LN229 Cells Research Articles

Cytotoxic compounds from Viscum coloratum (Kom.) Nakai.

Two new compounds (1-2) and a new artificial product (3) together with fifteen known compounds (4-18) were isolated from Viscum coloratum (Kom.) Nakai. Their structures were established by analysis of their spectroscopic data including MS, 1D and 2D NMR spectra, and comparison with existing literature. All the compounds were assessed for their cytotoxic activity against 4T1 and LN229 cells (with cisplatin as the positive control), and the anti-LN229 cytotoxicity of compounds 3, 4 and 5 were greater than that of 4T1 cells.

CSIG-23. CYTOMEGALOVIRUS-INFECTED HUMAN GLIOBLASTOMA CELL LINES SHOW AN AGGRESSIVE PHENOTYPE ASSOCIATED WITH UPREGULATION OF IL-6/STAT3 AND AKT SIGNALING

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

A Cancer-Specific Anti-Podoplanin Monoclonal Antibody, PMab-117-mG2a Exerts Antitumor Activities in Human Tumor Xenograft Models

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

Evidence to Support the Collaboration of SP1, MYC, and HIF1A and Their Association with microRNAs

This study provides evidence to support the concept proposed by Kimura et al. in 2023 that the inhibitors of SP1, MYC, and HIF1A should induce strong anticancer activity by reducing the expression of stem cell-related proteins. In LN229 and U87MG glioblastoma cells, either tetra-methyl-O-nordihydroguaiaretic acid (M4N) or tetra-acetyl-O-nordihydroguaiaretic acid (A4N) suppressed SP1 and only a few stem cell-related proteins and induced only a small amount of cell death; in contrast, the combination treatment of M4N with A4N greatly suppressed the expression of SP1, MYC, and HIF1A, as well as all of the stem cell-related proteins examined, and greatly induced cell death. The bioinformatic analysis showed that the proteins associated with SP1, MYC, and HIF1A were specifically involved in the regulation of transcription and that various microRNAs (miRNAs) that had been shown to induce either anti- or procancer activity were associated with SP1, MYC, and HIF1A, which suggested that the inhibition of SP1, MYC, and HIF1A could modulate the transcription of both coding and noncoding RNAs and affect cancers. These data overall supported our concept.

Bioinformatics investigation of the prognostic value and mechanistic role of CD9 in glioma

In recent years, CD9 has been extensively studied as a potential biomarker for cancer. However, the biological role of CD9 in gliomas remains unclear. This study investigates the function of CD9 in gliomas and its molecular mechanisms. Utilizing pan-cancer analysis with TCGA, CGGA, and GEO databases, differential expression of CD9 was observed in 11 tumor types within the TCGA cohort, and it was associated with patient survival rates. Analysis of the CGGA glioma database revealed that patients with high CD9 expression had lower survival rates. The area under the ROC curve (AUC) for GSE16011 was greater than 0.7, indicating a high discriminative ability. Through gene set enrichment analysis (GSEA), immune-related analysis, and CD9 mutation detection, CD9 was found to have the strongest correlation with neutrophil involvement (cor = 0.30, P < 0.05), and the high CD9 expression group exhibited higher rejection responses and TIDE scores, suggesting a lower likelihood of successful immunotherapy. The high CD9 expression group was more sensitive to 81 drugs, indicating potential therapeutic effects for gliomas. Furthermore, overexpression of CD9 in gliomas may be associated with gene mutations. Down-regulation or up-regulation of CD9 expression in the glioblastoma cell line LN229 showed that CD9 could positively regulate the migratory ability of LN229 cells. Further, several marker genes, such as VEGFR-2, TGF-β1, CASP1 and PI3K, were down regulated in CD9 knockdown cell lines and up regulated in CD9 overexpression cell lines, compared with control cell line. This study preliminarily explores the role of CD9 in gliomas and its prognostic value, providing new insights for personalized treatment strategies in glioma therapy.

SENP7 inhibits glioblastoma metastasis and invasion by dissociating SUMO2/3 binding to specific target proteins.

The poor surgical efficacy and recurrence of glioblastoma (GBM) are due to its lack of visible infiltrative features. Our bioinformatics study suggests that low expression of small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) indicates poor prognosis in GBM. This study investigated the effect of SENP7 expression on the invasion, migration, and proliferation of GBM cells and aims to identify the SUMO target proteins affected by SENP7. SENP7 expression was analyzed in eight GBM tumor samples and four GBM cell lines, comparing them to normal brain tissue. The effect of SENP7 overexpression on GBM LN229 cell migration, invasion, and proliferation was examined through in vitro assays. Furthermore, four SUMO target proteins involved in tumor invasion and proliferation (CDK6, matrix metalloproteinase-9 [MMP9], AKT, and HIF-1α) were studied to explore SENP7's molecular mechanism. SENP7 expression was significantly lower in GBM tumors compared to normal tissue. SENP7 overexpression in LN229 cells inhibited migration and invasion without affecting proliferation. Overexpression reduced the levels of MMP9, AKT, and HIF-1α, but not CDK6. Immunohistochemical analysis showed decreased MMP9 and CD31 levels, suggesting reduced tumor invasion and angiogenesis. However, SENP7 overexpression did not affect tumor growth in vivo. SENP7 inhibits GBM invasion by dissociating proteins associated with tumor invasion from SUMO2/3, providing a potential target for future GBM therapies.

Rosmarinic acid attenuates glioblastoma cells and spheroids’ growth and EMT/stem-like state by PTEN/PI3K/AKT downregulation and ERK-induced apoptosis

BackgroundGlioblastoma (GB) is a highly malignant type of brain cancer with a poor prognosis. Therapeutic strategies for GB are still limited. Rosmarinic acid (RA), a polyphenolic compound, is a promising experimental anticancer agent, but its specific protein targets for GB remain unclear. PurposeThis study aimed to elucidate the anticancer effects of RA in 2D- and 3D-GB cells and the underlying mechanisms. Methods3D-tumor spheroids (mimics in vivo tumors) were obtained by the hanging-drop/agarose method. RA's anti-glioma activity on U-87MG (p53-wt/PTEN-mt) and LN229 (p53-mt/PTEN-wt) cells was evaluated through cell viability, colony-formation, migration/invasion/angiogenesis assays, fluorescence imaging, and spheroid growth analysis. The underlying mechanism of the anticancer effects of RA was investigated by Western blot and immunofluorescence analysis. The MEK inhibitor U0126 was used to block ERK phosphorylation. ResultsRA treatments exerted anti-proliferative and pro-apoptotic effects on human GB cells. RA dose-dependently reduced angiogenesis and intracellular ROS levels, suppressed glioma growth, and migration/invasion in 2D-culture and cancer stem cell (CSC)-like 3D-spheroid culture (SPC). Repeated therapy in SPC was more effective by leading to disrupted structure than a single treatment. Treatments in SPC also suppressed epithelial-mesenchymal transition (EMT) and CSC-like properties. Strikingly, RA downregulated the SIRT1/FOXO1/NF-κB axis independently of p53 or PTEN function in both gliomas. Immunofluorescence labeling revealed decreased SIRT1 and NF-κB-p65 and increased FOXO1 and GAPDH proteins in nuclear location (associated with apoptosis). Surprisingly, RA increased p-ERK1/2 levels, but priming with U0126 abolished RA-mediated p-ERK upregulation; thus, autophagy and apoptosis induction in GB cells were prevented, and the growth of GB spheroids accelerated. Specifically, RA also inhibited the PTEN/PI3K/AKT pathway in U-87MG cells. Due to genetic differences in cells, U-87MG cells were more sensitive to RA treatments than LN229 cells. Meanwhile, our positive control drug trial results with FDA-approved temozolomide (TMZ) used in GB treatment showed that our test compound rosmarinic acid exhibited higher therapeutic effects than TMZ at lower doses. ConclusionSuppression of EMT, downregulation of SIRT1/FOXO1/NF-κB axis, inhibition of PTEN/PI3K/AKT signaling pathway, and ERK-induced apoptosis and autophagy were determined to be involved in stopping glioma progression. Our findings for the first time, revealed that RA may have potential therapeutic use by having multiple targets in human brain cancer with further clinical studies.

Inhibitory effects of Gracilaria edulis and Gracilaria salicornia against the MGMT and VEGFA biomarkers involved in the onset and advancement of glioblastoma using insilico and invitro approaches.

Glioblastoma (GBM), an aggressive primary brain tumor originating from glial cells, poses significant treatment challenges due to its rapid growth and invasiveness. The exact mechanisms of GBM's brain damage remain unclear. This study examines primary molecular markers commonly assessed in GBM patients, including brain-derived neurotrophic factor (BDNF), platelet-derived growth factor receptor A (PDGFRA), O6-methylguanine DNA methyltransferase (MGMT), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor A (VEGFA) using computational approaches. The study revealed significant differences (p ≤ 0.05) in PDGFRA, EGFR, and VEGFA expression rates, which are particularly interesting. Additionally, MGMT and VEGFA showed higher hazard ratios. Expression levels of MGMT and VEGFA were visualized in immune and malignant cells using single-cell RNA datasets GSE103224 and GSE148842. From a total of 48 compounds in Gracilaria edulis and 86 in Gracilaria salicornia, we identified 15 compounds capable of crossing the blood-brain barrier. Notably, 2-tridecanone (binding affinity [BA] = -4.2kcal/mol; root mean square deviation [RMSD] = 1.479Å) and decanoic acid, ethyl ester (BA = -4.2kcal/mol; RMSD = 1.702Å) from G. edulis interacted with MGMT via hydrogen bonds. The compound alpha-terpineol interacted with MGMT (BA = -5.7kcal/mol; RMSD = 0.501Å) and VEGFA (BA = -4.7kcal/mol; RMSD = 2.483Å). Ethanolic and methanolic extracts from G. edulis and G. salicornia demonstrated mild anti-cell proliferation properties in the GBM LN-229 cell line, suggesting potential therapeutic benefits. This study highlights the significance of molecular markers and natural compounds in understanding and potentially treating GBM.

The Proteasome Inhibitor Marizomib Evokes Endoplasmic Reticulum Stress and Promotes Apoptosis in Human Glioblastoma Cells.

Proteasomes play an important role in the physiology of cancer cells, and inhibition of their activity may be used as a promising therapeutic strategy against glioblastoma (GBM). Although certain proteasome inhibitors (PIs) have been approved for the treatment of other malignancies, they have limited effectiveness against GBM due to low brain bioavailability. Marizomib (MZB) is an irreversible, second-generation proteasome inhibitor, which unlike other PIs can penetrate through the blood-brain barrier, making it a promising therapeutic tool in brain malignancies. The antitumor activity of MZB was investigated in LN229 and U118 cells. The MTT test and the ATP-based assay were performed to evaluate cytotoxicity. Flow cytometry analysis was used to determine the apoptotic death of GBM cells. Luminescent assays were used to assess levels of reactive oxygen species (ROS) and the activity of caspase 3/7. RT-qPCR and Western blot analyses were used to determine gene and protein expressions. Marizomib decreased the viability and caused apoptotic death of GBM cells. The proapoptotic effect was accompanied by activation of caspase 3 and overexpression of cl-PARP, Noxa, Cyt C, and DR5. Moreover, treatment with MZB triggered endoplasmic reticulum (ER) stress, as shown by increased expressions of GRP78, IRE1α, p-EIF2α, p-SAPK/JNK, CHOP, ATF6α, and ATF4. On the contrary, overproduction of ROS or increased expressions of ERO1α, LC3 II, Beclin 1, and ATG5 were not detected, suggesting that neither oxidative stress nor autophagy were involved in the process of MZB-induced cell death. Thus, marizomib represents a potentially promising compound for facilitating further progress in brain cancer therapy.

Periplocin Inhibited Gliomas Migration by Targeting Migrasome Related Gene TSPAN6

Glioma is a life-threatening brain tumor characterized by rapid growth and resistance to treatment. Periplocin, the monomeric compound, exhibits anti-tumor activities. However, the action of periplocin in gliomas remain unclear. Migrasome, a vesicle-like structure derived from the tips or intersections of contractile filaments in migrating cells, was investigated in this study to explore the antitumor activity of periplocin in glioma and elucidate its mechanism. The proliferation, apoptosis, migration and MMP in periplocin-treated LN229 and KNS-89 cells were examined. In order to revealed the mechanism of Periplocin inhibition of migration. Additionally, we revealed the relation of Migrasome-related genes with glioma based on TCGA database analysis, and we found TSPAN6 may be the direct target in glioma, Periplocin could inhibited LN229 and KNS-89 cell proliferation and migration, and promoted their apoptosis. Moreover, targeting TSPAN6 resulted in reduced cell viability and migration, as well as increased apoptosis in glioma cells.

Metabolomic insights and bioactive efficacies of Tragopogon dubius root fractions: Antioxidant and antiproliferative assessments

Tragopogon dubius is commonly consumed as a vegetable and used in traditional medicine for treating inflammatory skin conditions and cutaneous swelling. Despite known pharmacological properties of its leaves and roots, many of its biological characteristics and active phytochemicals remain unexplored. The present study investigates the phytochemical composition, antioxidant, and anticancer properties of methanolic root extracts and isolated fractions (TdRM-1 and TdRM-2) of T. dubius. Utilizing preparative thin-layer chromatography, the crude extract was successfully separated into TdRM-1 and TdRM-2, characterized by GC-MS and FTIR analysis, revealing a diverse range of bioactive compounds including terpenes, flavonoids, and phenolic acids. Qualitative phytochemical screening indicated the presence of carbohydrates, tannins, alkaloids, and other phytoconstituents. Advanced UPLC-ESI-QTOF-MS analysis identified 54 metabolites, significantly contributing to the chemical profiling of the extract. The antioxidant activities of the fractions were quantitatively assessed using ABTS, DPPH, and superoxide radical scavenging assays, where TdRM-2 exhibited superior activity with IC50 values ranging from 51.29 to 60.03 μg/mL. Anticancer potential was evaluated against A549, LN-18, and MCF-7 cancer cell lines, demonstrating that TdRM-2 significantly inhibited cell proliferation with GI50 values as low as 31.62 μg/mL for A549 cells. Additionally, fluorescence microscopy revealed that TdRM-2 induces apoptosis, indicated by changes in nuclear morphology and loss of mitochondrial membrane potential. Annexin V-FITC/PI double staining indicate that the TdRM-2 fractions from T. dubius can significantly inhibit the growth of A-549, LN-18, and MCF-7 cancer cell lines by inducing apoptosis These findings suggest that T. dubius root extracts, particularly the TdRM-2 fraction, hold promising therapeutic potential due to their significant antioxidant and anticancer activities, underpinned by their rich phytochemical composition. This study underscores the importance of T. dubius as a source of natural bioactive compounds with potential health benefits.

Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Potential Mechanism and Involvement of p120-Catenin in the Malignant Biology of Glioma.

This study analyzed the influence of p120-catenin (catenin [cadherin-associated protein], delta 1 [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 polymerase chain reaction (QT-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-blank control (BC) group (no RNA sequence was transfected), siRNA-negative control (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 (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzymelabeled 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<0.0001) 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.

Novel tetrahydroquinoline derivatives induce ROS-mediated apoptosis in glioblastoma cells

Current treatment for Glioblastoma Multiforme (GBM) is not efficient due to its aggressive nature, tendency to infiltrate surrounding brain tissue, and chemotherapy resistance. Tetrahydroquinoline scaffolds are emerging as a new class of drug for treating many human cancers including GBM. This study investigates the cytotoxicity effect of eight novel derivatives of 2-((3,4-dihydroquinolin-1(2H)-yl)(aryl)methyl)phenol, containing substitute 1 with reduced dihydroquinoline fused with cyclohexene ring and substitute 2 with phenyl and methyl group. The 4-position of the aryl ring was determinant for the desired cytotoxicity, and out of the 8 synthesized compounds, the 4-trifluoromethyl substituted derivative (4ag) exhibited the most anti-GBM potential effect compared to the standard chemotherapeutic agent, temozolomide (TMZ), with IC50 values of 38.3 μM and 40.6 μM in SNB19 and LN229 cell lines, respectively. Our results demonstrated that 4ag triggers apoptosis through the activation of Caspase-3/7. In addition, 4ag induced intracellular reactive oxygen species (iROS) which in turn elevated mitochondrial ROS (mtROS) and causes the disruption of the mitochondrial membrane potential (Δψmt) in both GBM cells. This compound also exhibited anti-migratory properties over the time in both the cell lines. Overall, these findings suggest that tetrahydroquinoline derivative, 4ag could lead to the development of a new drug for treating GBM.

Combining the constitutive TRAIL-secreting induced neural stem cell therapy with the novel anti-cancer drug TR-107 in glioblastoma

Tumor-homing neural stem cell therapy (tNSC) is emerging as a promising treatment for aggressive cancers of the brain. Despite their success, developing tNSC therapies that maintain durable tumor suppression remains a challenge. Herein, we report a synergistic combination regimen where the novel small molecule TR-107 augments NSC-TRAIL therapy (hiNeuroS-TRAIL) in models of incurable brain cancer glioblastoma (GBM) in vitro. We report that the combination of hiNeuroS-TRAIL and TR-107 synergistically upregulated caspase markers and restored sensitivity to the intrinsic apoptotic pathway by significantly downregulating inhibitory pathways associated with chemoresistance and radioresistance in the TRAIL-resistant LN229 cell line. This combination also showed robust tumor suppression and enhanced survival of mice bearing human xenografts of both solid and invasive GBM. These findings elucidate a novel combination regimen and suggest combination of these clinically relevant agents may represent a new therapeutic option with increased efficacy for GBM patients.

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.

Co-treatment of human GBM cell lines with imipridone ONC206 and tumor treating fields: Effect on on cell death and p-AKT.

e14033 Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of cleaved PARP following cotreatment of ONC206 and TTFields. Methods: We investigated the effect of ONC206 at IC50s plus TTFields in LN229, U138, U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. At 72 h time point co treatment with ONC206 and TTFields resulted in synergistic upregulation of cleaved PARP. Conclusions: Here we showed cotreatment with TTFields and ONC206 results in inhibition of p-AKT, the key node in GBM growth pathway. This is the first report of cleaved PARP upregulation following TTFields and ONC206 in Human GBM cell line. Further studies to investigate the in vivo efficacy of TTField treatment in GBM with ONC206 are warranted. More studies are needed to understand the exact mechanism of synergy.

Identification and validation of COL6A1 as a novel target for tumor electric field therapy in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive primary brain malignancy. Novel therapeutic modalities like tumor electric field therapy (TEFT) have shown promise, but underlying mechanisms remain unclear. The extracellular matrix (ECM) is implicated in GBM progression, warranting investigation into TEFT-ECM interplay. T98G cells were treated with TEFT (200 kHz, 2.2 V/m) for 72 h. Collagen type VI alpha 1 (COL6A1) was identified as hub gene via comprehensive bioinformatic analysis based on RNA sequencing (RNA-seq) and public glioma datasets. TEFT intervention models were established using T98G and Ln229 cell lines. Pre-TEFT and post-TEFT GBM tissues were collected for further validation. Focal adhesion pathway activity was assessed by western blot. Functional partners of COL6A1 were identified and validated by co-localization and survival analysis. TEFT altered ECM-related gene expression in T98G cells, including the hub gene COL6A1. COL6A1 was upregulated in GBM and associated with poor prognosis. Muti-database GBM single-cell analysis revealed high-COL6A1 expression predominantly in malignant cell subpopulations. Differential expression and functional enrichment analyses suggested COL6A1 might be involved in ECM organization and focal adhesion. Western blot (WB), immunofluorescence (IF), and co-immunoprecipitation (Co-IP) experiments revealed that TEFT significantly inhibited expression of COL6A1, hindering its interaction with ITGA5, consequently suppressing the FAK/Paxillin/AKT pathway activity. These results suggested that TEFT might exert its antitumor effects by downregulating COL6A1 and thereby inhibiting the activity of the focal adhesion pathway. TEFT could remodel the ECM of GBM cells by downregulating COL6A1 expression and inhibiting focal adhesion pathway. COL6A1 could interact with ITGA5 and activate the focal adhesion pathway, suggesting that it might be a potential therapeutic target mediating the antitumor effects of TEFT.

Modeling Absorption Dynamics of Differently Shaped Gold Glioblastoma and Colon Cells Based on Refractive Index Distribution in Holotomographic Imaging.

Herein, it is demonstrated that the toxic effect of gold nanoparticles (Au NPs) on three different cancer cell lines (U-118 and LN-299 glioblastoma and HCT-116 colon) depends on their absorption dynamics by cells, related to the shapes of the NPs. This hypothesis is confirmed by showing that i) based on refractive index (RI) values, typical for cell components and gold nanoparticles, it is possible to show the absorption dynamics and accumulation locations of the latter ones inside and outside of the cells. Moreover, ii) the saturation of the accumulated Au NPs volume in the cells depends on the nanoparticle shape and is reached in the shortest time for star-shaped Au NPs (AuS NPs) and in the longest time for spherical Au NPs (AuSph NPs) and on the cancer cells, where the longest and the shortest saturation are noticed for HCT-116 and LN-229 cells, respectively. A physical model of Au NPs absorption dynamics is proposed, where the diameter and shape of the Au NPs are used as parameters. The obtained theoretical data are consistent with experimental data in 85-98%.

Exosomal HMGB3 released by glioma cells confers the activation of NLRP3 inflammasome and pyroptosis in tumor-associated macrophages

BackgroundPrevious evidences has highlighted the pivotal role of NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated inflammasomes and pyroptosis activation in driving tumor malignancy and shaping the tumor microenvironment. Herein, we aimed to elucidate the impact of high-mobility group box 3 (HMGB3) released in glioma-derived exosomes on macrophage infiltration in gliomas, NLRP3 inflammasome activation and polarization. MethodsTranscripts and protein levels of HMGB3, and cytokines associated with macrophage phenotypes and pyroptosis were assessed in glioma tissues and cell lines (U251, LN229, T98G, A172) using qRT-PCR and/or Western blot analysis. Exosomes secreted from LN229 and NHA cells were isolated via differential ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and analysis of exosome-related markers. PKH67 staining was employed to examine exosomes uptake by THP-1 differentiated macrophages. Flow cytometry was utilized to assess macrophage pyroptotic rates and polarization-related markers. ResultsHMGB3 expression was elevated in glioma tissues, serum samples and tumor cell lines. Kaplan-Meier curves revealed a positive correlation between higher HMGB3 expression and poor overall survival and recurrence-free survival. Moreover, glioma tissues with increased HMGB3 expression exhibited significant upregulation of M2 macrophages markers (CD68, CD206, Arg1) and NLRP3 inflammasome components (NLRP3, IL-1β, ASC), suggesting that HMGB3 was closely associated with macrophage infiltration and NLRP3 inflammasome activation. Notably, HMGB3 was found to be enriched in glioma cell- secreted exosomes and could be internalized by macrophages. Knockdown of HMGB3 in glioma cell exosomes could restrain M2 macrophage polarization, NLRP3 inflammasome activation and pyroptosis. ConclusionThese findings suggested that glioma cells secreted exosomal HMGB3 could facilitate macrophage M2 polarization, pyroptosis and inflammatory infiltration, indicating HMGB3 might be a poor prognosis factor for glioma.