LN229 Cells Research Articles

Inhibition of STAT3 signaling as critical molecular event in HUC-MSCs suppressed Glioblastoma Cells.

Objective: We investigated the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) supernatants on proliferation, migration, invasion, and apoptosis in glioblastoma (GBM) cell lines RG-2, U251, U87-MG, and LN-428, as well as their apoptosis and autophagy-mediated through IL-6/JAK2/STAT3 signaling pathway to explore the molecular mechanisms. Methods: In this study, RG-2, U251, U87-MG, and LN-428 cells were treated with 9 mg/ml HUC-MSCs supernatants. Their responses to HUC-MSCs supernatants treatment and the status of STAT3 signaling were analyzed by multiple experimental approaches to elucidate the importance of HUC-MSCs supernatants for GBM. Results: The results demonstrated that after treatment with HUC-MSCs supernatants, in vitro proliferation of RG-2, U251, U87-MG, and LN-428 cells were inhibited, and their sustained growth was also blocked. RG-2, U251, and U87-MG cells showed significant S phase accumulation, while LN-428 cells were blocked in G0/G1 phase. Their migratory invasive capacities were inhibited, and their apoptosis and autophagy ratios were increased. These effects were mediated through the IL-6/JAK2/STAT3 and its downstream signaling pathway. Conclusion: Our data showed that HUC-MSCs supernatants had anti-tumor effects on GBM cells. It inhibited the proliferation, migration, and invasion of GBM cells and promoted their apoptosis. Negative regulation of the IL-6/JAK2/STAT3 signaling pathway enhanced apoptosis and autophagy in tumor cells, thereby improving the therapeutic effect on GBM.

Enhanced Glycolysis Confers Resistance Against Photon but Not Carbon Ion Irradiation in Human Glioma Cell Lines.

Metabolic reprogramming is a key hallmark in various malignancies and poses a challenge in achieving success with various therapies. Enhanced glycolysis is known to confer resistance against photon irradiation while the tumor response to carbon ion irradiation (CII) has not been investigated. This study aimed to investigate the effects of enhanced glycolysis on the response of human glioma cell lines to CII compared to the response to X-rays. Glycolysis was stimulated using Dinitrophenol (DNP), a mild OXPHOS inhibitor, in three human glioma cell lines (U251, U87, and LN229) and assessed by monitoring glucose uptake and utilization as well as expression of regulators of glycolysis (glucose transporter protein type 1(Glut1), hexokinase-II (HKII), and Pyruvate Kinase-2 (PKM2). Radiation (X-rays and CII) induced loss of clonogenic survival growth inhibition and perturbations in cell cycle progression (G2+M block), cytogenetic damage (micronuclei formation), apoptosis, necrosis (reflecting interphase death), and cell migration (Scratch assay) were investigated as parameters of radiation response. DNP (1 mM) enhanced the expression levels of GLUT1, HKII, and PKM2 by 30-60% and glucose uptake as well as usage by nearly 3 folds in U251 cells suggesting the stimulation of glycolysis. Enhanced glycolysis attenuated the loss of clonogenic survival with D10 doses increasing by 20% to 65% in these cell lines, while no significant changes were noted following CII. Concomitantly, dose-dependent growth inhibition, and cytogenetic damage as well as apoptosis and necrosis induced by X-rays were also reduced by elevated glycolysis in U251 and LN229 cells by 20-50%. However, stimulation of glycolysis enhanced the X-ray-induced cell migration, while it had negligible effect on migration following CII. Our results suggest that enhanced glycolysis confers resistance against X-ray-induced cell death and migration, while it may not significantly alter the cellular responses to carbon ion irradiation.

Evaluating glioblastoma tumour sphere growth and migration in interaction with astrocytes using 3D collagen-hyaluronic acid hydrogels.

Glioblastoma (GB) is an astrocytic brain tumour with a low survival rate, partly because of its highly invasive nature. The GB tumour microenvironment (TME) includes its extracellular matrix (ECM), a variety of brain cell types, unique anatomical structures, and local mechanical cues. As such, researchers have attempted to create biomaterials and culture models that mimic features of TME complexity. Hydrogel materials have been particularly popular because they enable 3D cell culture and mimic TME mechanical properites and chemical composition. Here, we used a 3D collagen I-hyaluronic acid hydrogel material to explore interactions between GB cells and astrocytes, the normal cell type from which GB likely derives. We demonstrate three different spheroid culture configurations, including GB multi-spheres (i.e., GB and astrocyte cells in spheroid co-culture), GB-only mono-spheres cultured with astrocyte-conditioned media, and GB-only mono-spheres cultured with dispersed live or fixed astrocytes. Using U87 and LN229 GB cell lines and primary human astrocytes, we investigated material and experiment variability. We then used time-lapse fluorescence microscopy to measure invasive potential by characterizing the sphere size, migration capacity, and weight-averaged migration distance in these hydrogels. Finally, we developed methods to extract RNA for gene expression analysis from cells cultured in hydrogels. U87 and LN229 cells displayed different migration behaviors. U87 migration occurred primarily as single cells and was reduced with higher numbers of astrocytes in both multi-sphere and mono-sphere plus dispersed astrocyte cultures. In contrast, LN229 migration exhibited features of collective migration and was increased in monosphere plus dispersed astrocyte cultures. Gene expression studies indicated that the most differentially expressed genes in these co-cultures were CA9, HLA-DQA1, TMPRSS2, FPR1, OAS2, and KLRD1. Most differentially expressed genes were related to immune response, inflammation, and cytokine signalling, with greater influence on U87 than LN229. These data show that 3D in vitro hydrogel co-culture models can be used to reveal cell line specific differences in migration and to study differential GB-astrocyte crosstalk.

In vitro comparative cytotoxic assessment of pristine and carboxylic functionalized multiwalled carbon nanotubes on LN18 cells.

Multiwalled carbon nanotubes (MWCNTs) have been used in biomedical applications due to their ability to enter the cells. Carboxylic functionalization of MWCNT (MWCNT-COOH) is used to mitigate the toxicity of MWCNTs. Our study focuses on comparing the toxicity of MWCNT and MWCNT-COOH on the neuronal cells, LN18. Concentrations of 5, 10, 20, and 40 µg ml-1 were used for the study, and cytotoxicity was determined at 0, 1, 3, 6, 12, 24, and 48 h of incubation. Cell viability was assessed by Trypan Blue, MTT, and Live dead cell assays, and the oxidative stress produced was determined by reactive oxygen species (ROS) and Lipid peroxidation assays. MWCNT-COOH showed higher cell viability than MWCNT for 20 and 40 µg ml-1 at 24 and 48 h. This was also visually observed in the live dead cell imaging. However, at 48 h, the morphology of the cells appeared more stretched for all the concentrations of MWCNT and MWCNT-COOH in comparison to the control. A significant amount of ROS production can also be observed at the same concentration and time. Viability and oxidative stress results together revealed that MWCNT-COOH is less toxic when compared to MWCNT at longer incubation periods and higher concentrations. However, otherwise, the effect of both are comparable. A concentration of 5-10 µg ml-1 is ideal while using MWCNT and MWCNT-COOH as the toxicity is negligible. These findings can further be extended to various functionalizations of MWCNT for wider applications.

ONX-0914 Induces Apoptosis and Autophagy with p53 Regulation in Human Glioblastoma Cells.

Glioblastoma is believed to be one of the most aggressive brain tumors in the world. ONX-0914 (PR957) is a selective inhibitor of proteasome subunit beta type-8 (PSMB8). Previous studies have shown that inhibiting PSMB8 expression in glioblastoma reduces tumor progression. Therefore, this study aimed to determine whether ONX-0914 has antitumor effects on human glioblastoma. The results indicated that ONX-0914 treatment inhibited survival in LN229, GBM8401, and U87MG glioblastoma cells. Cell cycle analysis showed that ONX-0914 treatment caused cell cycle arrest at the G1 phase and apoptosis in glioblastoma cells. The protein expression of BCL-2 was reduced and PARP was cleaved after ONX-0914 treatment. Furthermore, the levels of p53 and phosphorylated p53 were increased by ONX-0914 treatment in glioblastoma cells. ONX-0914 also induced autophagy in glioblastoma cells. Furthermore, the p53 inhibitor pifithrin attenuated apoptosis but enhanced autophagy caused by ONX-0914. In an orthotopic mouse model, TMZ plus ONX-0914 reduced tumor progression better than the control or TMZ alone. These data suggest that ONX-0914 is a novel therapeutic drug for glioblastoma.

EXTH-65. SYNERGISTIC ONCOLYTIC EFFECTS OF TEMOZOLOMIDE AND ONCOMAGNETIC THERAPIES IN GLIOBLASTOMA

Abstract The noninvasive Oncomagnetic device developed in our laboratory selectively kills glioblastoma (GBM) and other cancer cells while sparing normal developing neurons and astrocytes in vitro. We have reported that brief 2 – 6 h of daily Oncomagnetic brain stimulation causes >30% reduction of contrast-enhanced tumor (CET) volume in an end-stage recurrent GBM patient after a ~30-day treatment. We have evidence that the spinning oscillating magnetic fields (sOMF) generated by this device disrupts electron transport in the mitochondrial respiratory chain, at least in part by inhibiting the activity of Complex II succinate dehydrogenase, leading to a marked increase in reactive oxygen species (ROS) and consequent cancer cell death. Here, we further studied the cellular effects of sOMF treatment alone and in combination with Temozolomide (TMZ) on GBM cell lines with other methods and investigated the response to Oncomagnetic monotherapy (OMT) in a mouse GBM model. We treated LN229 and U87 GBM cells with sOMF with and without a sub-therapeutic dose of TMZ. To study the anticancer effect of sOMF in vivo, we developed syngeneic GBM tumors in BALB/c mice by stereotactic intracranial injection of GL261WT cells. Our results show that cytotoxic effects of sOMF or TMZ alone, as measured by the MTT assay and reduced cell survival, are significantly potentiated by combining the two treatments. OMT of tumor-bearing mice using a whole-body sOMF stimulation method causes substantial retardation of tumor growth and reduction of the CET volume in 9.4 T MRI scans. To study the molecular pathways involved in the treatment response we are mapping alterations in various key biomarkers in treated tumor tissues and comparing them with those in untreated controls. These experiments will unravel the molecular mechanism of action underlying the response to OMT in GBM and help develop a sound rationale for an effective therapeutic protocol.

Carbon Ion Irradiation Downregulates Notch Signaling in Glioma Cell Lines, Impacting Cell Migration and Spheroid Formation.

Photon-based radiotherapy upregulates Notch signaling in cancer, leading to the acquisition of the stem cell phenotype and induction of invasion/migration, which contributes to the development of resistance to therapy. However, the effect of carbon ion radiotherapy (CIRT) on Notch signaling in glioma and its impact on stemness and migration is not explored yet. Human glioma cell lines (LN229 and U251), stable Notch1 intracellular domain (N1ICD) overexpressing phenotype of LN229 cells, and Notch inhibitor resistant LN229 cells (LN229R) were irradiated with either photon (X-rays) or (carbon ion irradiation) CII, and expressions of Notch signaling components were accessed by RT-PCR, Western blotting, and enzymatic assays and flow cytometry. Spheroid forming ability, cell migration, and clonogenic assay were used to evaluate the effect of modulated Notch signaling by irradiation. Our results show that X-ray irradiation induced the expression of Notch signaling components such as Notch receptors, target genes, and ADAM17 activity, while CII reduced it in glioma cell lines. The differential modulation of ADAM17 activity by CII and X-rays affected the cell surface levels of NOTCH1 and NOTCH2 receptors, as they were reduced by X-ray irradiation but increased in response to CII. Functionally, CII reduced the spheroid formation and migration of glioma cells, possibly by downregulating the N1ICD, as stable overexpression of N1ICD rescued these inhibitory effects of CII. Moreover, LN229R that are less reliant on Notch signaling for their survival showed less response to CII. Therefore, downregulation of Notch signaling resulting in the suppression of stemness and impaired cell migration by CII seen here may reduce tumor regrowth and disease dissemination, in addition to the well-established cytotoxic effects.

Spermidine/Spermine N1-Acetyltransferase 1 (SAT1)-A Potential Gene Target for Selective Sensitization of Glioblastoma Cells Using an Ionizable Lipid Nanoparticle to Deliver siRNA.

Spermidine/spermine N1-acetyltransferase 1 (SAT1) responsible for cell polyamine catabolism is overexpressed in glioblastoma multiforme (GB). Its role in tumor survival and promoting resistance towards radiation therapy has made it an interesting target for therapy. In this study, we prepared a lipid nanoparticle-based siRNA delivery system (LNP-siSAT1) to selectively knockdown (KD) SAT1 enzyme in a human glioblastoma cell line. The LNP-siSAT1 containing ionizable DODAP lipid was prepared following a microfluidics mixing method and the resulting nanoparticles had a hydrodynamic size of around 80 nm and a neutral surface charge. The LNP-siSAT1 effectively knocked down the SAT1 expression in U251, LN229, and 42MGBA GB cells, and other brain-relevant endothelial (hCMEC/D3), astrocyte (HA) and macrophage (ANA-1) cells at the mRNA and protein levels. SAT1 KD in U251 cells resulted in a 40% loss in cell viability. Furthermore, SAT1 KD in U251, LN229 and 42MGBA cells sensitized them towards radiation and chemotherapy treatments. In contrast, despite similar SAT1 KD in other brain-relevant cells no significant effect on cytotoxic response, either alone or in combination, was observed. A major roadblock for brain therapeutics is their ability to cross the highly restrictive blood-brain barrier (BBB) presented by the brain microcapillary endothelial cells. Here, we used the BBB circumventing approach to enhance the delivery of LNP-siSAT1 across a BBB cell culture model. A cadherin binding peptide (ADTC5) was used to transiently open the BBB tight junctions to promote paracellular diffusion of LNP-siSAT1. These results suggest LNP-siSAT1 may provide a safe and effective method for reducing SAT1 and sensitizing GB cells to radiation and chemotherapeutic agents.

Tumor Treating Fields (TTFields) Enhance the Efficacy of Temozolomide and Lomustine in Glioblastoma Cell Lines

<h3>Purpose/Objective(s)</h3> Temozolomide (TMZ) is part of the standard of care treatment for newly diagnosed glioblastoma (ndGBM), the most common primary malignant brain tumor in adults. However, 50% of patients do not respond to TMZ due to expression of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme involved in repair of TMZ-induced DNA damage. Tumor Treating Fields (TTFields) are alternating electric fields that display anti-mitotic effects on cancerous cells, and have also been shown to lower expression levels of proteins from the DNA repair BRCA pathway. Concurrent treatment with TMZ and TTFields demonstrated a major advance in treatment of patients with ndGBM, and was approved by the FDA in 2014. Recently, the addition of lomustine (CCNU) to TMZ demonstrated clinical benefit in ndGBM patients, with improved overall and progression free survival. The aim of the current study was to examine in GBM cells the effect of TTFields in conjunction with TMZ and CCNU. <h3>Materials/Methods</h3> U-87 MG, LN229, U118, and LN18 human GBM cell lines were treated with TTFields at an intensity of 0.83 V/cm RMS and frequency of 200 kHz using the inovitro system. Expression levels of MGMT in each cell line were confirmed by Western Blot. Effectiveness of TTFields concomitant with TMZ and/or CCNU was tested by measuring cell counts, colony formation, and apoptosis. Fluorescence microscopy detection of ɣH2AX was employed to measure levels of DNA double strand breaks. <h3>Results</h3> Application of TTFields with concurrent TMZ was additive in all cell lines, irrespective of MGMT expression levels. Effect of concomitant TTFields with CCNU was additive in cell lines with high MGMT expression, whereas it displayed tendency to synergism in cell lines with limited or null MGMT expression. TTFields also elevated the levels of DNA double strand breaks. TTFields in conjunction with both TMZ and CCNU further increased the efficacy of TMZ and CCNU or TTFields apart. <h3>Conclusion</h3> TTFields enhance the efficacy of TMZ and CCNU, with synergism seen for the later in cells with limited MGMT expression. The outcomes are in line with the BRCAness state induced by TTFields, as DNA damage induced by CCNU requires the BRCA pathway for repair, especially in the absence of MGMT. The results suggest a potential benefit of TTFields as concomitant treatment with TMZ/CCNU combination therapy in GBM.

Therapeutic Potential of Small Molecule Inhibitors of TBK1 in Glioma

<h3>Purpose/Objective(s)</h3> Previously, we identified TBK1 as a therapeutic target in glioma using a high-throughput molecular approach. TBK1 has been shown to play a critical role in both the inflammatory response and tumor progression through multiple signaling pathways, including interferon, NF-κB, PI3K-AKT, and Ras-ERK pathways. The goal of this study was to assess the therapeutic potential of targeting TBK1. We hypothesize that the use of small molecule inhibitors of TBK1 alone or in combination with temozolomide (TMZ) and/or radiation may be a promising novel therapeutic strategy in glioma. <h3>Materials/Methods</h3> TBK1 siRNAs and commercially available small molecule inhibitors of TBK1, Amlexanox (AML) and GSK-8612, were purchased for <i>in vitro</i> characterization. Cell survival and colony formation assays were performed using glioma cell lines (LN18, U87, GBM30). Radiation was delivered one hour post GSK-8612/AML/TMZ treatment. pTBK1, pIRF3, pIkBa, pp65, and pAKT expression were evaluated using Western blot. Cleaved PARP (Western blot) and Annexin –V staining (flow cytometry) were used to evaluate the effect on cell apoptosis. <h3>Results</h3> Knockdown of TBK1 via siRNAs or small molecular inhibitor (AML and GSK-8612) resulted in decreased cell viability and colony formation and increased apoptosis in LN18 and GBM30 cells in a dose- and time-dependent manner. Importantly, a synergistic effect was observed when cell lines were treated with AML or GSK-8612 in combination with TMZ and/or radiation. Further molecular analysis of interferon signaling pathway or the NF-κB signaling pathway revealed that GSK-8612 decreased phosphorylation of IRF3, p65, IkBa, and AKT <h3>Conclusion</h3> In conclusion, these results suggest that TBK1 may be a promising therapeutic target in gliomas by regulating the interferon and/or NF-κB signaling pathways. Importantly, these TBK1 inhibitors were found to enhance the efficacy of the current clinical standard of care (TMZ and radiation) <i>in vitro</i>. Due to the critical need for better therapeutic strategies for glioma patients, it is our hope that further evaluation and optimization of these inhibitors will result in an improved treatment option with enhanced patient outcomes. Future work will focus on <i>in vivo</i> studies with AML or GSK-8612 alone and in combination with TMZ and radiation, as well as characterizing the toxicity profile of this combination therapy.

Emodin coupled with high LET neutron beam-a novel approach to treat on glioblastoma.

The primary motivation of this investigative study is trying to find an alternative treatment that can be used to slow down or treat glioblastoma due to the witnessed toxic side effects of the current drugs coupled with limited effectiveness in overall treatment. Consequently, a Chinese plant extract emodin proves to play a critical role in this investigative study since results from the Western blot and the other accompanying assays for anti-cancer effects indicate that it cannot work a lot to suppress cell migration and possible invasion, but rather emodin can be combined with radiation to give desired outcomes. Our result shows that the kind of radiation which acts well with emodin is neutron radiation rather than gamma radiation. Emodin significantly enhanced the radiosensitivity of LN18 and LN428 cells to γ-rays through MTT assay and cell counting. Accordingly, exposure to neutron radiation in the presence of emodin induced apoptotic cell death and autophagic cell death to a significantly higher extent, and suppressed cell migration and invasiveness more robustly. These effects are presumably due to the ability of emodin to amplify the effective dose from neutron radiation more efficiently. Thus, the study below is one such trial towards new interventional discovery and development in relation to glioblastoma treatment.

UBE2T promotes glioblastoma malignancy through ubiquitination-mediated degradation of RPL6.

Glioblastoma (GBM) is the most frequent and aggressive malignant glioma. Due to patients' poor prognosis, it is of great clinical significance to determine new targets that may improve GBM treatment. In the present study, we showed that ubiquitin (Ub)-conjugating enzyme E2T (UBE2T) was significantly overexpressed in GBM and could promote proliferation, invasion, and inhibit apoptosis of GBM cells. Mechanistically, UBE2T functioned as the Ub enzyme of ribosomal protein L6 (RPL6) and induced the ubiquitination and degradation of RPL6 in an E3 ligase-independent manner through direct modification by K48-linked polyubiquitination, thus contributing to the malignant progression of GBM cells. Furthermore, inhibiting the expression of RPL6 by UBE2T could not only reduce the expression of wild-type p53,but also enhance the gain-of-function of mutant p53. Moreover, knockdown of UBE2T in LN229 cells obviously suppressed tumor growth in LN229 xenograft mouse models. Collectively, our study demonstrated that UBE2T promotes GBM malignancy through ubiquitination-mediated degradation of RPL6 regardless of the p53 mutation status. It will provide new candidates for molecular biomarkers and therapeutic targets for clinical application in GBM.

H3F3A K27M Mutation Promotes the Infiltrative Growth of High-Grade Glioma in Adults by Activating β-Catenin/USP1 Signaling.

Simple SummaryGliomas is a primary type of tumor in the central nervous system. High-grade glioma is a malignant cancerous disease and grows rapidly. This study reports the expression of H3.3K27M in high-grade glioma tissues and the association with malignant glioma cell behavior. Moreover, the results suggested that a high expression of H3.3K27M promotes the migration and invasion of glioma cells, leading to a poor prognosis by promoting the infiltration of glioma through aggravating aberrant activation of β-catenin signaling-driven pathway. H3F3A K27M (H3.3K27M) is a newly identified molecular pathological marker in glioma and is strongly correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating evidence has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade glioma in adults. We found that H3.3K27M was highly expressed at high levels in some high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not affect the growth of glioma cells in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we confirmed that H3.3K27M overexpression increased the levels of the β-catenin and p-β-catenin (Ser675) proteins, the ubiquitin-specific protease 1 (USP1) mRNA and protein levels, and the enhancer of zeste homolog 2 (EZH2) protein level. In addition, the β-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins and inhibited the increased migration and invasion caused by the H3.3K27M mutation. Overall, the H3.3K27M mutation in high-grade glioma is a potential biomarker for poor prognosis mainly due to the infiltration of glioma cells that is at least partially mediated by the β-catenin/USP1/EZH2 pathway.

Saikosaponin D improves chemosensitivity of glioblastoma by reducing the its stemness maintenance

ObjectiveChemotherapy is one of the important adjuvant methods for the treatment of glioblastoma (GBM), and chemotherapy resistance is a clinical problem that neurooncologists need to solve urgently. It is reported that Saikosaponin D (SSD), an active component of Bupleurum chinense, had various of antitumor activities and could also enhance the chemosensitivity of liver cancer and other tumors. However, it is not clear whether it has an effect on the chemosensitivity of glioma and its specific mechanism. MethodsThe CCK8 assay, Wound healing assay and Matrigel invasion assay were used to detect the effect of SSD on the phenotype of GBM cells. We detected the effect of SSD on the chemosensitivity of GSM by Flow cytometry, LDH content and MTT assay. Then, we used cell plate cloning, semi-quantitative PCR and western blotting experiments to detect the effect of SSD on the stem potential of GBM cells. Finally, the effect of SSD on the chemosensitivity of GBM and its potential mechanism were verified by nude mouse experiments in vivo. Resultsfirstly, we found that SSD could partially inhibit the malignant phenotype of LN-229 cells, including inhibiting migration, invasion and apoptosis, and increasing the apoptosis rate and lactate dehydrogenase (LDH) release of LN-229 cells under the treatment of temozolomide (TMZ), that is to say, increasing the chemotherapy effect of TMZ on the cells. In addition, we unexpectedly found that SSD could partially inhibit the colony forming ability of LN-229 cells, which directly related to the stemness maintenance potential of cancer stem cells. Subsequently, our results showed that SSD could inhibit the gene and protein expression of stemness factors (OCT4, SOX2, c-Myc and Klf4) in LN-229 cells. Finally, we verified that SSD could improve the chemotherapy effect of TMZ by inhibiting the stem potential of glioblastoma in vivo nude mice. Conclusionthis research can provide a certain theoretical basis for the application of SSD in the chemotherapy resistance of GBM and its mechanism of action, and provide a new hope for the clinical treatment of glioblastoma.

Expression and estrogen regulation of G protein-coupled estrogen receptor in human glioblastoma cells.

Glioblastoma (GB) is the most frequent primary brain tumor with a very poor prognosis. Sex hormones are crucial players in the development of GBs. 17 β-estradiol (E2) signaling is involved through its corresponding intracellular receptors [estrogen receptor α (ERα) and β (ERβ)] in GB cell proliferation and progression. E2 activates G-protein coupled estrogen receptor (GPER), leading to rapidly occurring effects, independently of gene transcription. GPER activation is involved in tumor progression in various cancer types. Currently, available data concerning the occurrence and role of GPER in GB are very limited. In the present study, it was observed that GPER was expressed in human brain tumor cell lines [U251 (astrocytoma-derived cell line), U87, LN229 and T98 (glioblastoma-derived cell line)]. Immunofluorescence assays revealed that GPER localizes in the plasma membrane, cytoplasm and nucleus. An in silico analysis identified two potential E2 response elements in the promoter region of the GPER gene. E2 increased GPER expression in the U251, U87 and LN229 cell lines. Molecular modeling data derived from in silico analysis predicted the three-dimensional conformation of GPER, and docking analysis identified potential binding sites of E2 and its specific agonist, G1. Taken together, these results indicate that GPER may be differentially expressed in human GB cell lines with E2 possibly upregulating GPER expression. The present study raises further questions about the implications of GPER-mediated E2 signaling in the biology of GBs.

Viral Particle-Mediated SAMHD1 Depletion Sensitizes Refractory Glioblastoma to DNA-Damaging Therapeutics by Impairing Homologous Recombination.

Simple SummaryGlioblastoma (GBM) is a lethal and common primary brain tumor that accounts for about 50% of all diagnosed malignant gliomas. Despite aggressive standard-of-care treatment of surgical resection followed by γ-irradiation (IR) and DNA alkylating agent temozolomide (TMZ), the average post-diagnosis survival time for a GBM patient remains at 15 months. This is mainly due to acquired resistance and limited therapeutic options. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) supports DNA double-strand break repair by promoting homologous recombination (HR) and it can be targeted to proteasomal degradation by viral protein X (Vpx). We aim to evaluate whether depleting SAMHD1 sensitizes refractory GBM to IR and TMZ, and the possibility of utilizing Vpx as therapeutic tool. We report that SAMHD1 is highly expressed in GBM. Vpx-mediated SAMHD1 depletion impaired HR and sensitized GBM cells to IR and TMZ. Our finding demonstrates the potential therapeutic benefit of targeting SAMHD1 with Vpx in GBM.The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1’s importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells’ sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.

ADAMDEC1 accelerates GBM progression via activation of the MMP2-related pathway.

The ADAM (a disintegrin and metalloprotease) gene-related family including ADAM, ADAMTS, and ADAM-like decysin-1 has been reported to play an important role in the pathogenesis of multiple diseases, including cancers (lung cancer, gliomas, colorectal cancer, and gastrointestinal cancer). However, its biological role in gliomas remains largely unknown. Here, we aimed to investigate the biological functions and potential mechanism of ADAMDEC1 in gliomas. The mRNA and protein expression levels of ADAMDEC1 were upregulated in glioma tissues and cell lines. ADAMDEC1 showed a phenomenon of “abundance and disappear” expression in gliomas and normal tissues in that the higher the expression of ADAMDEC1 presented, the higher the malignancy of gliomas and the worse the prognosis. High expression of ADAMDEC1 was associated with immune response. Knockdown of ADAMDEC1 could decrease the proliferation and colony-forming ability of LN229 cells, whereas ADAMDEC1 overexpression has opposite effects in LN229 cells in vitro. Furthermore, we identified that ADAMDEC1 accelerates GBM progression via the activation of the MMP2 pathway. In the present study, we found that the expression levels of ADAMDEC1 were significantly elevated compared with other ADAMs by analyzing the expression levels of ADAM family proteins in gliomas. This suggests that ADAMDEC1 has potential as a glioma clinical marker and immunotherapy target.

LACTB suppresses migration and invasion of glioblastoma via downregulating RHOC/Cofilin signaling pathway

Glioblastoma (GBM) is the most malignant tumor in human brain. High invasiveness of this tumor is the main reason causing treatment failure and recurrence. Previous study has found that LACTB is a novel tumor suppressor in breast cancer. Moreover, the function of LACTB in other tumors and mechanisms involving LACTB were also reported. However, the role and relevant mechanisms of LACTB in GBM invasion remains to be revealed. Our aim is to investigate the role LACTB in GBM migration and invasion. We found that LACTB was downregulated in gliomas compared to normal brain tissues. Overexpression of LACTB suppressed migration and invasion of LN229 and U87 cell lines. Mechanistically, LACTB overexpression downregulated the mesenchymal markers. Moreover, LACTB overexpression downregulated the expression of RHOC and inhibited RHOC/Cofilin signaling pathway. The study suggests that LACTB suppresses migration and invasion of GBM cell lines via downregulating RHOC/Cofilin signaling pathway. These findings suggest that LACTB may be a potential treatment target of GBM.

P10.10.A Tumor Treating Fields (TTFields), temozolomide and lomustine co-application is efficacious in glioblastoma cancer cell lines

Abstract Background Temozolomide (TMZ) is the standard of care chemotherapy for newly diagnosed glioblastoma (ndGBM), the most common primary malignant brain tumor in adults. However, 50% of patients do not respond to TMZ due to expression of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme involved in repair of TMZ-induced damage. Tumor Treating Fields (TTFields) are alternating electric fields that display anti-mitotic effects on cancerous cells, and have been shown to induce a state of BRCAness in various cancer types. Concurrent treatment with TMZ and TTFields demonstrated a major advance in treatment of patients with ndGBM, and was approved by the FDA in 2014. Recently, the addition of lomustine (CCNU) to TMZ demonstrated clinical benefit in ndGBM patients, with improved overall and progression free survival. The aim of the current study was to examine in GBM cells the effect of TTFields in conjunction with TMZ and CCNU. Materials and Methods U-87 MG, LN229, U118 and LN18 human GBM cell lines were tested for their MGMT expression levels, and treated with TTFields (200 kHz, of 0.83 V/cm RMS) for 72 h using the inovitro system. Efficacy of concomitant application of TTFields with TMZ and/or CCNU was tested by measuring cell count, colony formation, and apoptosis levels. Results U-87 MG and LN229 displayed no expression of MGMT, while U118 and LN18 expressed low and high levels of MGMT, respectively. Application of TMZ and TTFields resulted in increased cytotoxicity compared with each treatment alone, with an additive interaction seen in all examined cell lines. The cytotoxic effect resulting from co-application of CCNU with TTFields suggested a synergistic interaction between the two modalities for U-87 MG, LN229, and U118, an additivity for LN18. Concurrent TTFields/TMZ/CCNU was more efficacious than TTFields or TMZ/CCNU separately in all cell lines. Conclusions Application of TTFields with TMZ was additive, irrespective of MGMT expression levels, while TTFields with CCNU was additive when MGMT was plentiful, but displayed tendency to synergism when MGMT was absent or limited. These outcomes are in line with the BRCAness state induced by TTFields, as in the absence of MGMT, DNA damage induced by CCNU requires the BRCA pathway for repair. Application of TTFields together with TMZ and CCNU demonstrated increased efficacy, suggesting potential benefit of such therapy for ndGBM treatment.

Anticancer effects of OSW-1 on glioma cells via regulation of the PI3K/AKT signal pathway: A network pharmacology approach and experimental validation in vitro and in vivo.

Background: Gliomas are the most common primary intracranial malignant tumors with poor prognosis, despite the remarkable advances in medical technology that have been made. OSW-1, isolated from Ornithogalum saundersiae, possesses anticancer activity against various malignant cancer cells. However, the effects of OSW-1 on gliomas and its potential mechanisms remain unclear. Methods: Network pharmacology was employed for predicting potential key targets and mechanisms of the anticancer effects of OSW-1 on glioma. Experiments, including the Cell Counting Kit-8, colony formation, and flow cytometry, were performed to investigate how OSW-1 affects the biological behavior of glioma cells in vitro. Western blotting was used to detect changes in related proteins, such as those involved in the cell cycle, apoptosis, and signaling pathways. The nude mouse xenograft model was used to detect the effect of OSW-1 on inhibiting the proliferation of glioma cells in vivo. Results: An “OSW-1-Targets-Glioma” intersection network consisting of 151 intersecting genes was acquired to construct a “Protein–Protein Interaction network” and predict the top 10 core targets. According to the Kyoto Encyclopedia of Genes and Genomes pathway analysis, the PI3K/AKT signaling pathway was the top 3-ranked pathway, with 38 enriched intersecting genes. The glioma T98G and LN18 cell lines were used to verify the predictions. OSW-1 significantly inhibited the viability and proliferation of glioma cells in a dose- and time-dependent manner. Flow cytometry showed that OSW-1 arrested the cell cycle at the G2/M phase, and the apoptotic ratio of glioma cells increased significantly with increasing concentrations. Western blotting revealed that the expression levels of p-PI3K and p-AKT1 in glioma cells treated with OSW-1 were significantly lower than those in the controls; however, 740Y-P, a PI3K activator, significantly reversed the inactivation of the PI3K/AKT signaling pathway caused by OSW-1. Furthermore, the mouse xenograft model confirmed the suppressive effect of OSW-1 on tumor growth in vivo. Conclusion: OSW-1 is a promising anti-glioma chemotherapeutic drug owing to its anticancer effects via downregulation of the PI3K/AKT signaling pathway. However, OSW-1 still has a long way to go to become a real anti-glioma drug.