U87 Glioblastoma Multiforme Research Articles

Nanoplatforms for Magnetic-Photo-Heating of Thermo-Resistant Tumor Cells: Singular Synergic Therapeutic Effects at Mild Temperature.

A self-assemble amphiphilic diblock copolymer that can incorporate iron oxide nanocubes (IONCs) in chain-like assemblies as heat mediators for magnetic hyperthermia (MHT) and tuneable amounts of IR780 dye as agent for photothermal therapy (PTT) is developed. MHT-heating performance of photobeads in viscous media have the same heat performances in water at magnetic field conditions of clinical use. Thanks to IR780, the photobeads are activated by infrared laser light within the first biological window (808nm) with a significant enhancement of photo-stability of IR780 enabling the raise of the temperature at therapeutic values during multiple PTT cycles and showing unchanged optical features up to 8 days. Moreover, the photobeads fluorescent signal is preserved once internalized by glioblastoma multiforme (GBM) cells. Peculiarly, the photobeads are used as toxic agents to eradicate thermo-resistant GBM cells at mild heat, as low as 41°C, with MHT and PTT both of clinical use. Indeed, a high U87 GBM cell mortality percentage is obtained only with dual MHT/PTT while each single treatment dose not provide the same cytotoxic effects. Only for the combined treatment, the cell death mechanism is assigned to clear sign of apoptosis as observed by structural/morphological cell studies and enhanced lysosome permeability.

Ubiquitination of p21 by E3 ligase RNF135 promotes the proliferation of human glioblastoma cells.

Glioblastoma multiforme (GBM) is a highly heterogeneous tumor of the central nervous system with a high mortality rate. The upregulation of RING finger protein 135 (RNF135), an E3 ligase, has been observed in GBM, but the associated mechanisms have not been fully elucidated. The aim of the present study was to identify the substrate of RNF135 and study its functions in GBM. Bioinformatics analyses were performed. In addition, RNF135 was overexpressed or knocked down in human U87 and U251 GBM cells, and the effect on cell proliferation was analyzed using Cell Counting Kit-8 and colony formation assays. Furthermore, the interaction of RNF135 with its potential substrate was analyzed using glutathione S-transferase, yeast two-hybrid, immunoprecipitation (IP), co-IP and immunoblotting assays. Bioinformatics analysis indicated that RNF135 serves as a marker of poor prognosis in GBM. The overexpression of RNF135 was demonstrated to promote the proliferation of GBM cells, while the knockdown of RNF135 inhibited GBM cell growth. In addition, the results of the interaction experiments indicate that RNF135 interacts with p21 and mediates the degradation of p21 by ubiquitination. The major site of RNF135-mediated p21 ubiquitination was identified as K163. Further experiments demonstrated that RNF135 promotes the proliferation of GBM cells mainly via p21. In summary, these findings suggest that RNF135 has potential as a therapeutic target for the treatment of GBM.

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell's fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Abstract 427: A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype

Abstract Background: Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. Objective: We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. Methods: In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-β-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Results: Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-β-induced EMT biomarker SNAIL and cell chemotaxis. Conclusion: Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets. Citation Format: Sima Torabidastgerdooei, Borhane Annabi. A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype [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 427.

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

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

Moraea sisyrinchium inhibits proliferation, cell cycle, and migration of cancerous cells, and decreases angiogenesis in chick chorioallantoic membrane.

Experimental studies reported that some plants in the genus of Moraea (Iridaceae family) show anticancer potential. This study aimed to evaluate the effects of Moraea sisyrinchium on U87 glioblastoma multiforme and HepG2 liver cancer cells. The cells were incubated for 24 hr with hydroalcoholic extract of the stem, flower, and bulb of M. sisyrinchium. Then, the cell proliferation (MTT) assay, cell cycle analysis (propidium iodide staining), cell migration test (scratch), Western blotting (Bax and Bcl-2 expression), and gelatin zymography (for matrix metalloproteinases, MMPs) were performed. Oxidative stress was evaluated by determining the levels of reactive oxygen species and lipid peroxidation. Angiogenesis was evaluated on chick embryo chorioallantoic membrane. The extracts of the flower, stem, and bulb significantly decreased the proliferation of HepG2 and U87 cells. This effect was more for U87 than HepG2 and for the bulb and stem than the flower. In U87 cells, the bulb extract increased oxidative stress, cell cycle arrest, and the Bax/Bcl-2 ratio. Also, this extract suppressed the migration ability of HepG2 and U87 cells, which was associated with the inhibition of MMP2 activity. In addition, it significantly reduced the number and diameter of vessels in the chorioallantoic membrane. Liquid chromatography-mass spectrometry revealed the presence of xanthones (bellidifolin and mangiferin), flavonoids (quercetin and luteolin), isoflavones (iridin and tectorigenin), and phytosterols (e.g., stigmasterol) in the bulb. M. sisyrinchium bulb decreased the proliferation and survival of cancer cells by inducing oxidative stress. It also reduced the migration ability of the cells and inhibited angiogenesis.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype.

Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-β-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-β-induced EMT biomarker SNAIL and cell chemotaxis. Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.

Hyaluronan decorated layer-by-layer assembled lipid nanoparticles for miR-181a delivery in glioblastoma treatment

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer. Current pharmacological interventions marginally increase the 12-month overall survival of patients with GBM. Among the novel therapeutic strategies being pursued, micro-RNAs, a class of non-coding RNAs, are receiving considerable attention for their regulation of several pathways implicated in tumorigenesis and survival. Notably, microRNA-181a-5p (miR-181a) has consistently been reported to be downregulated in GBM clinical samples, and its overexpression negatively affects tumor growth both in vitro and in vivo. To improve the delivery of miR-181a to GBM cells, we sought to develop a modified lipid-based nanocarrier capable of encapsulating and delivering miR-181a to GBM cells in vitro and in vivo. Optimized ionizable-lipid containing lipid nanoparticles (LNP) were constructed by covering the miR-181a-loaded LNP with alternating layers of miR-181a, poly-l-arginine and hyaluronic acid through the layer-by-layer technique. The resulting hyaluronan-decorated lipid nanoparticles (HA-LNP) targeted GBM cells more efficiently than non-modified LNP and mediated siRNA and miRNA transfection in vitro. Finally, delivery of miR-181a by HA-LNP induced significant cellular death of U87 GBM cells in vitro and delayed tumor growth in an in vivo subcutaneous tumor model.

P02.20.B PLASMA MEMBRANE-ASSOCIATED 70 KDA HEAT SHOCK PROTEIN (HSP70) INVOLVED IN GLIOBLASTOMA CELLS INVASION AS A TARGET FOR TUMOR THERANOSTICS

Abstract BACKGROUND Heat shock proteins (HSPs) represent a large family of highly conserved proteins serving as molecular chaperones that play an important role in intracellular homeostasis. Apart from their intracellular localization, members of various HSPs families such as HSP70 have been reported to be expressed on the plasma membrane of malignant cells, including glioblastoma multiforme (GBM). However, the role of the membrane-bound protein Hsp70 has not yet been investigated in the pathophysiology of GBM. MATERIAL AND METHODS mHsp70 expression as related to GBM cell invasive potential was analyzed in biopsies obtained from neurooncological patients using CellVoyager CQ1 Benchtop High-Content Analysis System (Yokogawa) and inverted confocal microscopy (Leica Microsystems). Application of membrane-associated chaperone for targeted therapies was assessed in the intracranial human NCH644, NCH421k, and U87 GBM models in immunodeficient mice employing various chaperone inhibitors (i.e., JG-98, MKT-077, pifithrin-μ (PES)) as a monotherapy or in combination with a single dose of stereotactic irradiation (10Gy) (SARRP). RESULTS Live-cell imaging of the patient-derived biopsies demonstrated the increased cellular density of mHsp70+ GBM cells particularly in the tissue along the tumor-brain margin. Time-lapse evaluation of the tumor sample further proved the migration of mHsp70+ cells into the surrounding normal brain tissues. Subsequent high-powered multiplex immunofluorescence analysis (Akoya Biosystems) confirmed significantly increased single-cell infiltration of mHsp70+ tumor cells (also positively stained for Nestin, Oct4, and SOX2) in the normal brain tissues. When mHsp70 inhibitors were applied in GBM animal models as a monotherapy or in combination with radiotherapy dramatic inhibition of tumor progression (as shown by MRI) and increased animal survival were observed. CONCLUSION Expression of the mHsp70 on the plasma membrane of GBM cells corresponds to the highly invasive potential of the tumor cells in neuro-oncological patients. Administration of the HSP70 small molecular inhibitors significantly decreased the GBM growth and invasion thus prolonging the overall survival of tumor-bearing mice. Further development of mHsp70-targeted agents represents a promising approach in treatment of multiforme glioblastoma.

SYST-12 LP-184, A NOVEL ACYLFULVENE-DERIVED TUMOR SITE ACTIVATED SMALL MOLECULE INHIBITS ADULT AND PEDIATRIC CNS TUMOR CELL GROWTH

Abstract Blood-brain barrier (BBB) permeable agents effective against recurrent, chemotherapy-resistant central nervous system (CNS) tumors are urgently needed, particularly in Glioblastoma multiforme (GBM) and atypical teratoid/rhabdoid tumors (ATRT). These represent extremely aggressive and lethal types of CNS malignancies. LP-184 is a next-generation acylfulvene class drug candidate and has received FDA orphan drug designations for the treatment of malignant gliomas and ATRT. LP-184 creates covalent DNA adducts that are selectively repaired via nucleotide excision repair (NER). Repair of LP-184-induced DNA damage is mediated by ERCC complexes. Consistent with this, we observed 3-6X decreased cancer cell viability in U87, M1123 and Mayo39 GBM cells treated with LP-184 in combination with the ERCC3 degrader Spironolactone (SP) relative to LP-184 alone. Mice bearing pre-established subcutaneous U87 xenografts were treated with SP alone, LP-184 alone or their combination. Tumors recurred after initial regression in 5/5 animals treated with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals treated with LP-184 + SP. Thus, LP-184 likely functions as a synthetically lethal agent in the presence of DNA repair deficiencies. LP-184 inhibited cell growth in CHLA06 and BT37 ATRT cell lines, with IC50s in the 20-25nM range. LP-184 induced apoptosis 96 hours after treatment in these ATRT cell lines as measured by immunofluorescence for cleaved caspase-3 and Western blot for c-PARP. LP-184 intravenous treatment at 4 mg/kg twice weekly for 2 weeks showed tumor regression in mice implanted with CHLA06 ATRT subcutaneous xenografts, with 112% tumor growth inhibition and 2/10 treated mice being tumor-free at study termination. LP-184 further showed favorable intracranial tumor bioavailability along with in vivo BBB permeability comparable to other CNS cancer drugs. These findings identify LP-184 as a promising new agent and support its further development for treating CNS tumors in adult and pediatric populations.

α-Solanine Suppresses Glioblastoma Multiforme Growth and Metastatic Properties by Modulating the Apoptosis-autophagy Axis

Introduction: Glioblastoma multiforme (GBM) has a poor prognosis despite optimal treatment. Recent studies have shown the potential of phytochemicals as anti-cancer agents. α-solanine, derived from plants of the Solanum genus, is a promising molecule in this regard. This study investigated the efficacy of α-solanine compared to temozolomide (TMZ) against GBM cell lines (U87MG, U251, and T98G) in vitro.
 Methods: In-vitro assays were conducted to assess the viability, migration, invasion, and mode of cell death of U87MG, U251 and T98G GBM cell lines following α-solanine treatment in comparison to TMZ. Rt-qPCR and proteome profiling were conducted to investigate the changes induced by α-solanine on a molecular level.
 Results: α-solanine demonstrated potent cytotoxicity on all GBM lines, with IC50 values ranging from 19.66 µM to 22.87 µM between cell lines tested, and significantly inhibited GBM cell migration compared to TMZ treatment. RNA and protein level assays indicated upregulation of both apoptotic and autophagy proteins, suggesting the involvement of BECN1 and BNIP3L in α-solanine-induced cell death.
 Discussion and Conclusion: These findings contribute to the search for effective GBM treatments. Future studies should increase the number of biological replicates, employ alternative methods to strengthen the findings, and conduct in vivo experiments and testing using patient-derived GBM tissue to better evaluate any therapeutic suitability of and fully understand the mode of action of α-solanine on GBM.

Targeting glioblastoma multiforme using a novel fusion protein comprising interleukin-13 and staphylococcal enterotoxin B in vitro

Targeting cell surface receptors with immunotoxins provides a novel, unique and highly potent treatment against cancers. A high expression of interleukin-13 (IL13) receptor α2 (IL13Rα2) has been reported in different types of cancers including glioblastoma multiforme (GBM). In this paper, to target IL13Rα2 on GBM cells, a fusion protein was generated comprising human IL13 and staphylococcal enterotoxin B (SEB), termed IL13-linker-SEB. The fusion protein was cloned into pET28a(+) and expressed in Escherichia coli strain BL21 (DE3); U251 (IL13Rα2-positive) and T98G (IL13Rα2-negative) GBM cell lines were employed and the functional activity of IL13-linker-SEB was evaluated by cell ELISA, cytotoxicity (MTT and LDH), apoptosis (flow cytometry and caspase-3 activity), adhesion, scratch and RT-PCR tests. SEB and chemotherapeutic drugs were employed to be compared to IL13-linker-SEB function. The IL13-linker-SEB exhibited higher binding affinity and cytotoxicity compared to SEB on U251 cells, although both recombinant proteins had shown similar behavior regarding T98G cells. Furthermore, the highest induction of apoptosis was observed in U251 cells treated with IL13-linker-SEB which was confirmed by Bax/Bcl-2 ratio. The expression of MMP2, MMP9 and VEGFR2 in U251 cells experienced a significant reduction after treatment with IL13-linker-SEB compared to SEB and T98G treated cells. The data showed that IL13-linker-SEB can be considered as a novel potential agent for GBM treatment; however, further research is needed to investigate the efficacy.

Targeted delivery of temozolomide by nanocarriers based on folic acid-hollow TiO2 -nanospheres for the treatment of glioblastoma

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Its standard treatment includes a combination of surgery, radiation, and chemotherapy. The last involves the oral delivery of free drug molecules to GBM such as Temozolomide (TMZ). However, this treatment has limited effectiveness owing to the drugs premature degradation, lack of cell selectivity, and poor control of pharmacokinetics. In this work, the development of a nanocarrier based on hollow titanium dioxide (HT) nanospheres functionalized with folic acid (HT-FA) for the targeted delivery of temozolomide (HT-TMZ-FA) is reported. This approach has the potential benefits of prolonging TMZ degradation, targeting GBM cells, and increasing TMZ circulation time. The HT surface properties were studied, and the nanocarrier surface was functionalized with folic acid as a potential targeting agent against GBM. The loading capacity, protection from degradation, and drug retention time were investigated. Cell viability was performed to assess the cytotoxicity of HT against LN18, U87, U251, and M059K GBM cell lines. The cell internalization of HT configurations (HT, HT-FA, HT-TMZ-FA) was evaluated to study targeting capabilities against GBM cancer. Results show that HT nanocarriers have a high loading capacity, retain and protect TMZ for at least 48 h. Folic acid-functionalized HT nanocarriers successfully delivered and internalized TMZ to glioblastoma cancer cells with high cytotoxicity through autophagic and apoptotic cellular mechanisms. Thus, HT-FA nanocarriers could be a promising targeted delivery platform for chemotherapeutic drugs for the treatment of GBM cancer.

Synthetic miR-21 decoy circularized by tRNA splicing mechanism inhibited tumorigenesis in glioblastoma in vitro and in vivo models

Glioblastoma multiforme (GBM) is the deadliest primary central nervous system tumor. miRNAs (miRs), a class of non-coding RNAs, are considered pivotal post-transcriptional regulators of cell signaling pathways. miR-21 is a reliable oncogene that promotes tumorigenesis of cancer cells. We first performed an in silico analysis on 10 microarray datasets retrieved from TCGA and GEO databases to elucidate top differentially expressed miRs. Furthermore, we generated a circular miR-21 decoy, CM21D, using the tRNA-splicing mechanism in GBM cell models, U87 and C6. The inhibitory efficacy of CM21D with that of a linear form, LM21D, was compared under invitro conditions and an intracranial C6 rat glioblastoma model. miR-21 significantly overexpressed in GBM samples and confirmed in GBM cell models using qRT-PCR. CM21D was more efficient than LM21D at inducing apoptosis, inhibiting cell proliferation and migration, and interrupting the cell cycle by restoring the expression of miR-21 target genes at RNA and protein levels. Moreover, CM21D suppressed tumor growth more effectively than LM21D in the C6-rat GBM model (p<0.001). Our findings validate miR-21 as a promising therapeutic target for GBM. The introduced CM21D by sponging miR-21 reduced tumorigenesis of GBM and can be considered a potential RNA-base therapy to inhibit cancers.

Psidium guajava induces cytotoxicity in human malignant glioblastoma cell line: Role of reactive oxygen species

One of the deadliest types of CNS primary brain cancers is glioblastoma multiforme (GBM), and the survival rate of patients is about 7.2%. The standard treatment for GBM is surgical interventions followed by temozolomide. We investigated for the first time, the cytotoxic impacts of Psidium guajava (P. guajava) on the U87 GBM cell line. We measured cell toxicity through the MTT test following 24 h, 48 h, and 72 h treatment with different concentrations of fruit and seed hydroalcoholic extracts of P. guajava (25–400 μg/ml). Lipid peroxidation assay, reactive oxygen species (ROS) production, and apoptosis rate were evaluated 24 h after treatment by extracts of P. guajava. Moreover, to determine the Bax/Bcl-2 and NF-κB genes expression, we performed a real-time polymerase chain reaction (RT-PCR). Our finding demonstrated that 50–400 μg/ml of P. guajava extracts dose-dependently decreased the viability of U87 cells. Also, treatment by extracts increased lipid peroxidation, ROS production, and apoptosis in a dose-dependent manner. Moreover, the RT-PCR demonstrated an up-regulation in Bax\\Bcl-2 and NF-κB. Thus, P. guajava inhibited the proliferation of U87 GBM cells and increased apoptosis probably through Bax/Bcl-2 and NF-κB regulation.

The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor with a poor median survival of less than 15 months. However, clinical strategies and effective therapies are limited. Here, we found that the second-generation small molecule multi-CDK inhibitor AT7519 is a potential drug for GBM treatment according to high-throughput screening via the Approved Drug Library and Clinical Compound Library (2718 compounds). We found that AT7519 significantly inhibited the cell viability and proliferation of U87MG, U251, and patient-derived primary GBM cells in a dose-dependent manner. Furthermore, AT7519 also inhibited the phosphorylation of CDK1/2 and arrested the cell cycle at the G1-S and G2-M phases. More importantly, AT7519 induced intrinsic apoptosis and pyroptosis via caspase-3-mediated cleavage of gasdermin E (GSDME). In the glioblastoma intracranial and subcutaneous xenograft assays, tumor volume was significantly reduced after treatment with AT7519. In summary, AT7519 induces cell death through multiple pathways and inhibits glioblastoma growth, indicating that AT7519 is a potential chemical available for GBM treatment.

DDEL-08. NOVEL COMBINATION THERAPY USING PI3KINASE INHIBITION AND IMMUNE BASED THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract Hurdles to effective treatments for glioblastoma multiforme (GBM), the most aggressive form of brain cancer, are resistance to chemotherapy, radiation, and immune checkpoint inhibitors. GBMs, like other “difficult to treat” tumors, often over-utilize the phosphatidyl inositol 3 kinase (PI3K) pathway for survival, cell growth, and cell division, conferring both chemotherapy and radiation resistance. Furthermore, the PI3K pathway has been shown to modulate expression of Major Histocompatibility Complex (MHC) molecules, key components of immune recognition. Based on these observations, we examined the potential of a novel, small molecule PI3K inhibitor GCT.Glio.1 (NPT520-337), which was designed to cross the blood brain barrier, to increase the sensitivity of GBM to immune based therapies and radiation. We hypothesized that targeting PI3K would result in growth arrest and increase cell surface expression of MHC molecules and the PD-L1 checkpoint target. Treatment of GL261 mouse and human U251 GBM cell lines with GCT.Glio.1 caused growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1 and MHC class II, and synergized with radiation to cause growth arrest and cell death. Based on these results we designed and performed in vivo animal studies, implanting luciferase-flagged GL261 into the caudate nucleus of C57BL6 mice. We treated the animals orally with GCT.Glio.1 followed 24 hours later by anti-PD1 therapy in repeating cycles. The treatments significantly delayed progression of the tumor when compared to no treatment or either treatment alone, with several animals exhibiting complete regression as measured by MRI and bioluminescence. In conclusion, these results indicate that GCT.Glio.1 may sensitize GBM to immune checkpoint inhibitor and/or radiation therapies. Based on our encouraging pre-clinical data and early results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using this combination therapy.

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

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

Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells.

Simple SummaryGlioblastoma multiforme (GBM) is the most aggressive and malignant type of central nervous system (CNS) tumour. Although several treatment options are available, patients generally succumb within 14 months after diagnosis. With the rapid progression of exosome bioengineering technologies, novel therapy opportunities are emerging for GBM treatment. The surface of GBM cells is characterised by the overexpression of transmembrane receptor integrins, which are essential for cell interactions with several proteins in the extracellular matrix. Therefore, integrin-binding drug delivery vehicles have been proposed as a potential strategy for glioblastoma therapy. Small extracellular vesicles possess several attractive characteristics for drug delivery: small size, biocompatibility, ability to cross the blood–brain barrier and capacity to be loaded with exogenous materials. Current bioengineering technologies further increase extracellular vesicle capabilities by loading them with anticancer drugs and incorporating targeting ligands. This study explored the capacity of Arginylglycylaspartic acid (RGD, or Arginine–Glycine–Aspartate)-polypeptide-engineered extracellular vesicles to internalise and deliver loaded cargo in GBM cells. The results demonstrate that introducing the RGD ligand to extracellular vesicles could significantly increase their internalisation by GBM cells and hence improve drug delivery efficacy. Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells.

Combinatorial delivery of CPI444 and vatalanib loaded on PEGylated graphene oxide as an effective nanoformulation to target glioblastoma multiforme: In vitro evaluation.

Glioblastoma multiforme (GBM) is known as the primary malignant and most devastating form of tumor found in the central nervous system of the adult population. The active pharmaceutical component in current chemotherapy regimens is mostly hydrophobic and poorly water-soluble, which hampers clinical implications. Nanodrug formulations using nanocarriers loaded with such drugs assisted in water dispersibility, improved cellular permeability, and drug efficacy at a low dose, thus adding to the overall practical value. Here, we successfully developed a water-dispersible and biocompatible nanocargo (GO-PEG) based on covalently modified graphene oxide (GO) with a 6-armed poly(ethylene glycol) amine dendrimer for effective loading of the two hydrophobic anticancer drug molecules, CPI444 and vatalanib. These drug molecules target adenosine receptor (A2AR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and type III stem cell receptor tyrosine kinase (c-KIT), which plays a crucial role in cancers. The effective cellular delivery of the drugs when loaded on GO-PEG is attributed to the increased permeability of the drug-nanoconjugate formulation. We observed that this combinatorial drug treatment with nanocargo resulted in a significant reduction in the overall cell survival as supported by reduced calcium levels and stem cell markers such as Oct4 and Nanog, which are two of the prime factors for GBM stem cell proliferation. Furthermore, reduced expression of CD24 upon treatment with nanoformulation impeded cellular migration. Cellular assays confirmed inhibition of cell proliferation, migration, and angiogenic potential of GBM treated with GO-PEG–Drug conjugates. Ultimately, GBM U87 cells assumed programmed cell death at a very low concentration due to nanocarrier-mediated drug delivery along with the chosen combination of drugs. Together, this study demonstrated the advantage of GO-PEG mediated combined delivery of CPI444 and vatalanib drugs with increased permeability, a three-pronged combinatorial strategy toward effective GBM treatment.