Human Glioblastoma Cell Lines Research Articles

Cyclopeptide moroidin inhibits vasculogenic mimicry formed by glioblastoma cells via regulating β-catenin activation and EMT pathways.

Glioblastoma (GBM) is a highly vascularized malignant brain tumor with poor clinical outcomes. Vasculogenic mimicry (VM) formed by aggressive GBM cells is an alternative approach for tumor blood supply and contributes to the failure of anti-angiogenic therapy (AAT). However, there are still a lack of the effective drugs that target VM formation in GBM. In the present study, we evaluate the effects of a plant cyclopeptide moroidin on VM formed by GBM cells and explore its underlying molecular mechanisms. Moroidin evidently suppressed migration, tube formation and expression of α-SMA and metalloproteinase-9 in human GBM cell lines at sub-lethal concentrations. RNA sequencing data suggested the involvement of EMT pathway in the mechanism of moroidin. Exposing GBM cells to moroidin, the expression of EMT marker N-Cadherin and Vimentin decreased in a concentration-dependent manner. Moreover, moroidin significantly reduced the level of phosphorylated ERK and inhibited the activation β-catenin. The plant cyclopeptide moroidin inhibited the VM formed by GBM cells by inhibiting ERK/β-catenin-mediated EMT. Our study indicates a promising application of moroidin as an anti-VM agent to the treatment of GBM.

Novel Acetamide-Based HO-1 Inhibitor Counteracts Glioblastoma Progression by Interfering with the Hypoxic-Angiogenic Pathway.

Glioblastoma multiforme (GBM) represents the deadliest tumor among brain cancers. It is a solid tumor characterized by uncontrolled cell proliferation generating the hypoxic niches in the cancer core. By inducing the transcription of hypoxic inducible factor (HIF), hypoxia triggers many signaling cascades responsible for cancer progression and aggressiveness, including enhanced expression of vascular endothelial growth factor (VEGF) or antioxidant enzymes, such as heme oxygenase-1 (HO-1). The present work aimed to investigate the link between HO-1 expression and the hypoxic microenvironment of GBM by culturing two human glioblastoma cell lines (U87MG and A172) in the presence of a hypoxic mimetic agent, deferoxamine (DFX). By targeting hypoxia-induced HO-1, we have tested the effect of a novel acetamide-based HO-1 inhibitor (VP18/58) on GBM progression. Results have demonstrated that hypoxic conditions induced upregulation and nuclear expression of HO-1 in a cell-dependent manner related to malignant phenotype. Moreover, our data demonstrated that the HO-1 inhibitor counteracted GBM progression by modulating the HIFα/HO-1/VEGF signaling cascade in cancer cells bearing more malignant phenotypes.

Chitosan-PLGA mucoadhesive nanoparticles for gemcitabine repurposing for glioblastoma therapy

Glioblastoma (GBM) is a highly deadly brain tumor that does not respond satisfactorily to conventional treatment. The non-alkylating agent gemcitabine (GEM) has been proposed for treating GBM. It can overcome MGMT protein-mediated resistance, a major limitation of conventional therapy with the alkylating agent temozolomide (TMZ). However, GEM's high systemic toxicity and poor permeability across the blood–brain barrier (BBB) pose significant challenges for its delivery to the brain. Thus, mucoadhesive poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with chitosan (CH), suitable for intranasal GEM delivery, were proposed in this work. A central composite design (CCD) was implemented for NPs optimization, and NPs with appropriate characteristics for intranasal administration were obtained. in vitro studies revealed that the NPs possess excellent mucoadhesive properties and the ability to selectively release GEM in the simulated tumor tissue environment. In vitro studies using two human GBM cell lines (U215 and T98G) revealed the NPs' ability to promote GEM's antiproliferative activity to sensitize cells to the effect of TMZ. The findings of this work demonstrate that the developed CH-GEM-NPs are suitable delivery systems for GEM, both as a single therapy and as a chemosensitizer to the GBM gold standard therapy.

Hypofractionated Radiation Therapy Suppresses Radioresistance in U87 Human Glioma Cells by Inhibiting Yap1 and Hsp90 Proteins.

Radiotherapy plays a vital role in the management of high-grade gliomas. However, the radio resistance of glioma cells limits the effect of radiation and drives recurrence inside the irradiated tumor volume leading to poor outcomes for patients. High-grade glioma cell radioresistance significantly contributes to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. An increasing body of evidence complies with the Yes Associated Protein 1 (Yap-1) and heat shock protein 90 (Hsp90) as biomarkers for radioresistance in glioma cells. A number of studies suggest the potential of radioresistance-associated factors as biomarkers and/ or novel therapeutic targets in glioma cells. Thus, it is essential for glioblastoma patients to identify robust druggable targets involved in radioresistance, optimizing irradiation protocol, and understanding their underlying molecular mechanisms. Therefore, in the present study, we hypothesized that hypofractionated Gamma Knife radiation therapy (HF-GKRT) could target Yap-1 and Hsp90 and downregulate the mechanism of radioresistance in high-grade glioma cells. For this purpose, expression levels of radioresistance markers Yap-1 and Hsp90 were evaluated after treatment with HF-GKRT, and this was compared with single fraction Gamma Knife radiation therapy (SF-GKRT) in U87MG primary human glioblastoma cell line model. This would help design a novel radiation therapy regimen for glioblastoma patients by reducing the risk of radioresistance.

Exploring neuroadaptive cellular pathways in chronic morphine exposure: An in-vitro analysis of cabergoline and Mdivi-1 co-treatment effects on the autophagy-apoptosis axis.

The complex impacts of prolonged morphine exposure continue to be a significant focus in the expanding area of addiction studies. This research investigates the effectiveness of a combined treatment using Cabergoline and Mdivi-1 to counteract the neuroadaptive changes caused by in vitro morphine treatment. The impact of Methadone, Cabergoline, and a combination of Cabergoline and Mdivi-1 on the cellular and molecular responses associated with Morphine-induced changes was studied in human Neuroblastoma (SK-N-MC) and Glioblastoma (U87-MG) cell lines that were exposed to prolong Morphine treatment. Cabergoline and Mdivi-1 combined treatment effectively influenced the molecular alterations associated with neuroadaptation in chronic morphine-exposed neural cells. This combination therapy normalized autophagy and reduced oxidative stress by enhancing total-antioxidant capacity, mitigating apoptosis, restoring BDNF expression, and balancing apoptotic elements. Our research outlines morphine's dual role in modulating mitochondrial dynamics via the dysregulation of the autophagy-apoptosis axis. This emphasizes the significant involvement of DRP1 activity in neurological adaptation processes, as well as disturbances in the dopaminergic pathway during in vitro chronic exposure to morphine in neural cells. This study proposes a novel approach by recommending the potential effectiveness of combining Cabergoline and Mdivi-1 to modulate the neuroadaptations caused by morphine. Additionally, we identified BDNF and PCNA in neural cells as potential neuroprotective markers for assessing the effectiveness of drugs against opioid toxicity, emphasizing the need for further validation. The study uncovers diverse effects observed in pretreated morphine glioblastoma cells under treatment with Cabergoline and methadone. This highlights the potential for new treatments in the DRD2 pathway and underscores the importance of investigating the interplay between autophagy and apoptosis to advance research in managing cancer-related pain. The study necessitates an in-depth investigation into the relationship between autophagy and apoptosis, with a specific emphasis on protein interactions and the dynamics of cell signaling.

Delineation of three-dimensional tumor margins based on normalized absolute difference mapping via volumetric optical coherence tomography

The extent of surgical resection is an important prognostic factor in the treatment of patients with glioblastoma. Optical coherence tomography (OCT) imaging is one of the adjunctive methods available to achieve the maximal surgical resection. In this study, the tumor margins were visualized with the OCT image obtained from a murine glioma model. A commercialized human glioblastoma cell line (U-87) was employed to develop the orthotopic murine glioma model. A swept-source OCT (SS-OCT) system of 1300 nm was used for three-dimensional imaging. Based on the OCT intensity signal, which was obtained via accumulation of each A-scan data, an en-face optical attenuation coefficient (OAC) map was drawn. Due to the limited working distance of the focused beam, OAC values decrease with depth, and using the OAC difference in the superficial area was chosen to outline the tumor boundary, presenting a challenge in analyzing the tumor margin along the depth direction. To overcome this and enable three-dimensional tumor margin detection, we converted the en-face OAC map into an en-face difference map with x- and y-directions and computed the normalized absolute difference (NAD) at each depth to construct a volumetric NAD map, which was compared with the corresponding H&E-stained image. The proposed method successfully revealed the tumor margin along the peripheral boundaries as well as the margin depth. We believe this method can serve as a useful adjunct in glioma surgery, with further studies necessary for real-world practical applications.

Metformin Reduces Viability and Inhibits the Immunoinflammatory Profile of Human Glioblastoma Multiforme Cells

Glioblastoma (GBM) is the predominant primary malignant brain tumor. Metformin, a well-known antidiabetic medication, has emerged as a potential therapeutic candidate in the treatment of GBM. We have herein investigated two aspects of the effect of MTF on GBM cells: the effect of MTF on GBM cell viability, as previous studies have shown that MTF can selectively affect human GBM tumors; and the immunomodulatory effect of MTF on GBM, as there is evidence that inflammation is associated with GBM growth and progression. The human GBM cell line (U87) was exposed to various doses of MTF (1 mM, 20 mM, and 50 mM), followed by examination of cell viability and inflammatory mediator secretion at various time points. We observed that MTF treatment exerted a dose-response effect on glioblastoma multiforme cell viability. It also had an immunomodulatory effect on GBM cells. Our study identified several mechanisms that led to the overall inhibitory effect of MTF on human GBM. Further inquiry is necessary to gain a better understanding of how these in vitro findings would translate into successful in vivo approaches.

Potential diagnostic and drug target markers in glioblastoma

Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.

Synthesis, biological evaluation and in-silico ADMET studies of novel chalcones and dihydro pyrazoles of 2H-Chromene as anticancer agents

2H-chromenes and chalcones are biologically important scaffolds with applications in drug discovery. Their ease of synthesis and biological importance have attracted the attention of medicinal chemists worldwide. Inspired by this, we synthesized various substituted chalcones and dihydropyrazole derivatives bearing various aryl and hetero-aryl 2H-chromene moieties. The resulting compounds were evaluated for cytotoxicity in vitro against doxorubicin in triple-negative breast cancer cell lines (MDA-MB-231), estrogen receptor-positive breast cancer cell lines (MCF-7), and human brain glioblastoma (U-87 MG) cell lines. Compounds 6b and 6j exhibited anticancer activity against triple-negative breast cancer cell line (MDA-MB-231), with IC50 values of 4.02 µM and 3.35 µM, respectively. Further investigation involving in silico pharmacokinetic (ADME) and toxicity studies also revealed pivotal features for the favourable bioavailability of these derivatives.

Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 μM and 19.92±1 μM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.

Abstract 7596: (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines

Abstract 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 p-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 CHOP and caspase-10 following cotreatment of ONC206 and TTFields. Materials & Methods: We investigated the effect of ONC206 (at IC50s) plus TTFields in 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. Neurospheres were generated using mentioned cell lines and used for investigating effect of co-treatment on 3D structure. 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 at all treatment conditions. 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. Conclusion: Here we showed cotreatment results in inhibition of p-AKT, the key node in GBM growth pathway. Also, the upregulation of CHOP and caspase-10 that has not been discussed before can be the key to focus on novel therapeutic targets for GBM More studies are needed to elaborate the exact mechanism of synergy but these new findings can be used to develop novel treatment combination Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines [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 7596.

CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

Abstract 696: Proton radiation and Seco-Duocarmycin SA: A synergistic strategy unveiling new frontiers in glioblastoma multiforme therapy

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive brain cancers with a 6.9% five-year survival rate. More effective therapies are needed as standard treatments, which include surgery, radiation, and chemotherapy are inefficient due to radio-chemo-resistance and relapse. The goal of this project is to investigate the efficacy of seco-duocarmycin SA (sDSA) as an anticancer drug, alone and the potential synergy of combining proton radiation and sDSA in human GBM cell lines. Proton therapy offers numerous advantages in treating cancers because of its superior dose distribution and biological effects. sDSA is an exceptionally potent agent that selectively alkylates DNA at the N3 position of adenine, making it of great interest as a therapeutic drug. We hypothesize that by pairing sDSA with proton radiation, a more dynamic effect will result, which will amplify their collective power, unleashing an enhanced cytotoxicity against human GBM cells. GBM cell lines (T98G and LN18) were exposed to graded doses and concentrations of protons and sDSA respectively in order to evaluate cellular changes in proliferation, survival, cell cycle, apoptosis/necrosis induction, and DNA damage. Results show that proton radiation and sDSA when used alone induce a dose-dependent decrease of cell proliferation, an increase in foci formation, and an increase in G2/M phase arrest in LN18 and T98G cells. The data also illustrates that the combination of proton radiation and sDSA synergistically enhances these effects. The utilization of proton radiation in conjunction with sDSA against GBM represents an innovative and previously unexplored approach. Currently, the cellular mechanisms of sDSA cytotoxicity are not well understood or studied, and even less is known regarding the effects when combined with proton radiation. This study serves as a vital cornerstone in unraveling the biological response of GBM cells when confronted with proton radiation and sDSA, paving the way for the future development of a more precise and potent combination therapy. Citation Format: Ann Morcos, Antonella Bertucci, Ryan Nicholaus Fuller, Nathan Robert Wall, Marcelo Eduardo Vazquez. Proton radiation and Seco-Duocarmycin SA: A synergistic strategy unveiling new frontiers in glioblastoma multiforme therapy [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 696.

The fabrication method impacts biological properties of microfibrous three-dimensional in vitro cell culture scaffolds and their applications in drug screening

Two-dimensional (2D) cell culture models cannot accurately emulate the three-dimensional (3D) in vivo microenvironment; hence, the shift from compound screening in traditional 2D well plates to 3D cell culture models is forthcoming. New scaffold designs must comply with the 3Rs principle to reduce the use of animal-derived products, such as natural proteins. In line with this motivation, we engineered and compared various fibrous scaffolds featuring diverse fibre networks (coarse and fine fibres) and fibre surface properties (porous vs. smooth fibres), and tested them in in vitro cell culture. We fabricated coarse solid fibre scaffolds via a melt-based electrohydrodynamic process, which resulted in fibres with a diameter of 10.6 ± 1.9 µm and a porosity of 87.1 ± 0.9%. Using solution-based cryo-electrohydrodynamic process, we fabricated fine porous fibre and fine solid fibre scaffolds that exhibited fibres of 2.01 ± 0.5 µm and 1.72 ± 0.6 µm in diameter, with porosities of 96.5 ± 0.3% and 94.4 ± 0.5%, respectively. The scaffolds underwent only chemical modification via an alkaline treatment to enhance their hydrophilicity. We used mouse fibroblast L929 cells and human triple-negative breast cancer MDA-MB-231 cells as cell models, and used the MTT assay to measure their viability. After seven days of cell culture, all scaffolds were thoroughly penetrated. However, the coarse scaffolds created by the melt-based electrohydrodynamic process and the porous fibre scaffolds produced by the solution-based cryo-electrohydrodynamic process outperformed the simpler morphology of smooth fine fibres. Furthermore, the 50% half-maximum effective concentration (EC50) of doxorubicin tested in human glioblastoma (U87-MG) and human triple-negative breast cancer (MDA-MB-231) cell lines in 3D cell culture models was approximately twice as high as that achieved in conventional 2D cell cultures. Therefore, we infer that poly (ɛ-caprolactone) (PCL) microfibre scaffolds, devoid of animal-derived product surface treatment, may provide a favourable 3D milieu for various cell types for in vitro studies.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [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 5564.

Abstract 4729: Incorporation of the antitumor compound 2OHOA and its metabolite into cell membranes

Abstract The compound 2-hydroxyoleic (2OHOA) is a bioactive lipid molecule that has demonstrated an anti-tumor effect and high safety in cellular and animal models of glioblastoma multiform (GBM), considered the most aggressive and lethal of primary tumors of the central nervous system. Due to the poor prognosis of patients, with a median survival of 15 months, it is of great importance to develop new therapeutic strategies that improve the efficacy and safety of treatments to achieve complete remission of GBM. Granted orphan designation for the treatment of glioma in both the European Union and the United States, 2OHOA completed a phase I/IIA study on advanced solid tumors in adults (NCT01792310) showing pharmacological efficacy and safety. It is currently undergoing a phase IIB/III clinical trial on glioma patients (NCT04250922) in combination with the standard of care for glioma and a pediatric study on glioma and other solid tumors (NCT04299191).Here, we seek to define the mechanism of action of 2OHOA which is based on Membrane Lipid Therapy, an innovative approach that proposes the use of new molecules designed to modify the composition and lipid structure of the membrane and reverse a pathological lipid state. Therefore, we further investigated the effect of 2OHOA on membrane lipid composition when applied to human glioblastoma cell line by gas chromatography. In addition, we defined the 2OHOA’s effect on different components of the cell signaling pathway by Western Blot and PK in glioma patients.Our results demonstrated that 2OHOA is mostly incorporated in glioma cell membranes and poorly integrated in non-tumor cells, which would explain its specific effects and low side effects in GBM treatment. Moreover, we observed that after its incorporation, 2OHOA is metabolized to produce the compound C17:1n-9 by α-oxidation pathway, a key process for its anti-tumor effect in glioma cells. In addition, the C17:1n-9 metabolite impairs cell growth, reinforcing the 2OHOA activity. Finally, we examined this metabolite as a biomarker to predict and trace 2OHOA treatment, studying the pharmacokinetics of glioma patients treated with the drug in phase I/IIA and its accumulation in mice xenograft tumors. In conclusion, 2OHOA shows a different incorporation into cell membranes between tumor cells and non-tumor cells. Moreover, both 2OHOA and its metabolite C17:1n-9 have an antiproliferative effect and exhibit an appealing potential in the treatment of patients that develop GBM with unmet effective therapy. Citation Format: Roberto Beteta-Göbel, Raquel Rodríguez-Lorca, Paula Fernández-García, Pablo V. Escribá, Victoria Lladó. Incorporation of the antitumor compound 2OHOA and its metabolite into cell membranes [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 4729.

Abstract 1996: Novel mechanism of temozolomide resistance in glioblastoma

Abstract Intrinsic expression of PD-1 has been discovered in different types of tumurs, exhibiting either oncogenic or tumor-suppressing function. In glioblastoma (GBM), intrinsic PD-1 has been found to play a role in promoting the proliferation and self-renewal of brain tumor-initiating cells through nuclear factor κB without involving PD-L1 ligation, with unknown factors activating this signalling pathway. Meanwhile, TMZ resistance significantly contributes to treatment failure and an extremely poor prognosis for GBM. This study investigated whether intrinsic PD-1 is also expressed in differentiated human GBM cell lines and its role in GBM proliferation and temozolomide (TMZ) resistance. In vitro studies demonstrated the presence of PD-1 in both U87 and U251 TMZ-sensitive and -resistant cell lines, with a higher expression on TMZ-resistant cell lines. PDCD1 knockdown led to decreased GBM progression, including cell proliferation, colony formation and cell migration, in TMZ-resistant U87 and U251 cell lines using cell viability assays. It also resensitizes these cell lines to TMZ. Overexpression of PDCD1 in the PDCD1-knockdown cell lines increases their proliferation and restores their resistance to TMZ. Treatment of U87 and U251 TMZ-resistant cell lines with TMZ upregulated PD-1 expression. An orthotopic xenograft mouse model is further deployed by injecting previously cultured U87 and U251 cell lines into the mouse brains. A smaller tumour size is observed in the model with PDCD1 knockdown model than the one without PDCD1 knockdown when treated with TMZ. This study reveals that intrinsic PD-1 expression contributes to TMZ resistance in GBM and TMZ may be a factor activating the PD-L1-independent PD-1 signalling. PD-1 and its downstream signalling pathway, such as SHP-2 and IKK, may be potential therapeutic targets for GBM patients with TMZ resistance. Co-administration of TMZ and agents inhibiting the intrinsic PD-1 signalling pathway can potentially produce synergistic therapeutic effects. The higher expression of PD-1 on TMZ-resistant GBM than on TMZ-sensitive GBM also suggests that the intrinsic PD-1 level can be a predictor of TMZ outcome. Citation Format: Yuet Yi Charmaine Hung, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Novel mechanism of temozolomide resistance in glioblastoma [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 1996.

Synthesis and Characterization of ZIF-90 Nanoparticles as Potential Brain Cancer Therapy.

Human glioblastoma is probably the most malignant and aggressive among cerebral tumors, of which it represents approximately 80% of the reported cases, with an overall survival rate that is quite low. Current therapies include surgery, chemotherapy, and radiotherapy, with associated consistent side effects and low efficacy. The hardness in reaching the site of action, and overcoming the blood-brain barrier, is a major limitation of pharmacological treatments. In this paper, we report the synthesis and characterization of ZIF-90 (ZIF, Zeolitic Imidazolate Framework) nanoparticles as putative carriers of anticancer drugs to the brain. In particular, we successfully evaluated the biocompatibility of these nanoparticles, their stability in body fluids, and their ability to uptake in U251 human glioblastoma cell lines. Furthermore, we managed to synthesize ZIF-90 particles loaded with berberine, an alkaloid reported as a possible effective adjuvant in the treatment of glioblastoma. These findings could suggest ZIF-90 as a possible new strategy for brain cancer therapy and to study the physiological processes present in the central nervous system.

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Comprehensive antifungal investigation of green synthesized silver nanoformulation against four agriculturally significant fungi and its cytotoxic applications

The present study reports the green synthesis of silver nanoparticles (AgNPs) in powder form using the leaf extract of Azadirachta indica. The synthesis of AgNPs was confirmed by UV–vis spectroscopy, FTIR, XRD, FESEM, and EDX. The synthesized AgNPs were in a powdered state and dispersed completely in 5% polyethylene glycol (PEG) and demonstrated prolonged shelf life and enhanced bioavailability over a year without any aggregation. The resulting silver nanoformulation demonstrated complete inhibition against Sclerotinia sclerotiorum and Colletotrichum falcatum and 68% to 80% inhibition against Colletotrichum gloeosporioides and Rhizoctonia solani respectively, at 2000 ppm. The EC50 values determined through a statistical analysis were 66.42, 157.7, 19.06, and 33.30 ppm for S. sclerotiorum, C. falcatum, C. gloeosporioides, and R. solani respectively. The silver nanoformulation also established significant cytotoxicity, with a 74.96% inhibition rate against the human glioblastoma cell line U87MG at 250 ppm. The IC50 value for the cancerous cell lines was determined to be 56.87 ppm through statistical analysis. The proposed silver nanoformulation may be used as a next-generation fungicide in crop improvement and may also find application in anticancer investigations. To the best of our knowledge, this is also the first report of silver nanoformulation demonstrating complete inhibition against the economically significant phytopathogen C. falcatum.