LN229 Glioma Cells Research Articles

Function of a complex of p-Y42 RhoA GTPase and pyruvate kinase M2 in EGF signaling pathway in glioma cells.

Epidermal growth factor (EGF) is known to be a critical stimulant for inducing the proliferation of glioma cancer cells. In our study, we observed that GST-RhoA binds to pyruvate kinase M2 (PKM2) invitro. While EGF reduced the levels of RhoA protein, it significantly increased p-Y42 RhoA, as well as PKM1 and PKM2 in LN18 glioma cell line. We determined that RhoA undergoes degradation through ubiquitination involving SCF1 and Smurf1. Interestingly, we observed that p-Y42 RhoA binds to PKM2, while the dephosphomimetic form, RhoA Y42F, did not. Additionally, our observation revealed that PKM2 stabilized both RhoA and p-Y42 RhoA. Importantly, RhoA, p-Y42 RhoA, and PKM2, but not RhoA-GTP, were localized in the nucleus upon EGF stimulation. Knockdown of RhoA with siRNA resulted in the reduced levels of phosphoglycerate kinase1 (PGK1) and microtubule affinity-regulating kinase 4 (MARK). Furthermore, we found that the promoter of PGK1 was associated with β-catenin and YAP. Notably, p-Y42 RhoA and PKM2 co-immunoprecipitated with β-catenin and YAP. Based on these findings, we proposed a novel mechanism by which p-Y42 RhoA and PKM2, in conjunction with β-catenin and YAP, regulate PGK1 expression, contributing to the progression of glioma upon EGF.

Inhibition of the TCF12/VSIG4 axis by palbociclib diminishes the proliferation and migration of glioma cells and decreases the M2 polarization of glioma-associated microglia.

The CDK4/CDK6 inhibitor palbociclib has shown the encouraging promise in the treatment of glioma. Here, we elucidated how palbociclib exerts suppressive functions in the M2 polarization of glioma-related microglia and the progression of glioma. Xenograft experiments were used to evaluate the function in vivo. The mRNA levels of transcription factor 12 (TCF12) and VSIG4 were detected by RT-qPCR, and their protein levels were assessed by immunoblotting. Cell migration was tested by wound-healing assay. Cell cycle distribution and M1/M2 microglia phenotype analysis were performed by flow cytometry. The levels of IFN-γ, TNF-α, IL-6，and TGF-β were measured by ELISA. The TCF12/VSIG4 association was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. In U251 and LN229 glioma cells, TCF12 and VSIG4 were overexpressed, and palbociclib reduced their expression levels. TCF12 upregulation enhanced the proliferation and migration of glioma cells and the M2 polarization of glioma-associated microglia in vitro as well as the tumorigenicity of U251 glioma cells in vivo, which could be reversed by palbociclib. Mechanistically, TCF12 could enhance VSIG4 transcription and expression by binding to the VSIG4 promoter. TCF12 deficiency led to repression in glioma cell proliferation and migration as well as microglia M2 polarization, which could be abolished by increased VSIG4 expression. Our study reveals the novel TCF12/VSIG4 axis responsible for the efficacy of palbociclib in combating glioma, offering a rationale for the application of palbociclib in glioma treatment.

Potential Mechanism and Involvement of P120-Catenin in the Malignant Biology of Glioma.

This study analyzed the influence of p120-catenin (CTNND1) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative PCR technology was employed to assess the expression of P120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups: siRNA-BC group (no RNA sequence was transfected), siRNA-NC group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzyme-labeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (P < 0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r = 0.906, P = 0.00) and QT-PCR (F=830.6, P＜0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (P < 0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (P < 0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

Regulation and molecular mechanism of adiponectin on the proliferation, apoptosis, autophagy, and chemosensitivity of LN18 Glioma cell line.

This study aimed to explore the regulatory effects and associated mechanisms of adiponectin on apoptosis and proliferation in the LN18 glioma cell line through the AMPK and Akt signaling pathways. Additionally, we sought to elucidate the impact of adiponectin on the chemosensitivity of the LN18 glioma cell line to temozolomide (TMZ). The proliferation rate of glioma cells treated with adiponectin was assessed using the cholecystokinin (CCK8) assay. The Western blot analysis was employed to assess the expression of p-Akt, p-AMPK, p-mTOR, cleaved caspase3, Bax, Cyclin D1, and Cyclin B1 following adiponectin treatment. Cell apoptosis was quantified using AnnexinV/PI flow cytometry, while changes in the cell cycle were detected using PI staining flow cytometry. The findings revealed that adiponectin upregulates p-AMPK expression and downregulates p-mTOR expression in the PTEN wild-type glioma cell line LN18, with no discernible effect on p-Akt expression. Moreover, adiponectin inhibits the proliferation rate of the PTEN wild-type glioma cell line LN18, enhances the expression of cleaved caspase3 and Bax, and significantly elevates the apoptosis rate, as evidenced by AnnexinV/PI flow cytometry. Adiponectin was observed to suppress the expression of Cyclin D1 and Cyclin B1, increase the number of cells in the G1 phase, and promote autophagy. Additionally, adiponectin augments the expression of Beclin1 and the ratio of LC3II/I in the PTEN wild-type glioma cell line LN18, while decreasing p62 expression. In conclusion, this study posits that adiponectin holds therapeutic promise for glioma treatment. Furthermore, adiponectin enhances the inhibitory effect of TMZ on the proliferation rate of LN18 cells when treated with 0.1 mM and 1 mM TMZ. These results collectively suggest that adiponectin impedes proliferation, encourages apoptosis and autophagy in the LN18 glioma cell line, and heightens its sensitivity to the chemotherapeutic drug TMZ.

Abstract 3162: Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin

Abstract Introduction: Epidemiological studies showed that beta-adrenergic antagonists caused a low incidence of several cancer types. Glioblastoma highly expresses beta-2-adrenergic receptors. GBM accounts for 50% of central nervous system cancers. After standard-of-care therapeutic intervention, there is a survival time of 15 months. Our goal is to develop a translational therapy that will trigger the death of GBM cells while preserving healthy cells Methodology: Salmeterol xinafoate is a biased agonist that activates the G-protein pathway. Biased agonists activate signaling through the G-protein or the beta-arrestin pathway of G-protein coupled receptors. Dose-response experiments were performed using beta1 and beta2-adrenergic receptor antagonists and biased beta2-adrenergic receptor agonists to determine loss of glioma cell viability in GBM5, GBM12, and LN229 glioma cell lines. Western immunoblotting gauged survivin expression levels in all three lines. Epoxomicin was used to inhibit the proteasome complex. Annexin V and propidium iodide were employed to measure apoptotic or necrotic cell death. Results: Disrupting beta2-adrenergic receptor signaling by salmeterol xinafoate led to cell death of glioma cells but spared healthy neuron-glia cells using similar doses. Salmeterol xinafoate revealed greater efficacy for killing glioma cells than beta1 and beta2-adrenergic receptor antagonists. To determine the mechanism of glioma cell death, LN229 cells were treated with escalating doses of Salmeterol xinafoate, and at 24 hours, annexin V staining showed early apoptosis, and Annexin V/propidium iodide co-staining also showed late apoptosis. Propidium iodide revealed minor necrosis in LN229 glioma cells. Western immunoblotting showed salmeterol xinafoate downregulated survivin in a dose-dependent manner. However, the epoxomicin's inhibition of the proteasome complex prevented dose-escalated downregulation of survivin for all three glioma cell lines. Conclusion: The bitopic structure of salmeterol enables this drug to bind to the beta2-adrenergic receptors ligand binding site and an exosite site by its aryloxyalkyl tail that may contribute to the increased efficacy. Salmeterol xinafoate triggers glioma cell death by apoptosis at least in one glioma cell line. Salmeterol Xinafoate down-regulates survivin through protease degradation. Promising results show that the FDA-approved drug salmeterol xinafoate enables a favorable transition to the clinic. Repurposing this drug for GBMs may be combined with the current standard-of-care chemotherapies to enhance more significant loss of tumor volume and increase patient survival. Citation Format: Orli Algranatti, Ji Yae Lee, James M. Angelastro. Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3162.

NRP1 Induces Enhanced Stemness and Chemoresistance in Glioma Cells via YAP.

Neuropilin-1 (NRP1), a transmembrane glycoprotein, plays an important role in the malignant progression of gliomas; however, its role in chemoresistance is not fully understood. In this study, we observed the effects of NRP1 on the stemness and chemoresistance of glioma cells and the mediating role of Yes-associated protein (YAP). We constructed NRP1 overexpressing LN-229 glioma cells. Cells were treated with recombinant NRP1 protein (rNRP1) and the YAP inhibitor Super-TDU when necessary. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the sensitivity of cells to temozolomide (TMZ). Sphere and clone formation assays were performed to detect the sphere- and clone-forming abilities of cells. Western blotting was performed to detect cellular CD133, CD44, p-LATS1, and p-YAP protein expression. Immunofluorescence and flow cytometry were used to detect the subcellular localization of YAP and apoptosis, respectively. We found that both NRP1 overexpression and rNRP1 treatment enhanced self-renewal, TMZ resistance, and CD133 and CD44 protein expression in LN-229 cells. NRP1 overexpression and rNRP1 treatment also induced LATS1 and YAP dephosphorylation and YAP nuclear translocation. Super-TDU inhibits NRP1 overexpression-induced enhanced self-renewal and TMZ resistance in LN-229 cells. Our study suggests that NRP1 induces increased stemness in glioma cells, resulting in chemoresistance, and that this effect is associated with YAP activation.

DCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma

BackgroundMalignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ.MethodsVia lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis.ResultsWe used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects.ConclusiondCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications.

CDC6 overexpression contributes to the malignant phenotype of glioma via IL6/JAK2/STAT3 signaling.

Glioma, a prevalent primary tumor of the central nervous system, is targeted by molecular therapies aiming to intervene in specific genes and signaling pathways to inhibit tumor growth and spread. Our previous bioinformatics study revealed that significant CDC6 overexpression in gliomas was closely correlated with poor patient prognosis. Through qPCR, western blotting, and immunohistochemistry, we will further validate CDC6 expression in clinical glioma specimens, while the effects of silencing and overexpressing CDC6 in the U87 and LN229 glioma cell lines on malignancy will be assessed through MTS, EdU, transwell, and migration assays. Luciferase reporter assays, ChIP, qPCR, and western blotting were used to explore the upstream and downstream molecular mechanisms of CDC6. Our study confirmed the abnormal overexpression of CDC6 in gliomas, particularly in glioblastomas. CDC6 promotes glioma cell activity, proliferation, invasion, and migration by activating the IL6-mediated JAK2/STAT3 signaling pathway. The transcription Factor E2F8 directly regulates CDC6 transcription, playing a crucial role in its abnormal overexpression in gliomas. This research provides vital evidence supporting CDC6 as a molecular target for glioma therapy.

PATH-21. GPX4 IS CRUCIALLY IMPLICATED IN THE PATHOBIOLOGY OF GLIOBLASTOMA AND A PUTATIVE ACTIONABLE TARGET

Abstract BACKGROUND Glioblastoma(GBM) is the most common malignant primary brain tumor with a dismal prognosis. Glutathione-mediated inhibition of the ferroptotic form of cell death is crucially implicated in various malignancies, which is principally regulated by glutathione peroxidase 4 (GPX4). However, there is limited data on GBM. The present study evaluated the expression of GPX4 in GBM and its implications in tumor biology, clinical outcome, and therapeutic potentials. METHODS The mRNA and protein expression was assessed by qRT-PCR and immunohistochemistry, respectively, in 75 cases of adult hemispheric GBMs (IDH wild type). For comparison, 21 lower-grade astrocytomas (IDH-mutant, grade-2/3) [LGA] and 20 cases were also evaluated. In-vitro analysis was performed on U87MG and LN229 glioma cell line. RESULTS The mRNA expression of GPX-4 was significantly higher in GBM than LGA, and normal brain (NB) [p-values 0.028, and 0.001, respectively]. Normal brain cases were negative for GPX4. Almost half of the GBMs (49.3%) showed strong immunoreactivity, which was higher than LGAs (23.8%) [p-value -0.04]. Inhibition of GPX-4 in-vitro by 1S-RSL3 &amp; FIN56 was associated with decreased cell survival, proliferation, migration, invasion, and increased cell death. GPX-4 expression was not significantly associated with overall survival in GBM. CONCLUSION GPX-4 is crucial for the tumor cells' survival and aggressive biological behavior. Considering its immune-positivity only in the neoplastic cell and strong positivity in GBM it may be a useful adjunct to the diagnosis. The present study emphasized its role as a potential therapeutic target in the form of 1S-RSL3 &amp; FIN56.

Temozolomide-based sonodynamic therapy induces immunogenic cell death in glioma

BackgroundIn our previous study, we found for the first time that temozolomide (TMZ), the first-line chemotherapeutic agent for glioblastoma (GBM), can generate a large amount of reactive oxygen species (ROS) under ultrasound irradiation. Sonodynamic therapy (SDT) using TMZ as the sonosensitizer produced more potent antitumor effects than TMZ alone. Here, we further evaluate the effects of TMZ-based SDT on subcellular structures and investigate the immunogenic cell death (ICD)-inducing capability of TMZ-based SDT. MethodsThe sonotoxic effects of TMZ were explored in LN229 and GL261 glioma cells. The morphology of endoplasmic reticulum and mitochondria was observed by transmission electron microscopy. The nuclear DNA damage was represented by γ-H2AX staining. Bone marrow-derived dendritic cells (BMDCs) were employed to assess ICD-inducing capability of TMZ-based SDT. A cyclic arginine-glycine-aspartic (c(RGDyC))-modified nanoliposome drug delivery platform was used to improve the tumor targeting of SDT. ResultsTMZ-based SDT had a greater inhibitory effect on glioma cells than TMZ alone. Transmission electron microscopy revealed that TMZ-based SDT caused endoplasmic reticulum dilation and mitochondrial swelling. In addition, endoplasmic reticulum stress response (ERSR), nuclear DNA damage and mitochondrial permeability transition pore (mPTP) opening were promoted in TMZ-based SDT group. Most importantly, we found that TMZ-based SDT could promote the “danger signals” produced by glioma cells and induce the maturation and activation of BMDCs, which was associated with the mitochondrial DNA released into the cytoplasm in glioma cells. In vivo experiments showed that TMZ-based SDT could remodel glioma immune microenvironment and provoke durable and powerful anti-tumor immune responses. What's more, the engineered nanoliposome vector of TMZ conferred SDT tumor targeting, providing an option for safer clinical application of TMZ in combination with SDT in the future. ConclusionsTMZ-based SDT was capable of triggering ICD in glioma. The discovery of TMZ as a sonosensitizer have shown great promise in the treatment of GBM.

RPL34-Divergent Transcript, a Novel Long NonCoding Ribonucleic Acid, Promotes Migration by Activating Epithelial-Mesenchymal Transition in Glioma

Glioma is one of the leading causes of death in patients with intracranial tumours. RPL34 divergent transcript (RPL34-DT) is a long non-coding ribonucleic acid that is significantly upregulated in glioma tissues. However, the role of RPL34-DT in glioma behavior remains to be elucidated. Therefore, in this study, we focused on the effect of RPL34-DT on the epithelial-mesenchymal transition in gliomas. Real-time quantitative reverse transcription polymerase chain reaction was used to detect the levels of RPL34-DT in glioma tissue and cell lines. We further used the LN229 and U251 glioma cell lines to assess the role of RPL34-DT. Wound healing and invasion assays were performed to determine the role of RPL34-DT in migration. Changes in protein levels were assessed by western blotting. We found that RPL34-DT was upregulated in glioma tissues and glioma cell lines. Knockdown of RPL34-AS1 blocked migration of glioma cell. This effect occurred through a decrease of epithelial-mesenchymal transition and β-catenin. This study suggests that RPL34-DT affects cell migration in glioma and therefore may serve as a valuable therapeutic target in patients with glioma.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth

Gliomas are the most prevalent primary tumor of the CNS. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 (HNK-1) capped O-Man core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of HNK-1 capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

BLM helicase overexpressed in human gliomas contributes to diverse responses of human glioma cells to chemotherapy

Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways to sustain tumour proliferation capacity. RECQL helicases family, including BLM, participate in DNA damage and repair, and prevent the replication stress. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in GBM therapy resistance have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9 system. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence, Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of GBM patients with high BLM expression. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in brain tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP1 dependent pathways.

Liquid-liquid phase separation-related gene in gliomas: FABP5 is a potential prognostic marker.

The glioma is the most malignant human brain tumor. Early glioma detection and treatment are still difficult. New biomarkers are desperately required to aid in the evaluation of diagnosis and prognosis. The single cell sequencing dataset scRNA-6148 for glioblastoma was obtained from the Chinese Glioma Genome Atlas database. Data were gathered for the transcriptome sequencing project. Genes involved in liquid-liquid phase separation (LLPS) were taken out of the DrLLPS database. To find the modules connected to LLPS, the weighted co-expression network was analyzed. Differential expression analysis was used to identify the differentially expressed genes (DEGs) in gliomas. Pseudo-time series analysis, gene set enrichment analysis (GSEA) and immune cell infiltration analysis were used to investigate the role of important genes in the immunological microenvironment. We examined the function of key glioma genes using polymerase chain reaction (PCR) testing, CCK-8 assays, clone generation assays, transwell assays and wound healing assays. FABP5 was identified as a key gene in glioblastoma by multiomics research. Pseudo-time series analysis showed that FABP5 was highly linked with the differentiation of many different types of cells. GSEA revealed that FABP5 was strongly linked to several hallmark pathways in glioblastoma. We looked at immune cell infiltration and discovered a significant link between FABP5, macrophages and T cell follicular helpers. The PCR experiment results demonstrated that FABP5 expression was elevated in glioma samples. Cell experiments showed that FABP5 knockdown dramatically decreased the viability, proliferation, invasion and migration of the LN229 and U87 glioma cell lines. Our study provides a new biomarker, FABP5, for glioma diagnosis and treatment.

Cytotoxic Steroidal Saponins Containing a Rare Fructosyl from the Rhizomes of Paris polyphylla var. latifolia.

A phytochemical investigation of the steroidal saponins from the rhizomes of Paris polyohylla var. latifolia led to the discovery and characterization of three new spirostanol saponins, papolatiosides A-C (1-3), and nine known compounds (4-12). Their structures were established via extensive spectroscopic data analysis and chemical methods. Interestingly, compounds 1 and 2 possessed a fructosyl in their oligosaccharide moiety, which is rare in natural product and was firstly reported in family Melanthiaceae. The cytotoxicity of these saponins against several human cancer cell lines was evaluated by a CCK-8 experiment. As a result, compound 1 exhibited a significant cytotoxic effect on LN229, U251, Capan-2, HeLa, and HepG2 cancer cells with IC50 values of 4.18 ± 0.31, 3.85 ± 0.44, 3.26 ± 0.34, 3.30 ± 0.38 and 4.32 ± 0.51 μM, respectively. In addition, the result of flow cytometry analysis indicated that compound 1 could induce apoptosis of glioma cells LN229. The underlying mechanism was explored by network pharmacology and western bolt experiments, which indicated that compound 1 could induce glioma cells LN229 apoptosis by regulating the EGFR/PI3K/Akt/mTOR pathway.

MicroRNA-608 Regulates Epithelial-To-Mesenchymal Transition to Inhibit Malignant Behaviors of Glioma Cells Through Nuclear Factor I-C Signaling Pathway

MiR-608 is expressed in bladder cancer, colon cancer, prostate cancer risk, and exerts inhibitory role in gastric cancer and invasion of three negative malignant breast cancer by inhibiting the NIFC, but its role in the biology of glioma is unclear, so the purpose of this study was to explore MiR-608’s role in glioma, and further explore whether NFIC signal pathways are involved in the role of MiR-608 in glioma. We obtain glioma tissues and normal tissues to detect the expression of miR-608 by PCR. miR-608 mimics transfection or joint with NFIC expression plasmid were transfected into A172, and LN229 glioma cells followed by analysis of cell proliferation, clone formation, invasion and migration, and cell apoptosis. Compared to normal tissue, miR-608 expression in glioma tissues was decreased. After transfection of miR-608 mimics, miR-608 level, cell proliferation activity, invasion and migration activity increased significantly, and apoptosis reduced, in addition, the dual luciferase report gene and protein imprinting analysis showed NFIC to be a target of miR-608. NFIC transfection reversed miR-608’s role in glioma cells. In conclusion, microRNA-608 inhibits malignant invasion and migration of glioma cells via NFIC signaling.

Functional characterization of TSPAN7 as a novel indicator for immunotherapy in glioma.

Glioma is the most common primary malignant tumor of the central nervous system in clinical practice. Most adult diffuse gliomas have poor efficacy after standard treatment, especially glioblastoma. With the in-depth understanding of brain immune microenvironment, immunotherapy as a new treatment has attracted much attention. In this study, through analyzing a large number of glioma cohorts, we reported that TSPAN7, a member of the tetraspanin family, decreased in high-grade gliomas and low expression was associated with poor prognosis in glioma patients. Meanwhile, the expression pattern of TSPAN7 was verified in glioma clinical samples and glioma cell lines by qPCR, Western Blotting and immunofluorescence. In addition, functional enrichment analysis showed that cell proliferation, EMT, angiogenesis, DNA repair and MAPK signaling pathways were activated in the TSPAN7 lower expression subgroup. Lentiviral plasmids were used to overexpress TSPAN7 in U87 and LN229 glioma cell lines to explore the anti-tumor role of TSPAN7 in glioma. Moreover, by analyzing the relationship between TSPAN7 expression and immune cell infiltration in multiple datasets, we found that TSPAN7 was significantly negatively correlated with the immune infiltration of tumor-related macrophages, especially M2-type macrophages. Further analysis of immune checkpoints showed that, the expression level of TSPAN7 was negatively correlated with the expression of PD-1, PD-L1 and CTLA-4. Using an independent anti-PD-1 immunotherapy cohorts of GBM, we demonstrated that TSPAN7 expression may had a synergistic effect with PD-L1 on the response to immunotherapy. Based on the above findings, we speculate that TSPAN7 can serve as a biomarker for prognosis and a potential immunotherapy target in glioma patients.

HNF4G accelerates glioma progression by facilitating NRP1 transcription.

Hepatocyte nuclear factor 4γ (HNF4G) is considered to be a transcription factor and functions as an oncogene in certain types of human cancer. However, the precise functions and the potential molecular mechanisms of HNF4G in glioma remain unclear. Therefore, the present study aimed to elucidate the role of HNF4G in glioma and the underlying mechanism. Western blotting and reverse transcription-quantitative PCR (RT-qPCR) demonstrated that HNF4G was highly expressed in glioma tissues and cell lines. The overexpression of HNF4G in LN229 and U251 glioma cells promoted cell proliferation and cell cycle progression, and inhibited apoptosis, while the knockdown of HNF4G suppressed cell proliferation, cell cycle progression and tumor growth, and induced apoptosis. A significant positive association was detected between HNF4G and neuropilin-1 (NRP1) mRNA expression in glioma tissues. Bioinformatics analysis, chromatin immunoprecipitation-RT-qPCR and promoter reporter assays confirmed that HNF4G promoted NRP1 transcription in glioma by binding to its promoter. NRP1 overexpression facilitated glioma cell proliferation and cell cycle progression, and suppressed apoptosis in vitro, while the knockdown of NRP1 inhibited cell proliferation and cell cycle progression, and facilitated apoptosis. NRP1 overexpression reversed the effects induced by HNF4G knockdown on glioma cell proliferation, cell cycle progression and apoptosis. In summary, the present study demonstrated that HNF4G promotes glioma cell proliferation and suppresses apoptosis by activating NRP1 transcription. These findings indicate that HNF4G acts as an oncogene in glioma and may thus be an effective therapeutic target for glioma.

The new oncogene transmembrane protein 60 is a potential therapeutic target in glioma.

Glioma is a malignant tumor with a high fatality rate, originating in the central nervous system. Even after standard treatment, the prognosis remains unsatisfactory, probably due to the lack of effective therapeutic targets. The family of transmembrane proteins (TMEM) is a large family of genes that encode proteins closely related to the malicious behavior of tumors. Thus, it is necessary to explore the molecular and clinical characteristics of newly identified oncogenes, such as transmembrane protein 60 (TMEM60), to develop effective treating options for glioma. We used bioinformatic methods and basic experiments to verify the expression of transmembrane protein 60 in gliomas and its relationship with 1p and 19q (1p19q) status, isocitrate dehydrogenase (IDH) status, patient prognosis, and immune cell infiltration using public databases and clinical samples. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to detect co-expressed genes. Thus, we inhibited the expression of transmembrane protein 60 to observe the proliferation and activity of glioma LN229 cells. We found transmembrane protein 60 was significantly upregulated in glioma compared with that in normal brain tissue at the mRNA. In the subgroups of World Health Organization high grade, isocitrate dehydrogenase wildtype, 1p and 19q non-codeletion, or isocitrate dehydrogenase wild combined with 1p and 19q non-codeletion, the expression of transmembrane protein 60 increased, and the prognosis of glioma patients worsened. In the transmembrane protein 60 high expression group, infiltration of immune cells and stromal cells in the tumor microenvironment increased, tumor purity decreased, and immune cells and pathways were activated. The immune cells mainly included regulatory T-cell, gamma delta T-cell, macrophages M0, neutrophils, and CD8+ T-cells. Overexpression of co-inhibitory receptors (CTLA4, PDL1 and CD96) may promote the increase of depletion of T-cell, thus losing the anti-tumor function in the transmembrane protein 60 high expression group. Finally, we found that transmembrane protein 60 silencing weakened the viability, proliferation, and colony formation of glioma LN229 cells. This is the 0 report on the abnormally high expression of transmembrane protein 60 in glioma and its related clinical features, such as tumor microenvironment, immune response, tumor heterogeneity, and patient prognosis. We also found that transmembrane protein 60 silencing weakened the proliferation and colony formation of glioma LN229 cells. Thus, the new oncogene transmembrane protein 60 might be an effective therapeutic target for the clinical treatment of glioma.

PROS1 shapes the immune-suppressive tumor microenvironment and predicts poor prognosis in glioma.

Glioma is the most malignant cancer in the brain. As a major vitamin-K-dependent protein in the central nervous system, PROS1 not only plays a vital role in blood coagulation, and some studies have found that it was associated with tumor immune infiltration. However, the prognostic significance of PROS1 in glioma and the underlying mechanism of PROS1 in shaping the tumor immune microenvironment (TIME) remains unclear. The raw data (including RNA-seq, sgRNA-seq, clinicopathological variables and prognosis, and survival data) were acquired from public databases, including TCGA, GEPIA, CGGA, TIMER, GEO, UALCAN, and CancerSEA. GO enrichment and KEGG pathway analyses were performed using "cluster profiler" package and visualized by the "ggplot2" package. GSEA was conducted using R package "cluster profiler". Tumor immune estimation resource (TIMER) and spearman correlation analysis were applied to evaluate the associations between infiltration levels of immune cells and the expression of PROS1. qRT-PCR and WB were used to assay the expression of PROS1. Wound-healing assay, transwell chambers assays, and CCK-8 assays, were performed to assess migration and proliferation. ROC and KM curves were constructed to determine prognostic significance of PROS1 in glioma. The level of PROS1 expression was significantly increased in glioma in comparison to normal tissue, which was further certificated by qRT-PCR and WB in LN-229 and U-87MG glioma cells. High expression of PROS1 positively correlated with inflammation, EMT, and invasion identified by CancerSEA, which was also proved by downregulation of PROS1 could suppress cells migration, and proliferation in LN-229 and U-87MG glioma cells. GO and KEGG analysis suggested that PROS1 was involved in disease of immune system and T cell antigen receptor pathway. Immune cell infiltration analysis showed that expression of PROS1 was negatively associated with pDC and NK CD56 bright cells while positively correlated with Macrophages, Neutrophils in glioma. Immune and stromal scores analysis indicated that PROS1 was positively associated with immune score. The high level of PROS1 resulted in an immune suppressive TIME via the recruitment of immunosuppressive molecules. In addition, Increased expression of PROS1 was correlated with T-cell exhaustion, M2 polarization, poor Overall-Survival (OS) in glioma. And it was significantly related to tumor histological level, age, primary therapy outcome. The results of our experiment and various bioinformatics approaches validated that PROS1 was a valuable poor prognostic marker. Increased expression of PROS1 was correlated with malignant phenotype and associated with poor prognosis in glioma. Besides, PROS1 could be a possible biomarker and potential immunotherapeutic target through promoting the glioma immunosuppressive microenvironment and inducing tumor-associated macrophages M2 polarization.