Human Glioma Cell Lines Research Articles

Biological analyses of the effects of TiO2 and PEG-b-PLA nanoparticles on three-dimensional spheroid-based tumor.

The aim of our study was to monitor the antiproliferative/ cytotoxic and genotoxic effects of both, poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) and titanium dioxide (TiO2) nanoparticles on the tumor (HT-29, MCF-7, U118MG) and healthy (HEK-293T) cell lines during 2D cultivation and during cultivation in the spheroid form (3D cultivation). Cells or spheroids were cultivated with nanoparticles (0.01, 0.1, 1, 10, 50, and 100 ?g/ml) for 72 hours. The cytotoxic effect was determined by the MTT test and the genotoxic effect by the comet assay. We found that 2D cultivation of tumor cell lines with PEG-b-PLA and TiO2 nanoparticles had an anti-proliferative effect on human colon cancer cell line HT-29, human breast cancer cell line MCF-7, human glioma cell line U-118MG during 72h cultivation, but not on control/healthy HEK-293T cells. At the concentrations used, the tested nanoparticles caused no cytotoxic effect on tumor cell lines. Nanoparticles PEG-b-PLA induced significant damage to DNA in HT-29 and MCF-7 cells, while TiO2 nanoparticles in MCF-7 and U-118MG cells. Only PEG-b-PLA nanoparticles caused cytotoxic (IC50 = 7 mikrog/ml) and genotoxic effects on the healthy cell line HEK-293T after 72h cultivation. The cells which were cultivated in spheroid forms were more sensitive to both types of nanoparticles. After 72h cultivation, we observed the cytotoxic effect on both, the tumor and healthy cell lines.

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. We identified ADAMTS3 as a potential therapeutic target for GBM.

Characterization of LTr1 derived from cruciferous vegetables as a novel anti-glioma agent via inhibiting TrkA/PI3K/AKT pathway.

Malignant glioma is the most fatal, invasive brain cancer with limited treatment options. Our previous studies show that 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr1), a major metabolite of indole-3-carbinol (I3C) derived from cruciferous vegetables, produces anti-tumour effect against various tumour cell lines. In this study we characterized LTr1 as a novel anti-glioma agent. Based on screening 134 natural compounds and comparing the candidates' efficacy and toxicity, LTr1 was selected as the lead compound. We showed that LTr1 potently inhibited the viability of human glioma cell lines (SHG-44, U87, and U251) with IC50 values of 1.97, 1.84, and 2.03 μM, respectively. Furthermore, administration of LTr1 (100,300 mg· kg-1 ·d-1, i.g. for 18 days) dose-dependently suppressed the tumour growth in a U87 xenograft nude mouse model. We demonstrated that LTr1 directly bound with TrkA to inhibit its kinase activity and the downstream PI3K/AKT pathway thus inducing significant S-phase cell cycle arrest and apoptosis in SHG-44 and U87 cells by activating the mitochondrial pathway and inducing the production of reactive oxygen species (ROS). Importantly, LTr1 could cross the blood-brain barrier to achieve the therapeutic concentration in the brain. Taken together, LTr1 is a safe and promising therapeutic agent against glioma through inhibiting TrkA/PI3K/AKT pathway.

New class of fused [3,2-b][1,2,4]triazolothiazoles for targeting glioma in vitro

Glioma is aggressive malignant tumor with limited therapeutic interventions. Herein we report the synthesis of fused bicyclic 1,2,4-triazolothiazoles by a one-pot multi-component approach and their activity against C6 rat and LN18 human glioma cell lines. The target compounds 2-(6-phenylthiazolo[3,2–b][1,2,4]triazol-2-yl) isoindoline-1,3-diones and (E)-1-phenyl-N-(6-phenylthiazolo[3,2–b][1,2,4]triazol-2-yl) methanimines were obtained by the reaction of 5-amino-4H-1,2,4-triazole-3-thiol with substituted phenacyl bromide, phthalic anhydride, and different aromatic aldehydes in EtOH/HCl under reflux conditions. In C6 rat glioma cell lines, compounds 4g and 6i showed good cytotoxic activity with IC50 values of 8.09 and 8.74 μM, respectively, resulting in G1 and G2-M phase arrest of the cell cycle and activation of apoptosis by modulating phosphorylation of ERK and AKT pathway.

TB-3 A GINGER EXTRACT COMPOUND A REVEALED ANTITUMOR ACTIVITY AGAINST GLIOMA

Abstract Purpose Malignant neoplasms arising in the brain and central nervous system have a poor prognosis and present with symptoms such as headache, epileptic seizures, and paralysis of the arms and legs. Treatment of glioma, the most frequent primary brain tumor, is based on surgical removal of the tumor, radiation therapy, and chemotherapy. However, since gliomas grow invasively, surgical removal of the entire tumor is considered difficult. In addition, the standard treatment, temozolomide, possesses the problem of resistance. Therefore, the risk of recurrence is high, and the development of noble therapeutic agents for Glioma is required. We have screened for compounds with anti-tumor activity against Glioma from our natural compound library and focused on ginger extract, Compound A. In this study, we investigated the effect of Compound A on Glioma cell lines. Methods and Results To evaluate the antitumor activity of Compound A on Glioma in vitro , MTT assay was performed. Human glioma cell lines U87-MG and U251 cells were treated with Compound A. After 96 hours, cell viability was evaluated using CCK-8. The IC50 value of Compound A in U87-MG cells was calculated to be 16.3 μM. The IC50 of Compound A in U251 cells was 10.8 μM. Next, to examine the effect of Compound A on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. The MTT assay showed no decrease in cell viability in primary cultured rat astrocytes at 15 μM, whereas an antitumor effect was observed in a human glioma cell line. Discussion In this study, Compound A showed antitumor activity against Glioma without affecting normal cells. In the future, we will examine the effects of Compound A in an immunocompromised mouse brain in which U87-MG-RFP cells are implanted, which may lead to the development of therapeutic agents against Glioma.

Combined bulk RNA-seq and single-cell RNA-seq identifies a necroptosis-related prognostic signature associated with inhibitory immune microenvironment in glioma.

Necroptosis is a programmed cell death playing a significant role in cancer. Although necroptosis has been related to tumor immune environment (TIME) remodeling and cancer prognosis, however, the role of necroptosis-related genes (NRGs) in glioma is still elusive. In this study, a total of 159 NRGs were obtained, and parameters such as mutation rate, copy number variation (CNV), and relative expression level were assessed. Then, we constructed an 18-NRGs-based necroptosis-related signature (NRS) in the TCGA dataset, which could predict the patient's prognosis and was validated in two external CGGA datasets. We also explored the correlation between NRS and glioma TIME, chemotherapy sensitivity, and certain immunotherapy-related factors. The two necroptosis-related subtypes were discovered and could also distinguish the patients' prognosis. Through the glioblastoma (GBM) scRNA-seq data analysis, NRGs' expression levels in different GBM patient tissue cell subsets were investigated and the relative necroptosis status of different cell subsets was assessed, with the microglia score culminating among all. Moreover, we found a high infiltration level of immunosuppressive cells in glioma TIME, which was associated with poor prognosis in the high-NRS glioma patient group. Finally, the necroptosis suppressor CASP8 exhibited a high expression in glioma and was associated with poor prognosis. Subsequent experiments were performed in human glioma cell lines and patients' tissue specimens to verify the bioinformatic analytic findings about CASP8. Altogether, this study provides comprehensive evidence revealing a prognostic value of NRGs in glioma, which is associated with TIME regulation.

TMIC-12. COMPLEMENT SIGNALING IN HYPOXIC NICHES OF THE GLIOMA MICROENVIRONMENT

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.

EXTH-74. ATR INHIBITION IN EXPERIMENTAL GLIOMA

Abstract Glioblastoma are incurable primary tumors of the central nervous system. Novel treatment options are urgently needed. Recently, we investigated molecular effects of disrupting the (b)HLH network, a frequently altered and tumor-promoting transcriptional network in glioma, by introducing a dominant-negative variant of the E47 protein. The identified downstream anti-tumor effects included the DNA damage response pathway. In the present study, we aimed at investigating the anti-glioma effects of ATR inhibition in vitro and in vivo with a particular focus on treatment-induced vulnerabilities and potential combination therapies. We performed acute cytotoxicity, clonogenic survival assays, flow cytometry-based determination of cell cycle status and apoptosis induction in human and murine glioma cell lines in vitro, and investigated tumor growth in the SMA560/VMDk syngeneic mouse model in vivo. Furthermore, we applied RNA sequencing, DigiWest protein profiling analyses and genome-wide CRISPR/Cas9 loss-of function and activation screens to determine potential combination therapies. Combination treatments were further analyzed for synergism by the Bliss and ZIP synergy models. ATR inhibition in glioma cells resulted in reduced viability and clonogenic survival. However, differential patterns of gene expression deregulation (e.g. p53 signaling) and differing modes of cell cycle arrest in distinct glioma model systems argue for a substantial heterogeneity with regard to response to ATR inhibition. Based on our data we designed combinatorial treatments. We will present novel insights into promising combinatorial treatments using ATR inhibition and their underlying molecular mechanisms.

IMMU-27. SIMULTANEOUS TARGETING OF THE PD-L1 AND THE TGF-Β PATHWAYS WITH BINTRAFUSP ALFA IN EXPERIMENTAL GLIOMAS

Abstract Currently, immunotherapy is one of the most promising research areas in oncology, but immunotherapeutic strategies do not have proven clinical activity against glioblastoma so far. The immunosuppressive microenvironment of gliomas limits an appropriate anti-tumor immune response and represents a major challenge in the context of immunotherapy. Therefore, novel therapeutic strategies that reactivate the immune system and allow for potent anti-glioma immune responses are needed. Bintrafusp alfa is a bifunctional fusion protein that is composed of the extracellular domain of human transforming growth factor-beta receptor type II (TGF-βRII) and a human IgG1 monoclonal antibody blocking programmed death-ligand 1 (PD-L1). It is designed to target the TGF-β and the PDL-1 pathways simultaneously by specifically localizing to the tumor via the anti-PD-L1 moiety and sequestering TGF-β within the tumor microenvironment. We analyzed interrelations between the immunosuppressive factors TGF-β and PD-L1 in glioblastoma samples from patients and observed a significant correlation that points towards a co-regulation of both targets as part of a gene expression network. Next, we assessed the activity of bintrafusp alfa in human and murine glioma cell lines in vitro and observed interference with TGF-β-induced Smad2 phosphorylation as a bona fide response marker of TGF-β pathway activation and an increase in immune cell-mediated glioma cell killing. In vivo, bintrafusp alfa prolonged the survival of glioma-bearing immunocompetent mice, whereas the survival benefit was absent in immunodeficient Rag1-/- mice lacking T and B cell populations. Characterization of the tumor microenvironment by immunohistochemistry and high-dimensional flow cytometry confirmed the immune-activating effects of bintrafusp alfa. In summary, these encouraging results demonstrate that novel treatment options such as bintrafusp alfa may have the potential to overcome the lack of immunogenicity and reinvigorate anti-tumor immune responses to efficiently combat glioblastoma.

Propofol inhibits glioma progression by regulating circMAPK4/miR-622/HOXA9 axis.

Propofol has a tumor-suppressive role in glioma, but the mechanism by which propofol is involved in glioma progression is largely unknown. This study aims to explore a potential circular RNAs (circRNAs)/microRNAs (miRNAs)/mRNA network in response to Propofol in glioma. Human glioma cell lines (U251 and LN229) were suffered from Propofol treatment (5μg/mL for 24h) and transfection. circRNA mitogen-activated protein kinase 4 (circMAPK4), miR-622, homeobox A9 (HOXA9) abundances were determined by quantitative reverse transcription polymerase chain reaction and western blot. Migration and invasion were analyzed via transwell analysis. Cell proliferation was evaluated using Cell Counting Kit-8 and colony formation analysis. Cell apoptosis and related protein expression were determined via flow cytometry and western blot. Target relationship was assessed via dual-luciferase reporter analysis, RNA pull-down and RNA immunoprecipitation. Propofol reduced circMAPK4 expression. Propofol inhibited cell proliferation, migration and invasion, while increased apoptosis via decreasing circMAPK4 in glioma cells. miR-622 was targeted via circMAPK4. circMAPK4 knockdown decreased glioma cell growth, migration and invasion by up-regulating miR-622. miR-622 knockdown reversed the effect of Propofol on glioma progression. HOXA9 was targeted by miR-622, and its expression was decreased by Propofol treatment. miR-622 overexpression restrained glioma progression via decreasing HOXA9. Propofol regulated circMAPK4/miR-622/HOXA9 axis in glioma cells. Propofol constrains glioma progression by regulating circMAPK4/miR-622/HOXA9 axis in vitro. Propofol restrains glioma cell growth, migration and invasion. circMAPK4 can regulate HOXA9 by sponging miR-622 in glioma cells. Propofol represses glioma progression via a circMAPK4/miR-622/HOXA9 axis.

Irreversible Electroporation Mediates Glioma Apoptosis via Upregulation of AP-1 and Bim: Transcriptome Evidence.

The heat-sink effect and thermal damage of conventional thermal ablative technologies can be minimized by irreversible electroporation (IRE), which results in clear ablative boundaries and conservation of blood vessels, facilitating maximal safe surgical resection for glioblastoma. Although much comparative data about the death forms in IRE have been published, the comprehensive genetic regulatory mechanism for apoptosis, among other forms of regulatory cell death (RCD), remains elusive. We investigated the electric field intensity threshold for apoptosis/necrosis (YO-PRO-1/PI co-staining) of the U251 human malignant glioma cell line with stepwise increased uniform field intensity. Time course samples (0-6 h) of apoptosis induction and sham treatment were collected for transcriptome sequencing. Sequencing showed that transcription factor AP-1 and its target gene Bim (Bcl2l11), related to the signaling pathway, played a major role in the apoptosis of glioma after IRE. The sequencing results were confirmed by qPCR and Western blot. We also found that the transcription changes also implicated three other forms of RCD: autophagy, necroptosis, and immunogenic cell death (ICD), in addition to apoptosis. These together imply that IRE possibly mediates apoptosis by the AP-1-Bim pathway, causes mixed RCD simultaneously, and has the potential to aid in the generation of a systemic antitumor immune response.

Prefoldin 6 promotes glioma progression via the AKT signalling pathway.

Gliomas are one of the most aggressive primary tumours, accounting for 81% of malignant brain tumours, and are associated with a significant mortality. Therefore, the elucidation of the molecular mechanism underlying glioma progression and identification of promising treatment targets are necessary. Here, the expression of prefoldin (PFDN) 6 in human glioma tissues and cell lines was evaluated using immunohistochemistry and quantitative polymerase chain reaction. Celigo and CCK-8 assays were performed for assessing cell viability. Flow cytometry was used to analyse apoptosis and cell cycle distribution. Wound-healing and transwell assays were performed to observe cell migration. Lastly, xenograft models were developed for the in vivo validation of the results, and a human phospho-kinase array was used to explore the downstream signalling pathways. PFDN6 was upregulated in gliomas, and PFDN6 overexpression was significantly correlated with a low survival rate, estimated glomerular filtration rate (EGFR) expression, and tumour grade and recurrence. Moreover, PFDN6 knockdown significantly attenuated cell proliferation and migration, induced apoptosis, and blocked cell cycle progression in the G2 phase, which was further confirmed in the in vivo experiments. Mechanistically, the effects of PFDN6 may be mediated via the AKT signalling pathway. In conclusion, we showed that PFDN6 promotes glioma development by activating AKT signalling and emphasised the potential of PFDN6 as a crucial target in glioma therapy.

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.

CEP-1347 Targets MDM4 Protein Expression to Activate p53 and Inhibit the Growth of Glioma Cells.

The development of pharmacological inhibitors targeting negative regulators of p53, such as murine double minute (MDM) 2 and, more recently, MDM4, has been actively pursued as a potential strategy to treat cancers with wild-type p53. We previously showed that CEP-1347, a small molecule kinase inhibitor originally developed for the treatment of Parkinson's disease, suppressed MDM4 expression and activated wild-type p53 in retinoblastoma cells. However, it remains unknown whether CEP-1347 acts as an MDM4 inhibitor and as such activates p53 in other types of human cancer cells. The effects of CEP-1347 and MDM4 knockdown on the mRNA and protein expression of components of the p53 pathway, including MDM4, in human glioma cell lines with and without p53 mutation were examined by RT-PCR and western blot analyses. Trypan blue dye exclusion was used to examine the effect of CEP-1347 on cell growth. CEP-1347 decreased the expression of MDM4, increase that of p53, and activated the p53 pathway in glioma cells with wild-type p53. Knockdown-mediated inhibition of MDM4 expression in a glioma cell line with wild-type p53 that overexpresses MDM4 resulted in increased p53 expression and activation of the p53 pathway. CEP-1347 preferentially inhibited the growth of glioma cells with wild-type p53 without showing toxicity to normal cells at clinically relevant concentrations. Our findings suggest CEP-1347 is a novel inhibitor of MDM4 protein expression and as such activates p53 to inhibit the growth of cancer cells with wild-type p53, including retinoblastoma and glioblastoma.

Lin-28 Homolog B-Activated Protein Disulfide Isomerase A4 Regulates Cell Proliferation, Migration and Invasion of Glioma

The goal of this study is to elucidate the role of protein disulfide isomerase A4 (PDIA4) in glioma, as well as its regulatory mechanism. Cell transfection was performed to adjust the expression level of PDIA4 and RNA-binding protein lin-28 homolog B (LIN28B). The expression of PDIA4 in human astrocytes and glioma cell lines was determined by quantitative real-time PCR and western blot. CCK-8, colony formation, Transwell and wound-healing assays were applied to determine the capabilities of cells to proliferate, invade and migrate. The connection between PDIA4 and LIN28B was demonstrated by RNA immunoprecipitation (RIP) and RNA pull down assays. As a result, PDIA4 was elevated in glioma. PDIA4 depletion hugely suppressed cell proliferative ability, which was characterized by the reduced cell viability and colony formation, and declined contents of PCNA and Ki67. Meanwhile, PDIA4 knockdown repressed the cell capabilities to migrate and invade, accompanied with downregulated MMP2 and MMP9. LIN28N was also found to be upregulated in glioma cells, and was verified to bind with PDIA4 and positively regulate PDIA4 expression. Additionally, LIN28B overexpression partly hindered the suppressive impacts of PDIA4 knockdown on cell abilities to proliferate, migrate and invade. In conclusion, this study delineates that LIN28B-mediated PDIA4 plays a critical role in the progression of glioma.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Effect of glioma-derived immunoglobulin on biological function of glioma cells

IntroductionGlioma is the most common and most invasive primary central nervous system tumour, and it is urgent to develop new specific therapeutic targets. Studies have confirmed that epithelial-derived tumour cells promote tumour cell proliferation and metastasis by secreting a large number of immunoglobulins (Igs), but the role of tumour-derived Igs in glioma has never been reported. MethodsThe Gene Expression Profiling Interactive Analysis and Chinese Glioma Genome Atlas databases were used to analyse the Ig transcription and its correlation with the prognosis of patients with glioma. Immunohistochemistry and immunofluorescence were used to detect the protein expression of IgG and IgM in the glioma tissues of patients and glioma cell lines. When IgG was knocked down by small interfering RNA or knocked out by CRISPR-Cas9, the function of proliferation and migration of glioma cells were analysed by CCK-8, clone formation, wound healing, and transwell assays. Changes in proteins and their phosphorylation in signalling pathways were detected by western blotting. The nude mouse subcutaneous tumour-bearing model was established to analyse the effect of IgG in vivo. ResultsThe transcriptional level of IgG was pretty high in glioma tissues and was positively correlated with high WHO grade, recurrence, and poor prognosis. The expression of IgG and IgM was found in tumour tissues and human glioma cell lines U87 and U251, and the main expression form was secreted. Decreased IgG inhibited the proliferation and migration of glioma cells. Knockout or knockdown of IgG downregulated the phosphorylation of the key molecules in the MAPK and PI3K/Akt pathway through the HGF/SF-Met or FAK/Src pathway. In vivo tumourigenesis mouse model confirmed that reduced IgG expression inhibited glioma growth. ConclusionIg was expressed in glioma tissues and cell lines, and a high expression level predicted a poor prognosis of patients. Glioma-derived IgG promoted glioma cell proliferation and migration through the HGF/SF-Met or FAK/Src pathway.

P10.19.B An immunotoxin targeting CD317 for the treatment of glioblastoma

Abstract Background CD317 is an interferon-inducible cell surface receptor expressed in several solid cancer types. HM1.24-ETA’ is a small immunotoxin with a CD317 single-chain variable fragment (svFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’) that is explored as a novel therapeutic approach in CD317-expressing tumors. Material and Methods CD317 mRNA expression in human gliomas and its association with survival was analyzed using the database of the Cancer Genome Atlas (TCGA). CD317 protein levels in human gliomas were assessed by immunohistochemistry. CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in 13 human glioma cell lines in vitro. Efficacy of HM1.24-ETA’ was analyzed in acute cytotoxicity assays in vitro. Finally, HM1.24-ETA’ was evaluated in the intracranial human LN-229 glioma xenograft nude mouse model after intravenous injection. Results Interrogation of the TCGA database showed that increased CD317 mRNA expression correlated with grade of malignancy among isocitrate dehydrogenase (IDH) wildtype and IDH-mutant gliomas. Enhanced CD317 mRNA expression was associated with inferior survival in glioblastoma and astrocytoma, IDH-mutant, WHO grade 4. Immunohistochemistry confirmed CD317 overexpression in human glioblastoma compared to lower grade astrocytomas. CD317 was expressed heterogeneously on mRNA and protein levels in glioma cell lines in vitro. HM1.24-ETA’ induced acute cytotoxicity in CD317-positive glioma cells in vitro. CD317 expression and susceptibility to HM1.24-ETA’-induced cell death were enhanced by interferon-β. HM1.24-ETA’ prolonged survival in the LN-229 xenograft nude mouse model. Conclusion These data define CD317 as a novel target for treatment of human gliomas with immunoconjugates.

CASP6 predicts poor prognosis in glioma and correlates with tumor immune microenvironment.

BackgroundGlioma is an aggressive tumor of the central nervous system. Caspase-6 (CASP6) plays a crucial role in cell pyroptosis and is a central protein involved in many cellular signaling pathways. However, the association between CASP6 and prognosis of glioma patients remains unclear.MethodsFour bioinformatic databases were analyzed to identify differentially expressed genes (DEGs) between glioma and healthy tissues. Eighty-one protein-coding pyroptosis-related genes (PRGs) were obtained from the GeneCards database. The pyroptosis-related DEGs (PRDEGs) were extracted from each dataset, and CASP6 was found to be aberrantly expressed in glioma. We then investigated the biological functions of CASP6 and the relationship between CASP6 expression and the tumor microenvironment and immunocyte infiltration. The half maximal inhibitory concentration of temozolomide and the response to immune checkpoint blockade in the high- and low-CASP6 expression groups were estimated using relevant bioinformatic algorithms. Quantitative real-time reverse transcription PCR and western blotting were carried out to confirm the different expression levels of CASP6 between human astrocytes and glioma cell lines (U251 and T98G). We determined the role of CASP6 in the tumorigenesis of glioma by knocking down CASP6 in U251 and T98G cell lines.ResultsWe found that CASP6 was overexpressed in glioma samples and in glioma cell lines. CASP6 expression in patients with glioma correlated negatively with overall survival. In addition, CASP6 expression correlated positively with the degree of glioma progression. Functional analysis indicated that CASP6 was primarily involved in the immune response and antigen processing and presentation. Patients with high CASP6 levels responded more favorably to temozolomide, while patients with low expression of CASP6 had a better response to immunotherapy. Finally, in vitro experiments showed that CASP6 knockdown inhibited glioma proliferation.ConclusionsThe pyroptosis-related gene CASP6 might represent a sensitive prognostic marker for patients with glioma and might predict their response of immunotherapy and temozolomide therapy. Our results might lead to more precise immunotherapeutic strategies for patients with glioma.

Chalcone 9X Contributed to Repressing Glioma Cell Growth and Migration and Inducing Cell Apoptosis by Reducing FOXM1 Expression In Vitro and Repressing Tumor Growth In Vivo.

Objective Natural and synthetic chalcones played roles in inflammation and cancers. Chalcone 9X was an aromatic ketone that was found to inhibit cell growth of hepatic cancer and lung cancer cells. In this study, we wanted to investigate the functions of Chalcone 9X in glioma. Materials and Methods Chemical Chalcone 9X was added in human glioma cell lines (U87 and T98G cells) and normal astrocyte cell lines (HA1800) with various concentrations (0 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L). CCK-8 assay was used to measure cell viability. Flow cytometric assay was used to measure cell apoptotic rates. Wound healing assay and transwell assay were used to measure cell invasion. RT-PCR was used to detect relative mRNA expressions, and the protein expressions were detected by western blot (WB) and immunohistochemical staining (IHC). Finally, nude mouse xenograft assay was performed to prove the effects of Chalcone 9X in vivo. Results Results revealed that Chalcone 9X treatment suppressed cell viability and cell migration capacity; it could also induce cell apoptosis in U87 and T98G cells with dose dependence. However, it had little cytotoxicity to normal astrocyte HA1800 cells. Moreover, Chalcone 9X treatment could repress the mRNA and protein expressions of FOXM1 in human glioma cell lines, which was an oncogene that could promote the progression and malignancy of glioma. In addition, FOXM1 overexpression dismissed the Chalcone 9X effects on cell proliferation, apoptosis, and migration in human glioma cell lines. Finally, in vivo assay showed that Chalcone 9X treatment repressed the expression of FOXM1, which inhibited the tumor growth of a xenograft model injected with U87 in nude mice. Conclusions In all, we found that Chalcone 9X could suppress cell proliferation and migration and induce cell apoptosis in human glioma cells, while it has little cytotoxicity to normal astrocyte cells. Therefore, we uncovered a novel way that Chalcone 9X could inhibit FOXM1 expression and repress the progression and biofunctions of glioma cells, which might be a potential therapeutic drug for treating human glioma.