LN229 Glioblastoma Cells Research Articles

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.

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.

Neuropilin-1 enhances temozolomide resistance in glioblastoma via the STAT1/p53/p21 axis.

Glioblastoma (GBM) is the most prevalent primary intracranial tumor. Temozolomide (TMZ) is the first-line chemotherapy for GBM. Nonetheless, the development of TMZ resistance has become a main cause of treatment failure in GBM patients. Evidence suggests that neuropilin-1 (NRP-1) silencing can attenuate GBM cell resistance to TMZ. This study aims to determine potential mechanisms by which NRP-1 affects TMZ resistance in GBM. The parental U251 and LN229 GBM cells were exposed to increasing concentrations of TMZ to construct TMZ-resistant GBM cells (U251/TMZ, LN229/TMZ). BALB/c nude mice were injected with U251/TMZ cells to establish the xenograft mouse model. Functional experiments were carried out to examine NRP-1 functions. Western blotting and real-time quantitative polymerase chain reaction were used to evaluate molecular protein and mRNA expression, respectively. Immunohistochemical staining showed NRP-1 and STAT1 expression in mouse tumors. The results showed that NRP-1 was highly expressed in TMZ-resistant cells. Moreover, knocking down NRP-1 attenuated the TMZ resistance of U251/TMZ cells, while upregulating NRP-1 enhanced TMZ resistance of the parental cells. NRP-1 silencing elevated GBM cell sensitivity to TMZ in tumor-bearing mice. Depleting NRP-1 reduced STAT1, p53, and p21 expression in U251/TMZ cells. STAT1 depletion offset NRP-1 silencing evoked attenuation of GBM cell resistance to TMZ. Collectively, our study reveals that NRP-1 enhances TMZ resistance in GBM possibly by regulating the STAT1/p53/p21 axis.

High-Resolution Microscopic Characterization of Tunneling Nanotubes in Living U87 MG and LN229 Glioblastoma Cells.

Tunneling nanotubes (TNTs) are fine, nanometer-sized membrane connections between distant cells that provide an efficient communication tool for cellular organization. TNTs are thought to play a critical role in cellular behavior, particularly in cancer cells. The treatment of aggressive cancers such as glioblastoma remains challenging due to their high potential for developing therapy resistance, high infiltration rates, uncontrolled cell growth, and other aggressive features. A better understanding of the cellular organization via cellular communication through TNTs could help to find new therapeutic approaches. In this study, we investigate the properties of TNTs in two glioblastoma cell lines, U87 MG and LN229, including measurements of their diameter by high-resolution live-cell stimulated emission depletion (STED) microscopy and an analysis of their length, morphology, lifetime, and formation by live-cell confocal microscopy. In addition, we discuss how these fine compounds can ideally be studied microscopically. In particular, we show which membrane-labeling method is suitable for studying TNTs in glioblastoma cells and demonstrate that live-cell studies should be preferred to explore the role of TNTs in cellular behavior. Our observations on TNT formation in glioblastoma cells suggest that TNTs could be involved in cell migration and serve as guidance.

Genotoxic and Cytotoxic Activity of Fisetin on Glioblastoma Cells.

Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells. We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis. Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells. Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.

Zinc oxide nanoparticles exhibit anti-cancer activity against human cell lines

Zinc oxide nanoparticles have garnered tremendous interest in bio and nanomedical applications and more so in cancer research. In the present work, zinc oxide nanoparticles were synthesized using the solution combustion method and characterized by UV–vis spectroscopy, FTIR, XRD, SEM and EDX. The infrared spectrum of the synthesized nanoparticles had a peak at 547 cm−1 which indicated the elongation frequency of the zinc bond to oxygen. The XRD spectrum was confirmed with the ICDD Data Card No: 36-1451 for zinc oxide and the average size of zinc oxide nanoparticles was estimated to be 17.51 nm. Scanning electron microscopy confirmed the presence of zinc oxide nanoparticles as Zincite with a hexagonal structure having an average diameter of 30.95 nm. The DNA binding interactions of the nanoparticles were studied using the absorption titration method. The nanoparticles could bind via the non-intercalation mode with a binding efficiency of 3.563×105 M−1. The synthesized zinc oxide nanoparticles exhibited cytotoxic activity against the mammalian cancer cell lines tested with the calculated IC50 values being 3.66 µgmL−1 for MCF-7 (Breast), 2.91 µgmL−1 for LN-18 (Glioblastoma–Brain), 23.14 µgmL−1 for A-549 (Lung) and 94.33 µgmL−1 for SHSY-5Y (Neuroblastoma–Bone). The highest cell death was observed for LN-18 glioblastoma cells of the brain. The study demonstrated a simple and conventional method of synthesis of zinc oxide nanoparticles that have demonstrated anticancer activity as evidenced by the in vitro cell viability assays. The outcome of this study could be applied to develop zinc oxide nanoparticles as anticancer agents with further validation in vivo.

HB007 Administration Inhibits LN-229 and Patient-Derived Neurospheroid Glioblastoma Cell Growth With the Degradation of SUMO1 and Cell Cycle Regulator CDK4

Background and Hypothesis: Glioblastoma is the most common and malignant brain cancer and there is no effective therapy currently available to patients with this malignancy. Small ubiquitin-related modifier 1 (SUMO1) is a key regulator of cancer cell proliferation through its role in its modification of cellular proteins in various human cancers, especially glioblastoma. Degradation of SUMO1 through small molecule degrader, HB007, has been shown to inhibit growth in cancer cell lines and xenografts. Here, we hypothesize that HB007 can inhibit the glioblastoma cell growth through degradation of SUMO1 protein in glioblastoma cells and the cancer stem cell enriched neurospheres.
 Experimental Design: LN-229 glioblastoma cell viability was measured in response to increasing concentrations of HB007. LN-229 and patient-derived neurospheroid glioblastoma cells were cultured and seeded in 4 different plates at 1000 cells/ml concentrations before being treated with HB007 at increasing concentrations encircling the previously described IC50. Cells were then subjected to a SUMO lysis buffer and analyzed via western blot with antibodies specific to SUMO1, CDK4, and actin.
 Results: HB007 treated LN-229 cells exhibited an IC50 of 1.470μM. Western blot analysis confirmed the dose dependent reduction in SUMO-1-ylated proteins in HB007 treated cells. A reduction in CDK4 confirmed that cell progression is halted in a dose dependent manner in LN-229 and patient-derived neurospheroid glioblastoma cells when treated with HB007. Specificity of HB007 is towards SUMO1 with no nonspecific degradation of SUMO2/3.
 Conclusion: The cell growth of LN-229 and patient-derived neurospheroid glioblastoma cells was confirmed, through western blot, to be inhibited in a dose dependent manner by HB007. These results further establish the therapeutic potential of SUMO1 degraders as a novel anticancer drug for glioblastoma therapy. In the future, it is hoped that the bioavailability, potency, and blood brain barrier permeability can be improved to make this drug a potential treatment for patients.

Upregulation of the canonical signaling pathway of interferon-gamma is associated with glioblastoma progression.

Glioblastoma is a brain malignant tumor grade IV, highly invasive. Alterations in several signaling pathways are involved in glioblastoma development. In this work, we evaluated the IFN-γ canonical signaling pathway in glioblastoma cells and its effect on cell viability and migration. The levels of STAT1/pSTAT1, IRF1, and PD-L1 in LN-18 glioblastoma cells were analyzed using western blotting. Cell viability was evaluated by calcein-AM/propidium iodide assays, and a wound healing assay was used to study the migration of glioblastoma cells treated with IFN-γ. Our aim was to determine the expression of IFN-γ signaling elements in cell lines and tissue from glioblastoma samples and examine the relationship between these elements and the survival of glioblastoma patients. The following platforms were utilized for analysis: the CCLE (Cancer Cell Line Encyclopedia), UALCAN (University of Alabama at Birmingham Cancer data analysis Portal), GEPIA (Gene Expression Profiling Interactive Analysis), and GENT2 (Gene Expression patterns across Normal and Tumor tissues). Our results evidenced that IFN-γ signaling increases non-phosphorylated and phosphorylated STAT1 levels and promotes the upregulation of IRF1 and PD-L1 in glioblastoma cells. The activation of IFN-γ signaling increased cell migration without affecting the viability of glioblastoma cells. Furthermore, in silico analysis showed that the elements of IFN-γ signaling pathways (IFNGR1/IFNGR2/STAT1/IRF1) are upregulated in human glioblastoma samples. The upregulation of IFN-γ signaling was associated with shorter survival in glioblastoma patients. IFN-γ signaling pathway is upregulated in glioblastoma, displaying pro-tumor activity. Thus, IFN-γ signaling elements may be potential biomarkers and targets for treating glioblastoma.

Rare germline variants in POLE and POLD1 encoding the catalytic subunits of DNA polymerases ε and δ in glioma families

Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy.

Synergism of d-limonene and temozolomide on migratory and apoptotic behaviors of human glioblastoma cell lines.

Glioblastoma (GBM), which is a heterogeneous and aggressive type of brain tumor, is known for its poor survival outcomes. The treatment of GBM remains challenging primarily due to the drug resistance to the current standard therapeutic option, temozolomide (TMZ). Researchers are currently focusing on developing an appropriate alternative combinatorial therapeutic to enhance treatment outcomes. D-limonene (DL) is a monoterpene derived from citrus fruit. This study aims to assess the impact of combining DL with TMZ and explore its potential mechanism of action in U87MG and LN229 GBM cells. The effects of the combined treatment of DL and TMZ were assessed on various cellular aspects, including cell viability, anchorage-independent cell growth, and DNA damage. Furthermore, the influence of this combination on cell cycle progression, cell migration, and cell death was also investigated. The combination of DL+TMZ demonstrated a synergistic effect, resulting in reduced cell proliferation and suppressing the colony formation ability of a single cell. Treatment with DL and TMZ arrested the cells in G0/G1 phase. Furthermore, the DL+TMZ combination induced apoptosis by upregulating the expression of Bax, and Caspase (CASP)-3, while reducing the expression of the Bcl-2 gene in GBM cells. In addition, the combined treatment of DL+TMZ significantly decreased the expression of matrix metalloproteinase (MMP)-2 and MMP-9, expression, indicating inhibition of cell migration in GBM cells. In conclusion, the combination of DL and TMZ demonstrated a synergistic effect in reducing cell proliferation, suppressing colony formation, inducing apoptosis, and inhibiting cell migration in GBM cells. These findings suggest the potential of DL+TMZ combination therapy as an effective treatment for GBM.

Comparative analysis of α-pinene alone and combined with temozolomide in human glioblastoma cells

α-Pinene (PEN) is a phyto compound present in terpene plants. In traditional medicine, PEN has been used for its anti-inflammatory, pain-relieving, and bronchodilator properties. The effect of PEN in combination with temozolomide (TMZ) in glioblastoma multiforme (GBM) cells has been evaluated. The action of the PEN + TMZ combination on cell migration, soft-agar, and cell death was determined in LN229 and U87MG human glioblastoma cells. In combination, PEN with TMZ showed a synergistic inhibitory effect in the GBM cells. The PEN + TMZ treatment showed a higher fluorescent intensity and reduced the percentage of wound area closure compared to the compound alone. The compounds in combination also resulted in a reduction in single-cell colony formation. To conclude, the study showed that plant-derived PEN enhanced the effectiveness of standard chemotherapeutic, TMZ, in LN229 and U87MG cells.

Bio-fabrication of gold nanoparticles from brown seaweeds for anticancer activity against glioblastoma through invitro and molecular docking approaches

This research aimed to investigate the mechanism of glioblastoma-killing action of brown seaweed Spatoglossum asperum fabricated gold nanoparticles (Sa-AuNPs) as a novel medication. UV spectroscopy revealed the surface plasmon resonance peak at 530 nm for the stability of bio-fabricated Sa-AuNPs. The XRD and SEM analysis affirmed the Sa-AuNPs were crystalline condition and cubic shapes with 20 nm size. The reducing and capping agents of organic compounds present in the Sa-AuNPs were identified by FTIR, NMR and Mass spectroscopic analysis. In addition, Sa-AuNPs showed excellent antioxidant activity through maximum inhibition of DPPH radicals (73.21%) and hydrogen peroxide radicals (68.9%). The biosynthesized Sa-AuNPs showed potent cytotoxic activity against LN-18 Glioblastoma cells (IC50 = 13.38 μg/mL). Furthermore, 11 genes involved in glioma cancer (as protein) and 10 compounds in the S. asperum (as Ligand) were picked for molecular docking. The docking result showed, among the 10 selected compounds, stigmast-5-en-3-ol has an effective binding affinity with 11 genes and effectively targeted EGFR, IDH and PTEN. This study concludes that Sa-AuNPs have potent antioxidant and anticancer activity due to the presence of bioactive molecules involved in the nanoparticle's reduction process. The research provides strong support for the prospective development of innovative treatments for glioblastoma.

RBBP4 regulates the expression of the Mre11-Rad50-NBS1 (MRN) complex and promotes DNA double-strand break repair to mediate glioblastoma chemoradiotherapy resistance

For treatment of glioblastoma (GBM), temozolomide (TMZ) and radiotherapy (RT) exert antitumor effects by inducing DNA double-strand breaks (DSBs), mainly via futile DNA mismatch repair (MMR) and inducing apoptosis. Here, we provide evidence that RBBP4 modulates glioblastoma resistance to chemotherapy and radiotherapy by recruiting transcription factors and epigenetic regulators that bind to their promoters to regulate the expression of the Mre11-Rad50-NBS1(MRN) complex and the level of DNA-DSB repair, which are closely associated with recovery from TMZ- and radiotherapy-induced DNA damage in U87MG and LN229 glioblastoma cells, which have negative MGMT expression. Disruption of RBBP4 induced GBM cell DNA damage and apoptosis in response to TMZ and radiotherapy and enhanced radiotherapy and chemotherapy sensitivity by the independent pathway of MGMT. These results displayed a possible chemo-radioresistant mechanism in MGMT negative GBM. In addition, the RBBP4-MRN complex regulation axis may provide an interesting target for developing therapy-sensitizing strategies for GBM.

Metabolic Adjustments following Glutaminase Inhibition by CB-839 in Glioblastoma Cell Lines.

Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes: GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.

Evaluating glioblastoma tumour sphere growth and migration in interaction with astrocytes using 3D collagen-hyaluronic acid hydrogels.

Glioblastoma (GB) is an astrocytic brain tumour with a low survival rate, partly because of its highly invasive nature. The GB tumour microenvironment (TME) includes its extracellular matrix (ECM), a variety of brain cell types, unique anatomical structures, and local mechanical cues. As such, researchers have attempted to create biomaterials and culture models that mimic features of TME complexity. Hydrogel materials have been particularly popular because they enable 3D cell culture and mimic TME mechanical properites and chemical composition. Here, we used a 3D collagen I-hyaluronic acid hydrogel material to explore interactions between GB cells and astrocytes, the normal cell type from which GB likely derives. We demonstrate three different spheroid culture configurations, including GB multi-spheres (i.e., GB and astrocyte cells in spheroid co-culture), GB-only mono-spheres cultured with astrocyte-conditioned media, and GB-only mono-spheres cultured with dispersed live or fixed astrocytes. Using U87 and LN229 GB cell lines and primary human astrocytes, we investigated material and experiment variability. We then used time-lapse fluorescence microscopy to measure invasive potential by characterizing the sphere size, migration capacity, and weight-averaged migration distance in these hydrogels. Finally, we developed methods to extract RNA for gene expression analysis from cells cultured in hydrogels. U87 and LN229 cells displayed different migration behaviors. U87 migration occurred primarily as single cells and was reduced with higher numbers of astrocytes in both multi-sphere and mono-sphere plus dispersed astrocyte cultures. In contrast, LN229 migration exhibited features of collective migration and was increased in monosphere plus dispersed astrocyte cultures. Gene expression studies indicated that the most differentially expressed genes in these co-cultures were CA9, HLA-DQA1, TMPRSS2, FPR1, OAS2, and KLRD1. Most differentially expressed genes were related to immune response, inflammation, and cytokine signalling, with greater influence on U87 than LN229. These data show that 3D in vitro hydrogel co-culture models can be used to reveal cell line specific differences in migration and to study differential GB-astrocyte crosstalk.

2,5-Bis(2,2,2-trifluoroethoxy)phenyl-tethered 1,3,4-Oxadiazoles Derivatives: Synthesis, In Silico Studies, and Biological Assessment as Potential Candidates for Anti-Cancer and Anti-Diabetic Agent.

In the present work, a series of new 1-{5-[2,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,3,4-oxadiazol-3-acetyl-2-aryl-2H/methyl derivatives were synthesized through a multistep reaction sequence. The compounds were synthesized by the condensation of various aldehydes and acetophenones with the laboratory-synthesized acid hydrazide, which afforded the Schiff's bases. Cyclization of the Schiff bases yielded 1,3,4-oxadiazole derivatives. By spectral analysis, the structures of the newly synthesized compounds were elucidated, and further, their anti-cancer and anti-diabetic properties were investigated. To examine the dynamic behavior of the candidates at the binding site of the protein, molecular docking experiments on the synthesized compounds were performed, followed by a molecular dynamic simulation. ADMET (chemical absorption, distribution, metabolism, excretion, and toxicity) prediction revealed that most of the synthesized compounds follow Lipinski's rule of 5. The results were further correlated with biological studies. Using a cytotoxic assay, the newly synthesized 1,3,4-Oxadiazoles were screened for their in vitro cytotoxic efficacy against the LN229 Glioblastoma cell line. From the cytotoxic assay, the compounds 5b, 5d, and 5m were taken for colony formation assay and tunnel assay have shown significant cell apoptosis by damaging the DNA of cancer cells. The in vivo studies using a genetically modified diabetic model, Drosophila melanogaster, indicated that compounds 5d and 5f have better anti-diabetic activity among the different synthesized compounds. These compounds lowered the glucose levels significantly in the tested model.

ONX-0914 Induces Apoptosis and Autophagy with p53 Regulation in Human Glioblastoma Cells.

Glioblastoma is believed to be one of the most aggressive brain tumors in the world. ONX-0914 (PR957) is a selective inhibitor of proteasome subunit beta type-8 (PSMB8). Previous studies have shown that inhibiting PSMB8 expression in glioblastoma reduces tumor progression. Therefore, this study aimed to determine whether ONX-0914 has antitumor effects on human glioblastoma. The results indicated that ONX-0914 treatment inhibited survival in LN229, GBM8401, and U87MG glioblastoma cells. Cell cycle analysis showed that ONX-0914 treatment caused cell cycle arrest at the G1 phase and apoptosis in glioblastoma cells. The protein expression of BCL-2 was reduced and PARP was cleaved after ONX-0914 treatment. Furthermore, the levels of p53 and phosphorylated p53 were increased by ONX-0914 treatment in glioblastoma cells. ONX-0914 also induced autophagy in glioblastoma cells. Furthermore, the p53 inhibitor pifithrin attenuated apoptosis but enhanced autophagy caused by ONX-0914. In an orthotopic mouse model, TMZ plus ONX-0914 reduced tumor progression better than the control or TMZ alone. These data suggest that ONX-0914 is a novel therapeutic drug for glioblastoma.

TMIC-34. INVESTIGATING THE EFFECT OF IRON IN GLIOBLASTOMA CELL POLARIZATION AND MIGRATION

Abstract Glioblastoma represents one of the most difficult-to-treat malignancies as evidenced by the poor prognosis associated with a diagnosis. The ability of glioblastoma cells to diffusely infiltrate into healthy brain tissue renders complete surgical resection challenging. Consequently, a large majority of glioblastoma patients end up with recurrent disease despite receiving maximally feasible surgical resection and rigorous chemoradiation. This work examined how modulation of cellular iron levels in T98G and LN229 glioblastoma cells impacted migratory capacity. Treatment of T98G or LN229 glioblastoma cells with iron in the form of ferric ammonium citrate (FAC) resulted in significantly reduced migration as assessed by time-lapse phase contrast imaging and wound healing assays. The iron-induced reduction in migration was able to be rescued by the addition of equimolar concentrations of deferoxamine, an iron chelator. Cellular proliferation in response to the iron treatments was quantified using both optical confluence and nucleic-acid-based proliferation assays and it was found that iron treatment at the concentrations used for the migration assays (0 – 300 µM FAC) did not result in reduced proliferation. Mechanistically probing iron’s impact on cell migration revealed that addition of iron resulted in decreased expression of Cdc42, a Rho GTPase that is essential to determining cellular polarity during migration. Functional cellular polarization assays further confirmed that reduced expression of Cdc42 corresponded to reduced cellular polarization. Bioinformatic analysis of CDC42 transcripts revealed the presence of potential iron-responsive-elements that may drive the iron-induced reduction in Cdc42 expression. This work highlights the importance of iron biology in impacting glioblastoma cell phenotype and potentially glioblastoma patient outcomes.

Integration of synthetic and natural derivatives revives the therapeutic potential of temozolomide against glioma- an in vitro and in vivo perspective

AimsMalignant gliomas constitute one of the deadly brain tumors with high degeneration rate. Though temozolomide (TMZ) is the first-line drug for glioma, its efficacy has decreased due to chemo-resistance. Repurposing synthetic and natural compounds have gained increasing interest in glioma. Hence, we combined chloroquine (CHL) a synthetic drug, naringenin (NAR) and phloroglucinol (PGL) (natural derivatives), to investigate whether the apoptotic effect of these drugs both alone and in combination, enhances the anti-tumor effects of TMZ in an in vitro and in vivo orthotopic xenograft glioma model. Main methodsThe cytotoxic effect of the drugs was assessed in C6 (murine) glioma cells, U-87 MG and LN229 (human) glioblastoma cells, primary astrocytes (isolated from rat brain tissues) and HEK-293 T cells. Mitochondrial depolarization and alterations in the cell cycle was determined by confocal imaging and flow cytometry. The expression of angiogenic and apoptotic markers was evaluated using qRT-PCR and ELISA. The efficacy of the combinatorial treatment was assessed in an orthotopic xenograft model using U-87 MG cells. Key findingsThe combinatorial treatment inhibited cell proliferation, induced apoptosis and contributed to cell cycle arrest in glioma cells. The quadruple combinatorial cocktail down-regulated BCL-2 with a concomitant decrease in VEGF. As observed in vitro, the quadruple combinatorial treatment enhanced the median survival of glioma-induced rats with lower cellularity rate. SignificanceThe combination of CHL, NAR and PGL synergistically potentiated the efficacy of TMZ on glioma in vitro and in vivo. Hence, this combination may characterize an advanced strategy for glioma treatment, thereby providing a possible translation to clinical trial.

SS-4 is a highly selective small molecule inhibitor of STAT3 tyrosine phosphorylation that potently inhibits GBM tumorigenesis in vitro and in vivo

Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of ∼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.