Therapeutic Agent For Glioblastoma Research Articles

Abstract 655: Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade

Abstract Glioblastoma is a highly aggressive brain tumor for which there is no cure. The dire prognosis of this disease is largely attributable to a high level of heterogeneity, including the presence of a sub-population of tumor initiating glioblastoma stem-like cells (GSCs), which are refractory to chemo- and radiotherapy. Here, in an unbiased marine-derived fungal extract screen, together with bioguided dereplication based on high resolution mass spectrometry, we identified malformin C to preferentially induce cell death in patient-derived GSCs, and explore the potential of this cyclic peptide as a therapeutic agent for glioblastoma. Malformin C significantly reduced tumor growth in an in vivo xenograft model of glioblastoma. Using transcriptomics and chemoproteomics, we found that malformin C binds to many proteins leading to aggregation, and rapidly induces unfolded protein response, including autophagy, in GSCs. Malformin C also appears to accumulate in lysosomes, disrupting autophagic flux and driving cells to death. Supporting this, malformin C synergizes with chloroquine, an inhibitor of autophagy. Strikingly, we observed that autophagic flux is differentially regulated in GSCs compared with normal astrocytes. The sensitivity of GSCs to malformin C highlights the relevance of proteostasis and autophagy as a therapeutic vulnerability in glioblastoma. Citation Format: Emma L. Phillips, Sizèd van Enk, Sara Kildgaard, Silja Schlue, Mona Göttmann, Victoria Jennings, Frederic Bethke, Gabriele Müller, Christel Herold-Mende, Daniel Pastor-Flores, Martin Schneider, Dominic Helm, Thomas Ostenfeld Larsen, Violaine Goidts. Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade [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 655.

DDDR-13. ST-401, A BRAIN PENETRATING MICROTUBULE TARGETING AGENT THAT KILLS GLIOBLASTOMA IN INTERPHASE AND BY DISRUPTING MITOCHONDRIA FUNCTION

Abstract The development of small molecular entities that can effectively penetrate the blood-brain barrier and kill glioblastoma (GBM) is crucial in Neuro-Oncology. ST-401, a unique microtubule depolymerizer, has shown remarkable potential as a therapeutic agent for GBM in PD-GBM isolates (nanomolar potencies) and in vivo preclinical GBM models (potencies DNA-damaging agents). This study aims to investigate the novel mechanism of action of ST-401 and its impact on GBM cell death. We recently showed that increasing concentrations of ST-401 disrupts mitochondrial function, leading to cell death initially in interphase and then in mitosis. Through single-cell RNA sequencing analysis, significant changes in mRNA related to mitochondrial functions were observed, including ribosomal proteins and OXPHOS genes. The study focuses on the role of mitochondria in GBM tumorigenesis and explores how ST-401 affects mitochondrial OXPHOS function. We identified three GBM cell lines (U251, SNB-19, and SF-539) with increasing vulnerability to ST-401, respectively, as measured by using WST-1, an index of mitochondria OXPHOS. Further investigation using the Seahorse platform demonstrated that SF-539 cells treated with ST-401 exhibited a loss of spare capacitance and significantly reduced mitochondrial function, including basal and maximal OXPHOS response, as well as ATP production. In conclusion, our results emphasize the existence of specific subtypes of GBM that show vulnerability or resistance to the antitumor effects of ST-401. Identifying these subtypes is crucial for advancing precision Neuro-Oncology and facilitating human trials. Our results also suggest that ST-401's inhibition of microtubule function and induction of cell death in interphase are associated with the loss of mitochondrial respiratory function. Given the limited treatment options for brain tumors and the challenges in delivering drugs across the blood-brain barrier, the development of brain-penetrant drugs like ST-401 holds great promise. Funded by NIH (CA244213 to NS and NS106924 to NS).

A functional carbon dots induce ferroptosis by suppressing PLPP4 activity to inhibit glioblastoma growth

Glioblastoma is a fatal primary brain cancer lacking effective therapeutic drugs. The presence of the blood–brain barrier (BBB) severely hinders the delivery of drugs to the brain. In recent years, nanoparticles, especially carbon dots (CDs), are promising drug delivery strategy for CNS diseases. Here, we synthesized a novel carbon dots（MGA-CDs）for glioblastoma treatment by hydrothermal method using metformin and gallic acid as precursor. MGA-CDs shows dominant BBB permeability and sensitive anti-tumor activity. In addition, MGA-CDs exhibits significant capability of targeting tumor cells mitochondria without the aid of anyextra targeting molecules, resulting in the shrunken of mitochondria and reduced numbers of mitochondrial cristae. Transcriptome profiling suggested that MGA-CDs disturbs the glycerophospholipid metabolism pathway by inhibiting the expression of PLPP4, thereby inducing ferroptosis. The efficient therapeutic potency of MGA-CDs is further confirmed in human-derived orthotopic glioblastoma mice model. MGA-CDs significantly inhibited the growth of intracranial tumors and prolonged the survival of tumor-bearing mice. This work presents a viable strategy that development of CDs-based novel therapeutic agent for glioblastoma.

The location of estrogen receptor variant ER-α36 is associated with the invasion of glioblastoma

Glioblastoma (GBM) is the most common central nervous system tumor and is associated with poor outcomes. There have been no significant improvements in GBM mortality in recent decades. ER-α36 is a variant of ER-α66 that may be involved in carcinoma growth and proliferation via genomic and nongenomic mechanisms. This variant might play an essential role in tamoxifen resistance of several tumors. Previously, our laboratory found that ER-α36 is expressed in GBM and participates in proliferation; nevertheless, the role of ER-α36 in GBM invasion remains unknown. This study aimed to determine the effects of the ER-α36 modulator SNG162 on GBM growth and invasion. U251 cells, U87cells, and U87-36KD cells with knockdown of ER-α36 expression were cultured under the two-dimensional and the three-dimensional (3D) environments. GBM cells growth was examined by cell counting, flow cytometry, western blot, and MTT assays. Invasiveness was measured using confocal microscopy in the 3D environment. Growth of U87 cells with downregulated EGFR and ER-α36 expression was significantly reduced after treatment with 1 µM, 3 µM, and 5 µM of SNG162; growth inhibition in U251 cells was more potent than in U87 cells, although the expression level of ER-α36 in U251 cells was lower than in U87 cells. We found that 1 μM SNG162 suppressed E2-induced MAPK/ERK pathway activation in U87 cells. We also showed that SNG162 inhibited U87 cells invasion; however, it did not significantly affect U251 and U87-36KD cells invasion using the 3D culture method. Finally, we determined that ER-α36 was expressed in the nucleus of invading GBM cells, and SNG162 significantly inhibited the expression of ER-α36 in these cells. SNG162 inhibited the expression of EGFR on cell membranes of non-invasive GBM cells. These results suggest that SNG162 could be a therapeutic agent for GBM by targeting ER-α36.

Artemether-loaded polymeric lipid-core nanocapsules reduce cell viability and alter the antioxidant status of U-87 MG cells

Glioblastomas are tumors that present a high mortality rate. Artemether (ART) is a lactone with antitumor properties, demonstrating low bioavailability and water solubility. In the present study, we developed lipid-core nanocapsules (LNC) containing pequi oil (Caryocar brasiliense Cambess) as the oily core for ART-loaded LNCs (LNCART) and evaluated their effect on human glioblastoma cells (U-87 MG). LNCs were developed by interfacial deposition of a preformed polymer, followed by physicochemical characterization. LNCART revealed a diameter of 0.216 µm, polydispersity index of 0.161, zeta potential of −12.0 mV, and a pH of 5.53. Furthermore, mitochondrial viability, proliferation, total antioxidant status, and antioxidant enzyme activity were evaluated. ART reduced cell viability after 24 h and proliferation after 48 h of treatment at concentrations equal to or above 40 µg . mL−1. LNCART, at 1.25 µg . mL−1, reduced these parameters after 24 h of treatment. Furthermore, superoxide dismutase (SOD) activity was elevated, while glutathione reductase (GR) activity was reduced. These findings suggest that ART loaded into LNC may be a promising alternative to improve its pharmacological action and possible application as a therapeutic agent for glioblastoma.

Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin).

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti–programmed cell death 1 and anti–cytotoxic T lymphocyte–associated protein 4; anti–PD-1 and anti–CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade–resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell–independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti–PD-1 and anti–CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

MiR-375 inhibits the proliferation and invasion of glioblastoma by regulating Wnt5a.

The aberrant expression of microRNA-375 (miR-375) has been proved to be associated with carcinogenesis. However, the role of miR-375 in glioblastoma (GBM) remains unknown. The aim of this study was to investigate biological functions and its molecular mechanisms of miR-375 in GBM cells. In this study, real-time PCR results showed that the level of miR-375 expression in GBM tissues and GBM cell lines (U87 and U251) was decreased. Using MTT assay, Transwell migration and invasion assay, we demonstrated that miR-375 overexpression significantly suppress cell proliferation, cell migration and cell invasion capacity in U87 and U251 cells. However, downregulation of miR-375 had reverse effects on cell proliferation, migration and invasion. Targeting association analysis, dual luciferase assay, RT-PCR and western blot analysis results confirmed that miR-375 could target the 3'UTR of Wnt5a mRNA and regulated its protein expression. Further studies also find overexpression of Wnt5a could significantly reverse miR-375-mediated proliferation, migration and invasion on U87 and U251 cells. Therefore, we concluded that miR-375 inhibited the proliferation and invasion of GBM by regulating Wnt5a and might be a possible therapeutic agent for GBM.

Euphol, a tetracyclic triterpene, from Euphorbia tirucalli induces autophagy and sensitizes temozolomide cytotoxicity on glioblastoma cells.

Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy.

Abstract 1163: Estrogen receptor beta (ESR2)-mediated tumor suppression of glioblastoma involves modulation of cell cycle and DNA damage response pathways

Abstract Purpose: Glioblastomas (GBM) are deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a role of estrogen in tumor suppression. Estrogen signaling is mediated by estrogen receptor alpha (ESR1) and beta (ESR2). Our earlier studies showed that GBM preferentially express ESR2. However, mechanisms by which E2-ESR2 signaling promote suppression of GBM is poorly understood. The objective of this study is to determine the mechanisms by which ESR2 promote tumor suppression. Experimental design: Established and patient derived primary GBM cells overexpressing or under expressing ESR2 were generated using lentiviral vectors. Three different ESR2 agonists (LY500307, Liquiritigenin, and Erb041) were used in the study and transcription changes elicited by ESR2 were profiled using RNA-seq. Cell cycle was analyzed using FACS analysis. In vivo efficacy of ESR2 agonists was tested in an orthotopic model using syngeneic murine GBM model (GL26), tumor growth was measured using bioluminescence, and survival was recorded. The synergy experiments using 119 FDA approved drugs was done using cell viability assay. Results: ESR2 agonist treatment or ESR2 overexpression significantly reduced the viability and colony formation and induced apoptosis of various GBM cell lines with minimal effect on normal astrocytes. Further, ESR2 agonist (LY500307 5mg/kg/day) treatment significantly improved survival of GL26 tumor-bearing mice. RNA-seq studies using ESR2 overexpression or ligands treatment revealed downregulation of a number of genes involved in DNA repair, DNA damage response and cell cycle. The canonical pathways modulated by ESR2 included cell cycle, DNA damage, p53 signaling, and checkpoint regulation. In addition, pathways related to molecular mechanism of cancer, ILK signaling, Wnt signaling and glioma invasion were also altered. ESR2 agonist treatment significantly increased the percentage of cells in G2/M phase in U87, U251 and LN229 GBM cells when compared to vehicle further supporting the RNA-seq pathway analysis of cell cycle and G2/M DNA damage checkpoint regulation. Using combinational screening of 119 FDA-approved drugs combined with a low dose of ESR2 agonists (below the IC50 dose), we discovered synergism of ESR2 agonist treatment with several DNA-damaging agents. We independently validated synergy using additional ESR2 agonists with currently used chemotherapy drugs in GBM including temozolomide. Conclusions: Our results demonstrate that ESR2 as tumor suppressor for GBM and ESR2 agonists have potential as a therapeutic agent for GBM. ERβ agonists’ ability to suppress pathways involved in DNA repair can be exploited to promote apoptosis of GBM cells. Citation Format: Ratna K. Vadlamudi, Gangadhara Reddy Sareddy, Xiaonan Li, Jinyou Liu, Lauren Garcia, Andrew Brenner. Estrogen receptor beta (ESR2)-mediated tumor suppression of glioblastoma involves modulation of cell cycle and DNA damage response pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1163.

Selective Estrogen Receptor β Agonist LY500307 as a Novel Therapeutic Agent for Glioblastoma.

Glioblastomas (GBM), deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a tumor suppressive role of estrogen; however, estrogen as a potential therapy for GBM is limited due to safety concerns. Since GBM express ERβ, a second receptor for estrogen, targeting ERβ with a selective agonist may be a potential novel GBM therapy. In the present study, we examined the therapeutic effect of the selective synthetic ERβ agonist LY500307 using in vitro and in vivo GBM models. Treatment with LY500307 significantly reduced the proliferation of GBM cells with no activity on normal astrocytes in vitro. ERβ agonists promoted apoptosis of GBM cells, and mechanistic studies using RNA sequencing revealed that LY500307 modulated several pathways related to apoptosis, cell cycle, and DNA damage response. Further, LY500307 sensitized GBM cells to several FDA-approved chemotherapeutic drugs including cisplatin, lomustine and temozolomide. LY500307 treatment significantly reduced the in vivo tumor growth and promoted apoptosis of GBM tumors in an orthotopic model and improved the overall survival of tumor-bearing mice in the GL26 syngeneic glioma model. Our results demonstrate that LY500307 has potential as a therapeutic agent for GBM.

Bee Venom Induces Unfolded Protein Response in A172 Glioblastoma Cell Line

Background: Glioblastoma is a type of brain tumor with poor response to available therapies, and shows high rate of mortality. Despite remarkable advancements in our knowledge about cytogenetic and pathophysiologic features of glioblastoma, current treatment strategies are mainly based on cytotoxic drugs; however, these therapeutic approaches are facing progressive failure because of the resistant nature of glioblastomas. In the recent years, however, promising results have emerged owing to targeted therapies toward molecular pathways within cancerous cells. Unfolded Protein Response (UPR) is a remarkable signaling pathway that triggers both apoptosis and survival pathways within cells, and therefore induces UPR-related apoptotic pathways in cancer cells by ER stress inducers. Objectives: Recently, the role of Bee venom (Bv), which contains powerful bioactive peptides, in inducing UPR-related apoptosis was revealed in cancer cell lines. Nevertheless, currently there are no reports of Bv potential ability in induction of UPR apoptotic routes in glioblastoma. The aim of current study was to evaluate possible role of Bee venome in inducing of UPR pathway within A172 glioblastoma cell line. Materials and Methods: We treated the A172 glioblastoma cell line with different Bv doses, and assessed UPR-related genes expression by real-time Polymerase Chain Reaction (PCR). Results: The IC50 of Bv for the studied cell line was 28 μg/mL. Furthermore, we observed that Bv can induce UPR target genes (Grp94 and Gadd153) over-expression through a dose-dependent mechanism. Conclusions: Our results suggest the potential role of Bv as a therapeutic agent for glioblastomas. Keywords: Glioblastoma; A172 Cell Line; Unfolded Protein Response; Bee Venom

P17.61 * ANTI-TUMOR EFFECTS OF S14161 IN GLIOBLASTOMA CELLS

BACKGROUND: Glioblastoma is the most common and aggressive form of malignant glioma and is very difficult to treat. Controlling glioblastoma cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. It has been reported that the phosphoinositide 3-kinase (PI3K) inhibitor exhibits potent antiumor effects to malignant tumors. The small chemical compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was recently identified as an inhibitor of PI3K. Our study aimed to evaluate the antitumor effects of S14167 on two human glioblastoma cell lines (U87MG, T98G) and the molecular mechanisms underlying these effects. METHODS: The anti-invasive effect of S14161 was analysed by an in vitro invasion assay. An in vitro angiogenesis assay was also performed. In vitro growth inhibition of glioblastoma cells by S14161 was determined by the WST-1 assay. The anti-angiogenic effect of S14161 was analysed by an in vitro angiogenesis assay. RESULTS: S14161 suppressed the proliferation of glioblastoma cells. S14161 also suppressed both the invasion of glioblastoma cells and tumor-induced angiogenesis. Molecular-based studies demonstrated that S14161 suppressed gene and protein expression of angiogenic factors. CONCLUSIONS: The results of the present study suggest that S14161 may be a new therapeutic agent for glioblastomas.

Involvement of Akt/NF-κB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo

BackgroundGlioblastoma is the most common and most aggressive form of malignant glioma and is very difficult to treat. Controlling tumour cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. Since constitutive activation of nuclear factor-κB (NF-κB) is necessary for tumour progression, NF-κB may be an important pharmacological target for this disease. Our study aimed to evaluate the antitumour effects of parthenolide, a NF-κB inhibitor, in two human glioblastoma cell lines (U87MG and U373) and in glioblastoma xenografts. Furthermore, we aimed to investigate the molecular mechanisms underlying these effects.MethodsThe anti-invasive and anti-angiogenic effects of parthenolide were analysed using in vitro invasion and angiogenesis assays. Parthenolide-induced growth inhibition of glioblastoma cells in vitro was determined using the MTT (methyl thiazolyl tetrazolium) assay. In addition, the effect of parthenolide on orthotropic implantation in vivo was evaluated using an intracerebral human glioblastoma xenograft model.ResultsWe found that parthenolide suppresses proliferation, invasion, and tumour- induced angiogenesis of glioblastoma cells. Molecular studies demonstrated that parthenolide suppresses gene and protein expression of angiogenic factors. Furthermore, parthenolide reduced Akt phosphorylation and activated mitochondrial signalling, suggesting that the antitumour function of parthenolide may be mediated not only by the inhibition of NF-κB but also by the inhibition of Akt signalling and the activation of apoptotic proteins. Parthenolide suppressed neovascularity and tumour growth in glioblastoma xenografts.ConclusionThe present study identified parthenolide as a new therapeutic agent for glioblastomas.

Abstract B171: Involvement of Akt/NF-κ B pathway in antitumor effect of parthenolide on glioblastoma cells.

Abstract Background: Glioblastoma is the most common and aggressive form of malignant glioma and is very difficult to treat. Controlling glioblastoma cell invasion and angiogenesis is essential to improve the prognosis of glioblastoma patients. Constitutive activation of Nuclear factor-κB (NF-κB) is crucial for tumor progression. Inhibition of NF-κB may be an important pharmacological target for the control of tumor progression. Our study aimed to evaluate the antitumour effects of parthenolide, NF-κB inhibitor, on two human glioma cell lines (U87MG, U373) and the molecular mechanisms underlying these effects. Methods: The anti-invasive effect of parthenolide was analysed by an in vitro invasion assay. An in vitro angiogenesis assay was also performed. In vitro growth inhibition of glioma cells by parthenolide was determined by the MTT assay. The effect of parthenolide on an orthotropic implantation model using athymic mice was also evaluated. Results: Parthenolide suppressed the proliferation of glioblastoma cells. Parthenolide also suppressed both the invasion of glioblastoma cells and tumor-induced angiogenesis. Molecular-based studies demonstrated that parthenolide suppressed gene and protein expression of angiogenic factors. Furthermore, parthenolide reduced the phosphorylation of Akt and activated mitochondrial cascade, suggesting that the antitumor effect of parthenolide may be mediated by the inhibition of the Akt signal and the activation of apoptotic proteins. An in vivo intracerebral human glioblastoma xenograft mouse model demonstrated that parthenolide suppressed neovascularity and tumor growth. Conclusions: The results of the present study suggest that parthenolide may be a new therapeutic agent for glioblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B171.