Migration Of U251 Cells Research Articles

The Large GTPase Guanylate-Binding Protein-1 (GBP-1) Promotes Mitochondrial Fission in Glioblastoma.

Glioblastomas (aka Glioblastoma multiformes (GBMs)) are the most deadly of the adult brain tumors. Even with aggressive treatment, the prognosis is extremely poor. The large GTPase Guanylate-Binding Protein-1 (GBP-1) contributes to the poor prognosis of GBM by promoting migration and invasion. GBP-1 is substantially localized to the cytosolic side of the outer membrane of mitochondria in GBM cells. Because mitochondrial dynamics, particularly mitochondrial fission, can drive cell migration and invasion, the potential interactions between GBP-1 and mitochondrial dynamin-related protein 1 (Drp1) were explored. Drp1 is the major driver of mitochondrial fission. While GBP-1 and Drp1 both had punctate distributions within the cytoplasm and localized to regions of the cytoplasmic side of the plasma membrane of GBM cells, the proteins were only molecularly co-localized at the mitochondria. Subcellular fractionation showed that the presence of elevated GBP-1 promoted the movement of Drp1 from the cytosol to the mitochondria. The migration of U251 cells treated with the Drp1 inhibitor, Mdivi-1, was less inhibited in the cells with elevated GBP-1. Elevated GBP-1 in GBM cells resulted in shorter and wider mitochondria, most likely from mitochondrial fission. Mitochondrial fission can drive several important cellular processes, including cell migration, invasion, and metastasis.

Anticancer Properties of Different Varieties of Date Palm (Phoenix dactylifera L.) Leaf Extracts in Human Tumor Cells: a Comparative Study.

Plant polyphenols are nutraceutical components with relevant biological effects on human health. They act against development of several diseases including cancer. In this study, the methanolic extracts of four date palm Phoenix dactylifera leaves (Deglet Noor (DN), Barhee (B), Khalas (KS) and Khunezi (KZ)) collected from south Tunisia were preliminary analyzed for their effects against U87 (human glioblastoma) and MDA-MB-231 (human breast cancer) cell line development. Results showed that Barhee extract (30μg/mL) was the most efficient to reduce the growth of both tumor cells to about 40% (p < 0.05) without inducing cytotoxicity. Significantly, KS, KZ, DN and B extracts (30μg/mL) decreased MDA-MB-231 and U87 cell adhesion towards fibrinogen and fibronectin. Using integrin blocking antibodies, leaf extracts competitively decreased human glioblastoma cell attachment to immobilized antibodies by interfering to αvβ3 and α5β1 integrin receptors. At the same concentration, extracts decreased MDA-MB-23 and U87 cell migration performed with wound healing assay. Particularly, Barhee and Deglet Noor leaf extracts (30μg/mL) significantly reduced U87 cell invasion by 52.92% (p < 0.01) and 74.56% (p < 0.01), respectively. Collegially, our findings revealed beneficial proprieties of four varieties of date palm leaf especially those displayed by DN and B extracts that may serve as active candidates against human glioblastoma and breast cancer progression.

The mitochondria-related gene risk mode revealed p66Shc as a prognostic mitochondria-related gene of glioblastoma.

Numerous studies have highlighted the pivotal role of mitochondria-related genes (MRGs) in the initiation and progression of glioblastoma (GBM). However, the specific contributions of MRGs coding proteins to GBM pathology remain incompletely elucidated. The identification of prognostic MRGs in GBM holds promise for the development of personalized targeted therapies and the enhancement of patient prognosis. We combined differential expression with univariate Cox regression analysis to screen prognosis-associated MRGs in GBM. Based on the nine MRGs, the hazard ratio model was conducted using a multivariate Cox regression algorithm. SHC-related survival, pathway, and immune analyses in GBM cohorts were obtained from the Biomarker Exploration of the Solid Tumor database. The proliferation and migration of U87 cells were measured by CCK-8 and transwell assay. Apoptosis in U87 cells was evaluated using flow cytometry. Confocal microscopy was employed to measure mitochondrial reactive oxygen species (ROS) levels and morphology. The expression levels of SHC1 and other relevant proteins were examined via western blotting. We screened 15 prognosis-associated MRGs and constructed a 9 MRGs-based model. Validation of the model's risk score confirmed its efficacy in predicting the prognosis of patients with GBM. Furthermore, analysis revealed that SHC1, a constituent MRG of the prognostic model, was upregulated and implicated in the progression, migration, and immune infiltration of GBM. In vitro experiments elucidated that p66Shc, the longest isoform of SHC1, modulates mitochondrial ROS production and morphology, consequently promoting the proliferation and migration of U87 cells. The 9 MRGs-based prognostic model could predict the prognosis of GBM. SHC1 was upregulated and correlated with the prognosis of patients by involvement in immune infiltration. Furthermore, in vitro experiments demonstrated that p66Shc promotes U87 cell proliferation and migration by mediating mitochondrial ROS production. Thus, p66Shc may serve as a promising biomarker and therapeutic target for GBM.

Isocucurbitacin B inhibits glioma growth through PI3K/AKT pathways and increases glioma sensitivity to TMZ by inhibiting hsa-mir-1286a.

Aim: Glioma accounts for 81% of all cancers of the nervous system cancers and presents one of the most drug-resistant malignancies, resulting in a relatively high mortality rate. Despite extensive efforts, the complete treatment options for glioma remain elusive. The effect of isocucurbitacin B (isocuB), a natural compound extracted from melon pedicels, on glioma has not been investigated. This study aims to investigate the inhibitory effect of isocuB on glioma and elucidate its underlying mechanisms, with the objective of developing it as a potential therapeutic agent for glioma. Methods: We used network pharmacology and bioinformatics analysis to predict potential targets and associated pathways of isocuB in glioma. Subsequently, the inhibitory effect of isocuB on glioma and its related mechanisms were assessed through Counting Kit-8 (CCK-8), wound healing, transwell, Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and other in vitro experiments, alongside tumor formation experiments in nude mice. Results: Based on this investigation, it suggested that isocuB might inhibit the growth of gliomas through the PI3K-AKT and MAPK pathways. Additionally, we proposed that isocuB may enhance glioma drug sensitivity to temozolomide (TMZ) via modulation of hsa-mir-1286a. The CCK-8 assay revealed that isocuB exhibited inhibitory effects on U251 and U87 proliferation and outperformed TMZ. Wound healing and transwell experiments showed that isocuB inhibited the invasion and migration of U251 cells by suppressing the activity of MMP-2/9, N-cadherin, and Vimentin. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) and flow cytometry (FCM) assays revealed that isocuB induced cell apoptosis through inhibition of BCL-2. Subsequently, we conducted RT-qPCR and WB experiments, which revealed that PI3K/AKT and MAPK pathways might be involved in the mechanism of the inhibition isocuB on glioma. Additionally, isocuB promoted the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a. Furthermore, we constructed TMZ-resistant U251 strains and demonstrated effective inhibition by isocuB against these resistant strains. Finally, we confirmed that isocuB can inhibit tumor growth in vivo through experiments on tumors in nude mice. Conclusion: IsocuB may protect against glioma by acting on the PI3K/AKT and MAPK pathways and promote the sensitivity of glioma U251 to TMZ by inhibiting hsa-mir-1286a.

Glioblastoma Cells Use an Integrin- and CD44-Mediated Motor-Clutch Mode of Migration in Brain Tissue.

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells. Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch. The online version contains supplementary material available at 10.1007/s12195-024-00799-x.

Auraptene inhibits migration, invasion and metastatic behavior of human malignant glioblastoma cells: An in vitro and in silico study.

The present work examined the anti-metastatic effects of auraptene and their underlying mechanisms of action in U87 Glioblastoma multiforme (GBM) cells. To test the hypothesis, cell culture, Matrigel invasion assay, scratch wound healing assay, gelatin zymography assay, qRT-PCR, and western blot experiments were conducted. At sublethal concentrations of 12.5 and 25 µg/ml, auraptene exhibited a significant reduction in cell invasion and migration of U87 cells, as assessed using scratch wound healing and Transwell tests, respectively. The qRT-PCR and zymography experiments demonstrated a significant decrease in both mRNA expression and activities of MMP-2 and MMP-9 following auraptene treatment. Western blot analysis also showed that MMP-2 protein level and phosphorylation of metastasis-related proteins (p-JNK and p-mTOR) decreased in auraptene-treated cells. Molecular docking studies consistently demonstrated that auraptene exhibits a significant affinity towards MMP-2/-9, the ATP binding site of mTOR and JNK1/2/3. Auraptene inhibited the migration and invasion of GBM cells. This inhibitory effect was induced by modulating specific mechanisms, including suppressing MMPs, JNK, and mTOR activities.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Radiosynthesis and in-vitro identification of a molecular probe 131I-FAPI targeting cancer-associated fibroblasts.

Fibroblast activation protein (FAP) is highly expressed in the mesenchyme of most malignant epithelial tumors, while its expression is low in normal tissues. FAP inhibitors (FAPIs) bind specifically to FAP and are used for tumor-targeted diagnosis and therapy. The aim of this study was to radiosynthesize a novel molecular probe 131I-FAPI and evaluate its in-vitro targeting and biological characteristics. The structurally modified FAPI was labelled with 131I through the chloramine-T method. The radiolabeling rate was then detected by thin-layer chromatography (TLC). The stability of 131I-FAPI was determined at PBS (room temperature) and serum (37°C). Its hydrophilicity was calculated by measuring its lipid-water partition coefficient. Pancreatic cancer PANC-1 cell line and glioma U87 cell line were cultured in vitro. Cell uptake assay was used to show the binding ability of 131I-FAPI. The CCK-8 assay was used to calculate the inhibitory effects of 131I-FAPI at different time points (4h, 8h, 12h, 24h, 48h) after comparing with the 131I and FAPI. The before-and-after-24h scratch areas of the two cells were determined in order to verify the effect of 131I-FAPI on the migration ability of the cells. The radiolabeling rate was (84.9 ± 1.02) %. The radiochemical purity of 131I-FAPI remained over 80% in both 25°C PBS and 37°C serum. The value of the lipid-water partition coefficient was -0.869 ± 0.025, indicating the hydrophilic of the probe. The cellular uptake assay showed that U87 cells had a specific binding capacity for 131I-FAPI. In cell inhibition assays, the inhibitory effect of 131I-FAPI on U87 cells increased with time. The results of cell scratch assay showed that 131I-FAPI had the strongest inhibitory effect on the migratory ability of U87 cells compared with 131I and FAPI (P<0.001). 131I-FAPI was synthesized with good in-vitro stability and hydrophilic properties. It can be specifically bound by U87 cells. The proliferation and migration of U87 cells can be effectively inhibited. 131I-FAPI is promising to become a therapeutic probe.

Graphitic carbon nitride as a novel anticancer agent: potential mechanisms and efficacy in prostate cancer and glioblastoma treatment.

Carbon-derived compounds are gaining traction in the scientific community because of their unique properties, such as conductivity and strength, and promising innovations in technology and medicine. Graphitic nitride carbon (g-C3N4) stands out among these compounds because of its potential in antitumor therapies. This study aimed to assess g-C3N4's antitumor potential and cytotoxic mechanisms. Prostate cancer (DU-145) and glioblastoma (U87) cell lines were used to evaluate antitumor effects, whereas RAW 264.7 and HFF-1 non-tumor cells were used for selectivity evaluation. The synthesized g-C3N4 particles underwent comprehensive characterization, including the assessment of particle size, morphology, and oxygen content, employing various techniques, such as X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, transmission electron microscopy, and atomic force microscopy. The results indicated that g-C3N4 significantly affected tumor cell proliferation and viability, exhibiting high cytotoxicity within 48 h. In non-tumor cells, minimal effects on proliferation were observed, except for damage to the cell membranes of RAW 264.7 cells. Moreover, g-C3N4 changed the cell morphology and ultrastructure, affecting cell migration in U87 cells and potentially enhancing migration in RAW 264.7 cells. Biochemical assays in Balb/C mice revealed alterations in alanine aminotransferase, aspartate aminotransferase, and amylase levels. In conclusion, g-C3N4 demonstrated promising antitumor effects with minimal toxicity to non-tumor cells, suggesting its potential in neoplasm treatment.

The tertiary lymphoid structure-related signature identified PTGDS in regulating PD-L1 and promoting the proliferation and migration of glioblastoma

BackgroundTertiary lymphoid structure (TLS) is a unique organ that carries out tumor cell elimination at tumor sites. It is continuously stimulated by inflammatory tumor signals and has been found to augment immunotherapy response. However, the detailed mechanisms behind it still need to be defined. MethodsTo explore and grasp the whole picture of TLS from a pan-cancer view, we collected nine TLS-related genes from previous studies. We performed a comprehensive analysis of 9637 samples across 33 tumor types accessed from The Cancer Genome Atlas (TCGA) database. EdU, Transwell, and flow cytometry were performed on the feature gene PTGDS in U251 cells. The regulatory role of PTGDS on PD-L1 expression and macrophage polarization was verified. ResultsAlteration analysis showed that mutations of TLS-related genes were widespread and relatively high. Clustering analysis based on the expression of these nine genes obtained two distinct clusters, with high EIF1AY and PTGDS in cluster 2 and better overall survival in cluster 1. To distinguish the two clusters, we utilized six machine learning algorithms and filtrated EIF1AY, PTGDS, SKAP1, and RBP5 as the characteristic genes, among which the former two genes were proved to be hazardous. PTGDS was found to regulate PD-L1 expression and also promoted the proliferation and migration of U251 cells. The knockdown of PTGDS could reduce the migration of macrophages and inhibit the polarization of macrophages into M2-phenotype. In addition, we established a TLS score to demonstrate patients’ TLS activity. The low TLS-score group overlapped with cluster 1 and displayed a better prognosis. Besides, the low TLS-score group was related to better immunotherapy responses. The HE staining of histopathological sections confirmed that the low TLS-score group exhibited higher infiltration of immune cells. ConclusionThis study reveals broad molecular, tumorigenic, and immunogenic signatures for further functional and therapeutic studies of tertiary lymphoid structure. The TLS score we established effectively predicted immunotherapy response and patients’ survival. Its future application and combination await more research.

A prognostic biomarker NRG1 promotes U-87 MG glioblastoma cell malignancy by inhibiting autophagy via ERBB2/AKT/mTOR pathway.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Studies have shown that autophagy-related (ATG) genes play important roles in regulating GBM malignancy. However, the mechanism still needs to be fully elucidated. Based on clinical and gene expression information of GBM patients downloaded from The The Cancer Genome Atlas database, Kaplan-Meier, univariate Cox regression, least absolute shrinkage and selection operator regression and multivariate Cox regression were applied to construct a risk signature for GBM prognosis, followed by validation using receiver operating characteristic analysis. Next, Cell Counting Kit-8, wound healing assay, flow cytometry, monodansyl cadaverine autophagy staining assay, immunofluorescence staining and western blot, either in the absence or presence of ERBB2/AKT/mTOR inhibitors, were carried out in GBM U87 cell line to explore molecular pathway underlying GBM malignancy. A three-ATG-gene signature (HIF1A, ITGA3, and NGR1) was constructed for GBM prognosis with the greatest contribution from NRG1. In vitro experiments showed that NRG1 promoted U87 cell migration and proliferation by inhibiting autophagy, and ERBB2/AKT/mTOR is a downstream pathway that mediates the autophagy-inhibitory effects of NRG1. We constructed an ATG gene prognostic model for GBM and demonstrated that NRG1 inhibited autophagy by activating ERBB2/AKT/mTOR, promoting GBM malignancy, thus providing new insights into the molecular contribution of autophagy in GBM malignancy.

Deciphering the molecular mechanism of the THBS1 gene in the TNF signaling axis in glioma stem cells

Glioma stem cells (GSCs) are thought to be responsible for the initiation and progression of glioblastoma (GBM). GBM presents highly invasive growth with a very high recurrence rate, so it has become a clinical problem to be solved urgently. RNAseq demonstrates that thrombospondin 1 (THBS1) acts not only in the angiogenic core of glioma but also with a high degree of invasiveness and infiltration. Nevertheless, defects in the signaling pathway research lead to a poor prognosis in glioma patients. To investigate the relevant molecular mechanism and signal pathway of glioma stem cell behavior mediated by THBS1, U251 astroglioma cells and GSCs were taken as model cells for in vitro experiments. The biological effects of THBS1 on glioma proliferation, migration, and adhesion were evaluated using Cell Counting Kit-8(CCK8) assays, EdU incorporation assays, migration assays, Transwell assays, Western blotting, and RNAseq. We found that the knockout of the THBS1 gene by CRISPR/Cas9 promoted proliferation and migration in U251 cells and GSCs, as well as influencing cell cycle progression by regulating the TNF/MAPK/NF-κB and TGF-β/Smad signaling pathways. Moreover, U251 cells and GSCs showed different responses to THBS1 knockout, suggesting specific and potential targets for GSCs in signaling pathways mediated by THBS1.

Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

Inhibition of glioblastoma proliferation, invasion, and migration by Urolithin Bthrough inducing G0/G1 arrest and targeting MMP-2/-9 expression and activity.

One kind of brain cancer with a dismal prognosis is called glioblastoma multiforme (GBM) due to its high growth rate and widespread tumor cell invasion into various areas of the brain. To improve therapeutic approaches, the objective of this research investigates the cytotoxic, anti-metastatic, and apoptotic effect of urolithin-B (UB) as a bioactive metabolite of ellagitannins (ETs) on GBM U87 cells. The malignant GBM cell line (U87) was examined for apoptosis rate, cell cycle analysis, cell viability, mRNA expressions of several apoptotic and metastasis-associated genes, production of reactive oxygen species (ROS), MMP-2, and MMP-9 activity and protein expression, and migration ability. The findings revealed that UB decreased U87 GBM viability in a dose-dependent manner and NIH/3T3 normal cells with the IC50 value of 30 and 55μM after 24 h, respectively. UB also induces necrosis and G0/G1 cell cycle arrest in U87 cells. UB also increases ROS production and caused down-regulation of Bcl2 and up-regulation of Bax apoptotic genes. Additionally, treatment of UB reduced the migration of U87 cells. The protein levels, mRNA expression, and the MMP-2 and MMP-9 enzyme activities also decreased concentration-dependently. So, due to the non-toxic nature of UB and its ability to induce apoptosis and reduce the U87 GBM cell invasion and migration, after more research, it can be regarded as a promising new anti-GBM compound.

Expression of eIF4E Gene in Glioma and Its Sensitivity to Oxidative Stress.

Objective Increased expression of eIF4E has been observed in various cancers, which makes eIF4E an attractive target of anticancer drugs. This study mainly discussed eIF4E gene expression in glioma and its sensitivity to oxidative stress (OS). Methods Relevant data from The Cancer Genome Atlas (TCGA) database regarding eIF4E gene expression and its prognostic significance in glioma samples were analyzed. Additionally, we measured eIF4E at mRNA and protein levels in clinical samples collected between July 2019 and September 2021, as well as glioma cell strains. U251 cells cultured in vitro were treated with OS injury induced by hydrogen peroxide (H2O2) and then transfected with si-eIF4E to determine changes in cell multiplication, invasiveness, and migration capacities as well as apoptosis rate. ELISA quantified cell malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) concentrations, and flow cytometry measured reactive oxygen species (ROS) level. Results In glioma samples from the TCGA database, eIF4E showed obviously elevated levels in LGG and GBM patients, which was usually associated with adverse patient prognosis (P < 0.05). eIF4E was also upregulated in glioma cell strains than in HBE cells. In comparison with the blank control group, transfection of si-eIF4E statistically suppressed the capacity of U251 cells to proliferate, invade and migrate, and enhance apoptosis rate, while reducing SOD and GSH-Px and increasing MDA and ROS. In addition, H2O2 induced the upregulation of eIF4E in U251 cells. H2O2 + si-eIF4E exhibited reduced multiplication and number of clone cell formation, invasion, and migration of U251 cells, as well as increased apoptosis rate than H2O2 + si-NC group. Conclusions eIF4E is highly expressed in glioma. Knocking down eIF4E can effectively inhibit the capacity of U251 to proliferate, invade and migrate, and significantly increase apoptosis. In addition, eIF4E knock-down is able to lower OS reaction under H2O2 inducement and enhance U251 cells' sensitivity to OS.

Nitrated Hsp90 Supports Glioblastoma Multiforme Cell Survival and Migration

Glioblastoma multiforme (GBM) is the most aggressive malignancy affecting the central nervous system. With no effective pharmacological options, patients undergo repeated surgeries to remove or debulk tumors. New targets for the development of safe and effective therapies are desperately needed. We and others have shown that solid tumors present high levels of protein tyrosine nitration, an oxidative post‐translational modification that occurs in pathological conditions involving oxidative stress but is absent in normal tissues. In many solid tumors tyrosine nitration correlates with poor prognosis, suggesting nitrated proteins may benefit tumor growth. We have previously shown that tyrosine nitration supports survival and migration of GBM cells. Here, we identified the first nitrated protein with pro‐tumorigenic activity, nitrated Heat shock protein 90 (Hsp90). Hsp90 is an essential molecular chaperone present in all cells. We showed that nitration of Hsp90 induces a gain‐of‐function that decreases mitochondrial metabolism in tumor cells. We detected endogenously nitrated Hsp90 (NO2Hsp90) in GBM‐patient derived cell culture models, and in 2D‐ and 3D‐ U87 cell culture models of GBM. To study the role of NO2Hsp90 in supporting GBM cell survival/proliferation, we prevented tyrosine nitration in U87 cells for 48 h to decrease the endogenous levels of nitrated proteins. In these conditions, cell viability decreases by ~25%, while the levels of nitrated proteins decrease by ~50%. We then intracellularly released NO2Hsp90 at levels comparable to those normally present in U87 cells (~1.5% of total cellular Hsp90 calculated by quantitative slot blot), and sustained prevention of endogenous tyrosine nitration for an additional 48 h. Cell viability was assessed at this final timepoint. If NO2Hsp90 is necessary and sufficient to support U87 cell survival/proliferation, intracellular release of the nitrated chaperone to cells containing decreased levels of nitrated proteins should rescue cell viability. Release of unmodified (UM) Hsp90 was used as control. Excitingly, the intracellular release of NO2Hsp90 but not UM Hsp90 significantly increased U87 cell survival/proliferation by ~50%. Intracellular release of NO2Hsp90 to untreated cells that contain endogenous NO2Hsp90 had no effect on cell proliferation but to our surprise, the extracellular release of NO2Hsp90 significantly increased cell numbers after 48 h incubation. This effect was significantly hindered by preventing endogenous tyrosine nitration, suggesting that intracellular nitrated proteins mediate the effect of extracellular NO2Hsp90. It is well described that extracellular Hsp90 (eHsp90) induces GBM motility and invasion. We detected eHsp90 in culture media conditioned by U87 cells by western blot. Using live transwell migration assays, we found that incubation with either extracellular UM‐ or NO2Hsp90 increased U87 cell migration versus control cells that did not receive protein. These results suggest that NO2Hsp90 plays multiple essential roles in GBM biology, posing NO2Hsp90 as a promising tumor‐directed therapeutic target.

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

Synthesis and biological evaluation of celastrol derivatives as potential anti-glioma agents by activating RIP1/RIP3/MLKL pathway to induce necroptosis

Celastrol, a quinone methide triterpenoid, possesses potential anti-glioma activity. However, its relatively low activity limit its application as an effective agent for glioma treatment. In search for effective anti-glioma agents, this work designed and synthesized two series of celastrol C-3 OH and C-20 COOH derivatives 4a–4o and 6a–6o containing 1, 2, 3-triazole moiety. Their anti-glioma activities against four human glioma cell lines (A172, LN229, U87, and U251) were then evaluated using MTT assay in vitro. Results showed that compound 6i (IC50 = 0.94 μM) exhibited substantial antiproliferative activity against U251 cell line, that was 4.7-fold more potent than that of celastrol (IC50 = 4.43 μM). In addition, compound 6i remarkably inhibited the colony formation and migration of U251 cells. Further transmission electron microscopy and mitochondrial depolarization assays in U251 cells indicated that the potent anti-glioma activity of 6i was attributed to necroptosis. Mechanism investigation revealed that compound 6i induced necroptosis mainly by activating the RIP1/RIP3/MLKL pathway. Additionally, compound 6i exerted acceptable BBB permeability in mice and inhibited U251 cell proliferation in an in vivo zebrafish xenograft model, obviously. In summary, compound 6i might be a promising lead compound for potent celastrol derivatives as anti-glioma agents.

OTME-18. Targeted CRISPR/Cas9 gene-editing regulates the brain tumor environment

Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.

A new insight into the role of aryl hydrocarbon receptor (AhR) in the migration of glioblastoma by AhR-IL24 axis regulation

Cancer occurrence and development are closely related to the environment. Aryl hydrocarbon receptor (AhR) is an important receptor mediating the toxic effects of many environmental compounds, and is also involved in regulating tumor cell migration. Glioblastoma is the most malignant glioma and exhibits high motility, but the effects of AhR on the migration of glioblastoma are still unclear. We aimed to understand the role of AhR in the migration of this type of tumor cell and to explore the underlying molecular mechanism. In cultured human neuroblastoma cells (U87), we found that AhR overexpression or knockdown increased or suppressed the migration ability of U87 cells, respectively. Furthermore, inhibition of basal activation of the AhR pathway suppressed migration ability, suggesting a positive correlation between endogenous activity of the AhR pathway and cell migration. When the AhR pathway was activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 6-formyl [3,2-b] carbazole (FICZ), the migration of U87 cells was inhibited by inducing the expression of a tumor suppressor, IL24, which is a downstream responsive gene of AhR activation. Moreover, a similar AhR-IL24-dependent mechanism for migration inhibition of TCDD was documented in a breast cancer cell line and a lung cancer cell line. This study demonstrated that AhR plays important roles in regulating the migration of glioblastoma, and the induction of the AhR-IL24 axis mediates the inhibition of migration in response to TCDD or FICZ treatment.