) and serious AEs (SAEs) were found. The median overall survival (OS) of patients receiving high-dose Cerebraca wafer have exceeded 17.4 months, and a 100% progression-free survival (PFS) rate at six month was achieved. In sum, these findings demonstrate that the Cerebraca wafer has superior therapeutic effects to Gliadel wafer in recurrent high-grade gliomas. In recurrent glioblastoma, Gliadel wafer implantation after surgery has been shown to result in incomplete chemical removal of residual tumor and development of brain edema. Furthermore, temozolomide (TMZ) resistance caused by O6-methylguanine-DNA-methyltransferase (MGMT) activation and programmed cell death-ligand 1 (PD-L1) expression leads to immune-cold lesions that result in poorer prognosis. Cerebraca wafer, a biodegradable polymer containing (Z)-n-butylidenephthalide (BP), is designed to eliminate residual tumor after glioma resection. An open-label, one-arm study with four dose cohorts, involving a traditional 3 + 3 dose escalation clinical trial, of the Cerebraca wafer combined with TMZ on patients with recurrent high-grade glioma, was conducted. Of the 12 patients who receive implantation of Cerebraca wafer, there were no drug-related adverse events (AEs) or serious AEs (SAEs). The median overall survival (OS) of patients receiving low-dose Cerebraca wafer was 12 months in the group with >25% wafer coverage of the resected tumor, which is longer than OS duration in previously published studies (Gliadel wafer, 6.4 months). Patients who received high-dose Cerebraca wafer treatment had not yet died at the data cut-off date; a 100% progression-free survival (PFS) rate at six month was achieved, indicating the median OS of cohort IV was more than 17.4 months. In vitro study of the primary cells collected from the patients revealed that the IC50 of BP against tumor stem cells was four times lower than that of bis-chloroethylnitrosourea (BCNU). A synergistic effect between BP and TMZ was demonstrated by a reduction in MGMT expression. Furthermore, BP inhibited PD-L1 expression, thereby activating T-cell cytotoxicity and increasing interferon-gamma (IFN-γ) secretion. The better therapeutic effect of Cerebraca wafer on recurrent high-grade glioma could occur through re-sensitization of TMZ and reduction of PD-L1. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastomas are the deadliest brain tumours. The standard of care associates surgery, radio- and chemotherapy with Temozolomide as the reference drug. Despite this treatment, most of the tumours recur. The characterization of resistance mechanisms is of paramount importance to enable the proposal of more effective therapies. In this work we aimed to evaluate the molecular changes occurring during and after Temozolomide treatment in a glioma cell line. A high plasticity in the integrin repertoire exists in these cells. As an example, variations of the α5β1 integrin expression were observed with a reduction during the treatment and re-expression after removal of the drug. The association of integrin antagonists with p53 reactivators appears to be efficient in recurrent tumours. Specific integrins may thus be particularly targetable at different time points of glioblastoma treatment and combination therapies evaluated according to their time-dependent expression. Despite extensive treatment, glioblastoma inevitably recurs, leading to an overall survival of around 16 months. Understanding why and how tumours resist to radio/chemotherapies is crucial to overcome this unmet oncological challenge. Primary and acquired resistance to Temozolomide (TMZ), the standard-of-care chemotherapeutic drug, have been the subjects of several studies. This work aimed to evaluate molecular and phenotypic changes occurring during and after TMZ treatment in a glioblastoma cell model, the U87MG. These initially TMZ-sensitive cells acquire long-lasting resistance even after removal of the drug. Transcriptomic analysis revealed that profound changes occurred between parental and resistant cells, particularly at the level of the integrin repertoire. Focusing on α5β1 integrin, which we proposed earlier as a glioblastoma therapeutic target, we demonstrated that its expression was decreased in the presence of TMZ but restored after removal of the drug. In this glioblastoma model of recurrence, α5β1 integrin plays an important role in the proliferation and migration of tumoral cells. We also demonstrated that reactivating p53 by MDM2 inhibitors concomitantly with the inhibition of this integrin in recurrent cells may overcome the TMZ resistance. Our results may explain some integrin-based targeted therapy failure as integrin expressions are highly switchable during the time of treatment. We also propose an alternative way to alter the viability of recurrent glioblastoma cells expressing a high level of α5β1 integrin. [ABSTRACT FROM AUTHOR]

Background: 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.Purpose: 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.Results: 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.Conclusion: 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. [ABSTRACT FROM AUTHOR]

Resistance to temozolomide poses a major clinical challenge in glioblastoma multiforme treatment, and the mechanisms underlying the development of temozolomide resistance remain poorly understood. Enhanced DNA repair and mutagenesis can allow tumour cells to survive, contributing to resistance and tumour recurrence. Here, using recurrent temozolomide-refractory glioblastoma specimens, temozolomide-resistant cells, and resistant-xenograft models, we report that loss of miR-29c via c-Myc drives the acquisition of temozolomide resistance through enhancement of REV3L-mediated DNA repair and mutagenesis in glioblastoma. Importantly, disruption of c-Myc/miR-29c/REV3L signalling may have dual anticancer effects, sensitizing the resistant tumours to therapy as well as preventing the emergence of acquired temozolomide resistance. Our findings suggest a rationale for targeting the c-Myc/miR-29c/REV3L signalling pathway as a promising therapeutic approach for glioblastoma, even in recurrent, treatment-refractory settings. [ABSTRACT FROM AUTHOR]

Glioblastomas (GBM) often acquire resistance against temozolomide (TMZ) after continuous treatment and recur as TMZ-resistant GBM (TMZ-R-GBM). Lomustine (CCNU) and nimustine (ACNU), which were previously used as standard therapeutic agents against GBM before TMZ, have occasionally been used for the salvage therapy of TMZ-R-GBM; however, their efficacy has not yet been thoroughly examined. Therefore, we investigated the antitumor effects of CCNU and ACNU against TMZ-R-GBM. As a model of TMZ-R-GBM, TMZ resistant clones of human GBM cell lines (U87, U251MG, and U343MG) were established (TMZ-R-cells) by the culture of each GBM cells under continuous TMZ treatment, and the antitumor effects of TMZ, CCNU, or ACNU against these cells were analyzed in vitro and in vivo. As a result, although growth arrest and apoptosis were triggered in all TMZ-R-cells after the administration of each drug, the antitumor effects of TMZ against TMZ-R-cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ-R-cells as well as parental cells. It was also demonstrated that TMZ resistance of TMZ-R-cells was regulated at the initiation of DNA damage response. Furthermore, survival in mice was significantly prolonged by systemic treatment with CCNU or ACNU but not TMZ after implantation of TMZ-R-cells. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against TMZ-R-GBM., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O 6 -methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.

Temozolomide (TMZ) resistance is an important cause of clinical treatment failure and poor prognosis in gliomas. Increasing evidence indicates that cancer-derived exosomes contribute to chemoresistance; however, the specific contribution of glioma-derived exosomes remains unclear. The aim of this study was to explore the role and underlying mechanisms of exosomal macrophage migration inhibitory factor (MIF) on TMZ resistance in gliomas. We first demonstrated that MIF was upregulated in the exosomes of TMZ-resistant cells, engendering the transfer of TMZ resistance to sensitive cells. Our results indicated that exosomal MIF conferred TMZ resistance to sensitive cells through the enhancement of cell proliferation and the repression of cell apoptosis upon TMZ exposure. MIF knockdown enhanced TMZ sensitivity in resistant glioma cells by upregulating Metalloproteinase Inhibitor 3 (TIMP3) and subsequently suppressing the PI3K/AKT signaling pathway. Additionally, exosomal MIF promoted tumor growth and TMZ resistance of glioma cells in vivo , while IOS-1 (MIF inhibitor) promotes glioma TMZ sensitive in vivo . Taken together, our study demonstrated that exosome-mediated transfer of MIF enhanced TMZ resistance in glioma through downregulating TIMP3 and further activating the PI3K/AKT signaling pathway, highlighting a prognostic biomarker and promising therapeutic target for TMZ treatment in gliomas., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background: Glioblastoma (GB) is one of the most common (~30%) and lethal cancers of the central nervous system. Although new therapies are emerging, chemoresistance to treatment is one of the major challenges in cancer treatment. Brain cytoplasmic 200 (BC200) RNA, also known as BCYRN1, is a long noncoding RNA (lncRNA) that has recently emerged as one of the crucial members of the lncRNA family. BC200 atypical expression is observed in many human cancers. BC200 expression is higher in invasive cancers than in benign tumors. However, the clinical significance of BC200 and its effect on GB multiforme is still unexplored and remains unclear. Methods: BC200 expression in GB patients and cell lines were investigated through RT-qPCR, immunoblotting, and immunohistochemistry analysis. The biological importance of BC200 was investigated in vitro and in vivo through knockdown and overexpression. Bioinformatic analysis was performed to determine miRNAs associated with BC200 RNA. Results: Our findings revealed that in GB patients, BC200 RNA expression was higher in blood and tumor tissues than in normal tissues. BC200 RNA expression have a statistically significant difference between the IDH1 and P53 status. Moreover, the BC200 RNA expression was higher than both p53, a prognostic marker of glioma, and Ki-67, a reliable indicator of tumor cell proliferation activity. Overexpression and silencing of BC200 RNA both in vitro and in vivo significantly modulated the proliferation, self-renewal, pluripotency, and temozolomide (TMZ) chemo-resistance of GB cells. It was found that the expressions of BC200 were up-regulated and that of miR-218-5p were down-regulated in GB tissues and cells. miR-218-5p inhibited the expression of BC200. Conclusions: This study is the first to show that the molecular mechanism of BC200 promotes GB oncogenicity and TMZ resistance through miR-218-5p expression modulation. Thus, the noncoding RNA BC200/miR-218-5p signaling circuit is a potential clinical biomarker or therapeutic target for GB. [ABSTRACT FROM AUTHOR]

• Chronic HCMV infection led to increased cell proliferation and cell viability of LN 229 glioblastoma cells. • Chronic HCMV infection increased resistance to the chemotherapeutic temozolomide (TMZ) in LN 229 glioblastoma cells. • Increased expression of the IE1 protein could contribute to TMZ resistance in HCMV infected glioblastoma cells. • Chronic HCMV infection of the non-glioblastoma stem cell line LN 229 changed the cell phenotype over time. Glioblastoma multiforme (GBM) is the most aggressive form of primary human gliomas. While chemotherapy using the DNA alkylating agent temozolomide (TMZ) is a first line treatment for GBMs, the development of resistance to TMZ is a common limitation to successful treatment. Human Cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that establishes a lifelong infection latent infection in host haematopoetic cells, where lytic replication of the virus is silenced. HCMV can also establish a persistent infection in hosts, where low levels of virus are lytically produced. Furthermore, multiple studies have identified HCMV DNA and/or proteins in human GBM samples, and have shown that acute infection with HCMV confers a glioblastoma stem cell (GSC) phenotype, further supporting an oncomodulatory role for HCMV in GBM progression and severity. In this current study, we examined the long-term effects of HCMV persistence to cell viability, cell proliferation, and the development of TMZ resistance over time using a glioblastoma cell line known as LN-229. Persistent HCMV infections were established and maintained in this cell line for 30 weeks without the addition of new virus. Here, we report that HCMV persistence in this cell line resulted in increased cell viability, increased cell proliferation, and a marked resistance to the DNA alkylating agent, TMZ, over time, suggesting that low levels of lytically replicating HCMV could contribute to tumor progression in GBM. [ABSTRACT FROM AUTHOR]

The current standard first-line treatment for adult patients with newly diagnosed glioblastoma includes concurrent radiotherapy and daily oral temozolomide (TMZ), followed by adjuvant TMZ. As a prodrug, TMZ undergoes spontaneous hydrolysis generating a methylating agent. O6-methylguanine is considered the most preponderant toxic damage mechanism at therapeutically relevant TMZ doses, whereas MGMT, which encodes the O6-methylguanine-DNA methyltransferase DNA repair enzyme, is the most relevant resistance mechanism. Speculations on clinically relevant TMZ concentrations, cytotoxic and cytostatic effects of TMZ, and resistance mechanisms exist in the literature. Here, we raise the following principal issues: What are the clinically relevant TMZ concentrations in glioma patients, and which TMZ-induced molecular lesion(s) and corresponding resistance mechanism(s) are important for TMZ therapeutic effects at clinically relevant concentrations? According to clinical data from patients with glioblastoma, the mean peak TMZ concentrations in the peritumoral tissue might be much lower (around 5 µM) than usually used in in vitro research, and may represent only 20% of systemic drug levels. According to in vitro reports, single-dose TMZ at concentrations around 5 µM have minimal, if any, effect on apoptosis and/or senescence of glioblastoma cell lines. However, the clinically relevant concentrations of TMZ are sufficient to radiosensitize both MGMT-positive and -negative cell lines in vitro. It is speculated that a single DNA repair protein, MGMT, is highly efficient in protecting cells against TMZ toxicity. However, an endogenous level of MGMT protein expression is not universally correlated with TMZ responsiveness, and MGMT-independent mechanisms of TMZ resistance exist. [ABSTRACT FROM AUTHOR]

Background: Acquired chemoresistance is a major challenge in the clinical treatment of glioblastoma (GBM). Circular RNAs have been verified to play a role in tumor chemoresistance. However, the underlying mechanisms remain unclear. The aim of this study was to elucidate the potential role and molecular mechanism of circular (circ)RNA ADP-ribosylation factor GTPase activating proteins with Src homology 3 domain, ankyrin repeat and Pleckstrin homology domain 1 (circASAP1) in temozolomide (TMZ) resistance of GBM., Methods: We analyzed circRNA alterations in recurrent GBM tissues relative to primary GBM through RNA sequencing. Real-time quantitative reverse transcription PCR verified the expression of circASAP1 in tissues and cells. Knockdown and overexpressed plasmids were used to evaluate the effect of circASAP1 on GBM cell proliferation and TMZ-induced apoptosis. Mechanistically, fluorescent in situ hybridization, dual-luciferase reporter, and RNA immunoprecipitation assays were performed to confirm the regulatory network of circASAP1/miR-502-5p/neuroblastoma Ras (NRAS). An intracranial tumor model was used to verify our findings in vivo., Results: CircASAP1 expression was significantly upregulated in recurrent GBM tissues and TMZ-resistant cell lines. CircASAP1 overexpression enhanced GBM cell proliferation and TMZ resistance, which could be reduced by circASAP1 knockdown. Further experiments revealed that circASAP1 increased the expression of NRAS via sponging miR-502-5p. Moreover, circASAP1 depletion effectively restored the sensitivity of TMZ-resistant xenografts to TMZ treatment in vivo., Conclusions: Our data demonstrate that circASAP1 exerts regulatory functions in GBM and that competing endogenous (ce)RNA-mediated microRNA sequestration might be a potential therapeutic strategy for GBM treatment., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Our previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown., Methods: The distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1-treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance., Results: Overexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy., Conclusions: Our results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Objective: To establish a mouse model bearing orthotopic temozolomide (TMZ)-resistant glioma that mimics the development of drug resistance in gliomas in vivo ., Methods: Seventy-eight adult C57BL/6 mice were randomly divided into 6 groups ( n =13), including 3 TMZ induced groups with low, medium and high doses (5, 25, and 50 mg/kg, respectively) and 3 control groups. In each group, 5 mice were used for evaluating tumor size, 5 for observing survival, and 3 for collecting tumor tissues for primary cell culture. In low-dose TMZ induced group, 3 mice bearing orthotopic murine glioma GL261 cell xenografts received intraperitoneal injections of 5 mg/kg TMZ for 5 days followed by a 10-day washout period before collecting glioma tissues. Tumor cell suspensions were prepared and injected in the mice in the medium-dose group, which were treated with the same protocol but with an increased TMZ dose, and the tumor cells harvested from 3 mice were injected in the high-dose group. The mice bearing GL261 cell xenografts in the 3 control groups received no treatment or were injected with medium- or high-dose TMZ. Cell colony forming assay was used to assess TMZ resistance of each generation of the tumor cells; CCK8 assay was used to determine drug resistance index of the cells., Results: The mouse models bearing TMZresistant glioma was successfully established. The cells from the high-dose induced group showed a significantly higher colony-forming rate than those from the high-dose control group ( P < 0.05), and had a drug resistance 4.25 times higher than that of the cells from untreated control group. High-dose TMZ significantly reduced the tumor volume in the control group (P < 0.05) but not in the high-dose induced group ( P < 0.01). The survival time of the tumor-bearing mice was significantly shortened in the high-dose induced group ( P =0.0018)., Conclusions: Progressive increase of TMZ doses in mice bearing orthotopic gliomas can effectively induce TMZ resistance of the gliomas.

Glioblastoma is one of the most difficult cancers to treat with a 5-year overall survival rate less than 5%. Temozolomide (TMZ) is an effective drug for prolonging the overall survival time of patients, while drug-resistance is an important clinical problem at present. Pennogenin-3-α-L-rhamnopyranosyl-(1→4)-[α-Lrhamno-pyranosyl-(1→2)]- β-D-glucopyranoside (N45), a steroidal saponin, was isolated from the rhizomes of Paris vietnamensis (Takht.), which is used as a Traditional Chinese Medicine and has been reported to possess preclinical anticancer efficacy in various cancer types. However, the mechanism of the inhibition of N45 on glioblastoma cells and its possible application in the treatment of chemotherapy-resistant glioblastoma cells are still unknown. In this study, we use cellular methodological experiments including cell counting kit-8 (CCK-8) assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assay, flow cytometry assay, transmission electron microscopy (TEM) and Western blot. The results show that N45 significantly suppresses the proliferation of glioblastoma cells and TMZ-resistant glioblastoma cells (U87R) by inducing mitochondrial apoptosis through reactive oxygen species (ROS)/phosphoinositide 3-kinase (PI3K)/Akt signal pathway, and the N-acetyl-L-cysteine (NAC) combined with N45 effectively reduced N45-mediated apoptosis and reversed the inhibition of PI3K/Akt signal pathway. In addition, N45 decreased the drug-resistance by down-regulation of nuclear factor kappa-B p65 (NF-κB p65) to attenuate O 6 -methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant glioblastoma cells (U87R). Our findings proved that N45 might be a potential therapeutic agent against glioblastoma and TMZ-resistant glioblastoma, promising to be a potential agent to reduce drug resistance.

Background/aim: Histone deacetylase 6 (HDAC6) is considered as one of the most promising targets in drug development for cancer therapy. Drug resistance is a major cause of treatment failure in many cancers including glioblastoma (GBM), the most lethal malignant tumor. The role of HDAC6 in GBM resistance and its underlying mechanisms have not been well elucidated. Herein, we investigated the function of HDAC6 in modulating GBM resistance., Materials and Methods: The anticancer effects of four structurally distinct selective HDAC6 inhibitors were addressed using western blot, flow cytometry, CCK-8 assay, and CI in temozolomide (TMZ)-resistant GBM cells., Results: We showed that HDAC6-selecitve inhibitors block activation of the EGFR and p53 pathways in TMZ-resistant GBM cells. Importantly, the inhibition of HDAC6 correlates with increased levels of MSH2 and MSH6, key DNA mismatch repair proteins, in TMZ-resistant GBM cells. In addition to the MSH, HDAC6 inhibitors decrease MGMT expression in TMZ-resistant GBM cells. Furthermore, HDAC6 inhibitors increase TMZ sensitivity and efficiently induce apoptosis in TMZ-resistant GBM cells., Conclusion: Selective inhibition of HDAC6 may be a promising strategy for the treatment of TMZ-resistant GBM., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

To investigate the underlying mechanism by which glioblastoma (GBM) cells gain temozolomide (TMZ) resistance and to clarify novel therapeutic targets and new prognostic biomarkers for GBM. A genome-wide hierarchical bi-clustering based on previously published microarray databases identified Nuclear Factor I A (NFIA) as one of the most significantly upregulated genes correlated to TMZ resistance in GBM. Then, the potential biological functions of NFIA in oncogenesis and chemoresistance were clarified by qRT-PCR, Western blotting and in vivo xenograft models with artificially induced TMZ-resistant U87 cells. Additionally, immunohistochemistry (IHC) assays were performed to explore the clinical significance of NFIA in glioma patients. Last, luciferase reporter assay was performed to study the transcriptional regulation of NFIA on the nuclear factor κb (NF-kB) pathway. NFIA was correlated with TMZ resistance in GBM. Clinically, elevated NFIA expression was significantly correlated with adverse outcomes of glioma patients, especially in GBM patients. Moreover, NFIA contributed to the acquired TMZ resistance of GBM cells, while suppression of NFIA via lentivirus reduced cell proliferation, tumorigenesis and resistance to TMZ of GBM. Additionally, NFIA promoted transcription activity that regulated the expression of NF-kB. Last, NFIA induced phosphorylation of NF-kB p65 at serine 536, thus inducing TMZ resistance in GBM cells. Altogether, our study suggests that NFIA-dependent transcriptional regulation of NF-kB contributes to acquired TMZ resistance in GBM. Abnormally activated NFIA-NF-kB signaling was strongly correlated with acquired TMZ resistance and poor prognosis in GBM, and it could be a new therapeutic target for TMZ-resistant GBM. [ABSTRACT FROM AUTHOR]

Temozolomide is the current first-line treatment for glioblastoma patients but, because many patients are resistant to it, there is an urgent need to develop antitumor agents to treat temozolomide-resistant glioblastoma. Gossypol, a natural polyphenolic compound, has been studied as a monotherapy or combination therapy for the treatment of glioblastoma. The combination of gossypol and temozolomide has been shown to inhibit glioblastoma, but it is not clear yet whether gossypol alone can suppress temozolomide-resistant glioblastoma. We find that gossypol suppresses the growth of temozolomide-resistant glioblastoma cells in both tumor sphere and adherent culture conditions, with tumor spheres showing the greatest sensitivity. Molecular docking and binding energy calculations show that gossypol has a similar affinity to the Bcl2 (B-cell lymphoma 2) family of proteins and several dehydrogenases. Gossypol reduces mitochondrial membrane potential and cellular ATP levels before cell death, which suggests that gossypol inhibits several dehydrogenases in the cell's metabolic pathway. Treatment with a Bcl2 inhibitor does not fully explain the effect of gossypol on glioblastoma. Overall, this study demonstrates that gossypol can suppress temozolomide-resistant glioblastoma and will be helpful for the refinement of gossypol treatments by elucidating some of the molecular mechanisms of gossypol in glioblastoma. [ABSTRACT FROM AUTHOR]

The management of glioblastomas (GBMs) is challenged by the development of therapeutic resistance and early disease recurrence, despite multi-modal therapy. This may be attributed to the presence of glioma stem cells (GSCs) which are known to survive radio- and chemotherapy, by circumventing death signals and inducing cell re-population. Recent findings suggest GSCs may be enriched by certain treatment modality. These necessitate the development of novel therapeutics capable of targeting GBM cell plasticity and therapy-resistant GSCs. Here, aided by computer-assisted structure characterization and target identification, we predicted that a novel 5-(2′,4′-difluorophenyl)-salicylanilide derivative, LCC-09, could target dopamine receptors and oncogenic markers implicated in GBMs. Bioinformatics data have indicated that dopamine receptor (DRD) 2, DRD4, CD133 and Nestin were elevated in GBM clinical samples and correlated to TMZ (Temozolomide) resistance and increased ALDH (Aldehyde dehydrogenase) activity (3.5–8.9%) as well as enhanced (2.1–2.4-fold) neurosphere formation efficiency in U87MG and D54MG GBM cell lines. In addition, TMZ-resistant GSC phenotype was associated with up-regulated DRD4, Akt, mTOR, β-catenin, CDK6, NF-κB and Erk1/2 expression. LCC-09 alone, or combined with TMZ, suppressed the tumorigenic and stemness traits of TMZ-resistant GBM cells while concomitantly down-regulating DRD4, Akt, mTOR, β-catenin, Erk1/2, NF-κB, and CDK6 expression. Notably, LCC-09-mediated anti-GBM/GSC activities were associated with the re-expression of tumor suppressor miR-34a and reversal of TMZ-resistance, in vitro and in vivo. Collectively, these data lay the foundation for further exploration of the clinical feasibility of administering LCC-09 as single-agent or combinatorial therapy for patients with TMZ-resistant GBMs. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme represents one of the deadliest brain tumor types, manifested by a high rate of recurrence and poor prognosis. The presence of glioma stem cells (GSCs) can repopulate the tumor posttreatment and resist therapeutics. A better understanding of GSC biology is essential for developing more effective interventions. We established a CD133 promoter-driven dual reporter, expressing green fluorescent protein (GFP) and firefly luciferase (CD133-LG), capable for in vitro and in vivo imaging of CD133+ GSCs. We first demonstrated the reporter enabled in vitro analyses of GSCs. DBTRG-05MG (Denver Brain Tumor Research Group 05) carrying CD133-LG (DBTRG-05MG-CD133-LG) system reported increased GFP/luciferase activities in neurospheres. Additionally, we identified and isolated CD133+/GFP+ cells with increased tumorigenic properties, stemness markers, Notch1, β-catenin, and Bruton's tyrosine kinase (Btk). Furthermore, prolonged temozolomide (TMZ) treatment enriched GSCs (reflected by increased percentage of CD133+ cells). Subsequently, Btk inhibitor, ibrutinib, suppressed GSC generation and stemness markers. Finally, we demonstrated real-time evaluation of anti-GSC function of ibrutinib in vivo with TMZ-enriched GSCs. Tumorigenesis was noninvasively monitored by bioluminescence imaging and mice that received ibrutinib showed a significantly lower tumor burden, indicating ibrutinib as a potential GSC inhibitor. In conclusion, we established a dual optical imaging system which enables the identification of CD133+ GSCs and screening for anti-GSC drugs. [ABSTRACT FROM AUTHOR]

Background: Dysregulation of numerous lncRNAs has been recently confirmed in glioma; however, the majority of their roles and mechanisms involved in this notorious disease remain largely unclear. This study aims to explore the roles and molecular mechanisms of LINC01198 implicated in the proliferation and chemoresistance in glioma., Results: LINC01198 was elevated in glioma, and this predicted a poorer prognosis for patients with glioma. LINC01198 knockdown inhibited, while LINC01198 overexpression promoted, glioma cell proliferation and resistance to temozolomide. Mechanistically, NEDD4-1 (neural precursor cell expressed, developmentally downregulated 4, E3 ubiquitin protein ligase) and phosphatase and tensin homolog (PTEN) were recruited by LINC01198, which functioned as a scaffold. Moreover, we showed that LINC01198 exerted its oncogenic activities by enhancing the NEDD4-1-dependent repression of PTEN., Conclusions: Our study elucidated the role of oncogenic LINC01198 in glioma proliferation and temozolomide resistance, and this role may serve as a promising target for glioma therapy., Methods: LINC01198 expression in glioma tissues and that in paired normal tissues were measured by qRT-PCR. The functional roles of LINC01198 in glioma were demonstrated by a series of in vitro experiments. CCK-8 assay, RNA pulldown, RNA immunoprecipitation and western blotting were used to demonstrate the potential mechanisms of LINC01198.

Aim: To assess the effect of zinc-doped copper oxide nanocomposites (nZn-CuO NPs) on glioblastoma therapy., Materials & Methods: nZn-CuO NPs were synthesized by sonochemical method and its antitumor effects and underlying molecular mechanisms were investigated both in vitro and in vivo., Results: After nZn-CuO NPs treatment, cell proliferation was significantly inhibited in dividing cancer cells but less toxicity was observed in normal cells. In vivo studies show that nZn-CuO NPs inhibited tumor growth in a dose-dependent manner. Further study found that nZn-CuO NPs trigger cell reactive oxygen species (ROS) generation and intrinsic apoptotic pathway. In temozolomide resistance glioblastoma, nZn-CuO NPs disturb cell growth and sphere formation by inhibiting AKT and ERK1/2 activation., Conclusion: nZn-CuO NPs possess the potential to be developed as a novel anti-tumor agent, especially to treat temozolomide resistance glioblastoma.