Growth Of Human Glioblastoma Cells Research Articles

SUZ12 inhibition attenuates cell proliferation of glioblastoma via post-translational regulation of CDKN1B.

Human gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. Differential expression of Polycomb repressive complex 2 (PRC2) has been reported in various subtypes of glioma. However, the role of PRC2 in uncontrolled growth in glioma and its underlying molecular mechanisms remain to be elucidated. We aimed to investigate the functional role of PRC2 in human glioblastoma cell growth by silencing SUZ12, the non-catalytic core component of PRC2. Knockdown of SUZ12 was achieved by infecting T98G cells with lentivirus carrying sequences specifically targeting SUZ12 (shSUZ12). Gene expression was examined by quantitative PCR and western analysis. The impact of shSUZ12 on cell growth was assessed using a cell proliferation assay. Cell cycle distribution was analyzed by flow cytometry, and protein stability was evaluated in cycloheximide-treated cells. Subcellular localization was examined through immunofluorescence staining and biochemical cytoplasmic-nuclear fractionation. Gene expression analysis was also performed on human specimens from normal brain and glioblastoma patients. SUZ12 knockdown (SUZ12 KD) led to widespread decrease in the PRC2-specific histone mark, accompanied by a slowdown of cell proliferation through G1 arrest. In SUZ12 KD cells, the degradation of CDKN1B protein was reduced, resulting from alterations in the MYC-SKP2-CDKN1B axis. Furthermore, nuclear localization of CDKN1B was enhanced in SUZ12 KD cells. Analysis of human glioblastoma samples yielded increased expression of EZH2 and MYC along with reduced CDKN1B compared to normal human brain tissue. Our findings suggest a novel role for SUZ12 in cell proliferation through post-translational regulation of CDKN1B in glioblastoma.

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.

Givinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics.

Simple SummaryGlioblastoma is the most common malignant brain tumor with a high grade of recurrence, invasiveness, and aggressiveness. Currently, there are no curative treatments; therefore, the discovery of novel molecules with anti-tumor activity or suitable drug delivery systems are important research topics. The aim of the present study was to investigate the anti-tumor activity of Givinostat, a pan-HDAC inhibitor, and to design an appropriate liposomal formulation to improve its pharmacokinetics profile and brain delivery. The present work demonstrates that the incorporation of Givinostat in liposomes composed of cholesterol and sphingomyelin improves its in vivo half-life and increases the amount of drug reaching the brain in a mouse model. Furthermore, this formulation preserves the anti-tumor activity of glioblastoma in 2D and 3D in vitro models. These features make liposome-Givinostat formulations potential candidates for glioblastoma therapy.Glioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time- and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.

Manganese oxide nanoparticles inhibit selectively the in vitro and in vivo growth of human colorectal SW620 adenocarcinoma cells

A promising area of oncotherapy is the use of nanomaterials for diagnostics and imaging, as well as for delivering drugs and direct effect agents to tumour cells. We used earlier manganese oxide nanoparticles (NP MnO) as magnetic resonance imaging agents for visualisation and suppression of in vitro and in vivo growth of human glioblastoma cells. The present study was to demonstrate the selective antitumor effect of NP MnO against human tumour cells of different tissue origins, in particular, cells SW620, human colorectal adenocarcinoma. It was shown that NP MnO can inhibit selectively in vitro growth of SW620 cells; the index of selective cytotoxicity against human colorectal adenocarcinoma cells was 20. The range of optimal NP MnO doses was determined using subcutaneous introduction of the nanoparticles to SCID mice; the doses no more than 0.96 mgMn kg−1 had practically no local toxic effect in the animals. The subcutaneous administration of NP MnO in the specified dose range inhibited the growth of SW620 xenografts in SCID mice and led to an increase in their life expectancy. With administered NP MnO in doses of 0.32 and 0.96 mgMn kg−1, the index of inhibition of tumour growth for 21 days from the beginning of the introduction of nanoparticles was 43.0% and 69.8%, respectively. NP MnO seem promising for developing nanotheranostics agents for the visualisation and treatment of human tumours of different tissue origins.

The eIF4A Inhibitor Silvestrol Blocks the Growth of Human Glioblastoma Cells by Inhibiting AKT/mTOR and ERK1/2 Signaling Pathway

The most frequently identified central nervous system tumor in adults is glioblastoma multiforme (GBM). GBM prognosis remains poor despite multimodal treatment, i.e., surgery and radiation therapy with concurrent temozolomide-based chemotherapy. Silvestrol, an eIF4A inhibitor, has been demonstrated to be able to kill tumor cells in previous studies. In this study, it was found that silvestrol considerably attenuated the proliferative potential of U251 and U87 glioma cells and reduced expression of cyclin D1. In addition, silvestrol reduced the level of ERK1/2 and decreased the levels of AKT phosphorylation. Unfortunately, the effect of silvestrol in inhibiting GBM cells was greatly reduced with hypoxia, and the downregulation in AKT/mTOR and ERK1/2 were also rescued with an upregulation of HIF1α, which warranted further research. Taken together, silvestrol exerted antitumor effects in GBM cells by inhibiting the AKT/mTOR and ERK1/2 signaling cascades.

Anti-proliferating effect of Ocimum sanctum and Centella asiatica plant extract on growth of human glioblastoma cells: An in vitro study

Glioblastoma is aggressive brain tumour with poor prognosis with conventional chemotherapy, hence there is need to find alternative targets for developing newer treatment. Advent of new treatment methods involving medicinal plants have shown to reduced Cancer mortalities and prevents development of drug resistance for chemotherapy. Present study aimed at investigates the anti-proliferating activity of two promising medicinal plants, Ocimum sanctum and Centella asiatica . We studied the effect of their plant extract on U87MG Glioblastoma cells proliferation, survival effect and apoptosis. Cytotoxic activity was assessed, after the plant extract treatment on U87MG using MTT assay with dose of 1 mg/mL to 25mg/mL and apoptosis assess was done using Annexin V assay with the three dose (1.5 mg/mL, 2 mg/mL and 2.5 mg/mL). Survivin gene expression was studied using QRT-PCR (Rotar gene Q, Qiagene) has a marker of proliferation. Ocimum sanctum and Centella asiatica treatment of U87MG cells with dosage of 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL showed increase in mean apoptotic cells 2.8 %, 4.9%, 10 % and 3.1%, 5.8% and 7.2%, respectively, compared to untreated U87MG cells. Survivin gene analysis of U87MG cells showed down-regulation in gene expression and differences was significant in comparison to untreated control group with both the plant extract, Centella asiatica showed more down-regulation (97% with 2.5 mg/mL) than Ocimum sanctum. Ocimum sanctum and Centella asiatica exhibited promising anti-proliferating activity and induces apoptosis by down regulation of survivin gene expression

Retraction.

The Anatomical RecordVolume 304, Issue 12 p. 2904-2904 RetractionFree Access Retraction This article retracts the following: RETRACTED: Alpinumisoflavone suppresses human Glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ Lijuan He, Dong Shen, Jianmei Li, Weidong Mao, Volume 303Issue 11The Anatomical Record pages: 2801-2810 First Published online: January 8, 2020 First published: 27 October 2021 https://doi.org/10.1002/ar.24816AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Alpinumisoflavone suppresses human glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ”, by Lijuan HE, Dong SHEN, Jianmei LI, and Weidong MAO. The above article, published in The Anatomical Record (DOI: 10.1002/ar.24350) is retracted by agreement among the authors and the Editor-in-Chief, Dr. Kurt H. Albertine, with notification to the American Association for Anatomy and Wiley Periodicals LLC. The decision is the result of an investigation that revealed issues of scientific integrity in the data that compromise the findings of the paper. REFERENCE He, L., Shen, D., Li, J., & Mao, W. (2020 Nov). Alpinumisoflavone suppresses human Glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ. Anat Rec (Hoboken)., 303(11), 2801– 2810. https://doi.org/10.1002/ar.24350Wiley Online LibraryCASPubMedGoogle Scholar Volume304, Issue12December 2021Pages 2904-2904 ReferencesRelatedInformation

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment.

Background: Glioblastoma (GBM) is one of the most aggressive types of brain cancer. GBM progression is closely associated with microglia activation; therefore, understanding the regulation of the crosstalk between human GBM and microglia may help develop effective therapeutic strategies. Elucidation of efficient delivery of microRNA (miRNA) via extracellular vesicles (EVs) and their intracellular communications is required for therapeutic applications in GBM treatment.Methods: We used human GBM cells (U373MG) and human microglia. MiRNA-124 was loaded into HEK293T-derived EVs (miR-124 EVs). Various anti-tumor effects (proliferation, metastasis, chemosensitivity, M1/M2 microglial polarization, and cytokine profile) were investigated in U373MG and microglia. Anti-tumor effect of miR-124 EVs was also investigated in five different patient-derived GBM cell lines (SNU-201, SNU-466, SNU-489, SNU-626, and SNU-1105). A three-dimensional (3D) microfluidic device was used to investigate the interactive microenvironment of the tumor and microglia.Results: MiR-124 EVs showed highly efficient anti-tumor effects both in GBM cells and microglia. The mRNA expression levels of tumor progression and M2 microglial polarization markers were decreased in response to miR-124 EVs. The events were closely related to signal transducer and activator of transcription (STAT) 3 signaling in both GBM and microglia. In 3D microfluidic experiments, both U373MG and microglia migrated to a lesser extent and showed less-elongated morphology in the presence of miR-124 EVs compared to the control. Analyses of changes in cytokine levels in the microfluidic GBM-microglia environment showed that the treatment with miR-124 EVs led to tumor suppression and anti-cancer immunity, thereby recruiting natural killer (NK) cells into the tumor.Conclusions: In this study, we demonstrated that EV-mediated miR-124 delivery exerted synergistic anti-tumor effects by suppressing the growth of human GBM cells and inhibiting M2 microglial polarization. These findings provide new insights toward a better understanding of the GBM microenvironment and provide substantial evidence for the development of potential therapeutic strategies using miRNA-loaded EVs.

Synergism of the Cytopathic Effect of Synchrotron Radiation and Manganese Oxide Nanoparticles on the Growth of Human Glioblastoma Cells In Vitro

Synergism of the Cytopathic Effect of Synchrotron Radiation and Manganese Oxide Nanoparticles on the Growth of Human Glioblastoma Cells In Vitro

Effect of CRISPR/Cas9-mediated PD-1-disrupted primary human third-generation CAR-T cells targeting EGFRvIII on human glioblastoma cell growth

Abstract Glioblastoma (GBM) is the most common and malignant brain tumor. As GBM is resistant to standard treatment, immunotherapy might be a promising alternative. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. The antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells are limited in GBM, on account of the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on target tumor cells. Antibodies against PD-1 have shown limitations, so permanent deletion of the PD-1 gene in CAR-T cells might be a promising solution. Therefore, in the present study, PD-1-disrupted EvCAR-T cells were established for the first time with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing tool. We determined the disruption efficiency of the human PD-1 gene in EvCAR-T cells and evaluated the antitumor effect of PD-1-disrupted EvCAR-T cells against human GBM cells. The results showed that our designed-sgRNA/Cas9 expression vectors precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. Further, the PD-1-disrupted EvCAR-T cells also had a growth inhibitory effect on EGFRvIII-expressing GBM cells. Thus, this study provides a simple, easy, and clinical applicable strategy for inducing PD-1-disrupted EvCAR-T cells, which could potentially be useful for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM, and even other cancers.

Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth.

Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM.

Alpinumisoflavone suppresses human Glioblastoma cell growth and induces cell cycle arrest through activating peroxisome proliferator-activated receptor-γ.

As a common subtype of malignant gliomas, glioblastoma multiforme (GBM) is associated with poor prognosis. This study is aimed to examine the anticancer activities of alpinumisoflavone (AIF) and its underlying mechanisms. Our results demonstrated that AIF inhibited the proliferation of GBM cells (U373 and T98G) in a time and dose-dependent manner. In addition, flow cytometry analysis not only confirmed AIF arrested cell cycle at the G0/G1 phase but also the induced apoptosis of U373 and T98G cells. Western blotting also confirmed that AIF altered the expression levels of cell cycle-related proteins. Further mechanism studies revealed that AIF inhibited cell proliferation, induced G0/G1 phase arrest and induced apoptosis of U373 and T98G cells through activating PPARγ, as evidenced by the fact that GW9662 (PPARγ inhibitor) could effectively reverse the effects of AIF on U373 and T98G cells. Furthermore, the in vivo study also revealed that AIF suppressed tumor growth and caused cell cycle arrest. Collectively, these results highlighted the potential use of AIF in the treatment of GBM.

Allicin suppresses human glioblastoma cell growth by inducing cell cycle arrest and apoptosis, and by promoting autophagy

Glioblastoma is the most aggressive cancer that occurs in the brain and spinal cord. In the present study, we investigated the effect of allicin, an organosulfur compound obtained from garlic (Allium sativum), on glioblastoma cell growth. When human glioblastoma DBTRG-05MG cells were incubated with different concentrations of allicin for 24 h, cell growth was suppressed in a dose-dependent manner. The results from image-based cytometer assays suggested that allicin caused S and G2/M phase cell cycle arrest and induced apoptosis. Autophagy detection studies showed that allicin also promoted this mechanism. Because cell migration is a key process during tumor formation, the effect of allicin on glioblastoma cell migration was also examined. After allicin treatment, the migration ability of cells decreased when compared with the control after 24 h. Taken together, the present results suggested that allicin inhibited human glioblastoma cell growth by inducing S and G2/M phase cell cycle arrest, apoptosis and autophagy. Our findings suggest that allicin suppressed glioblastoma cell growth through multiple target pathways. Therefore, allicin potentially serves as an alternative therapeutic candidate or could be synergistically used in combination with the standard drug for the treatment of glioblastoma multiforme.

Overexpression of Tripartite Motif-Containing 48 (TRIM48) Inhibits Growth of Human Glioblastoma Cells by Suppressing Extracellular Signal Regulated Kinase 1/2 (ERK1/2) Pathway.

BackgroundHerein, we found that tripartite motif-containing 48 (TRIM48) was reduced in human glioblastoma (GBM) cell lines. We investigated whether and how TRIM48 functions in human GBM in vitro.Material/MethodsHuman GBM cells (U87 MG and U138 MG) were infected with lentivirus to overexpress TRIM48, and 1 human GBM cell line (T98G) was infected with siRNAs to knock down TRIM48 expression. Techniques used included cell proliferation assay, measured by CCK-8 and BrdU-ELISA method, and cell cycle assay, determined using flow cytometry. Curcumin, a specific activator of extracellular signal regulated kinases (ERK1/2), or PD98059, a specific inhibitor of ERK1/2, was used to activate or block the ERK1/2 pathway, respectively. Expression of phosphorylated (p)-ERK1/2, and its downstream targets (Cyclin D1) were measured to assess the mechanism.ResultsOur data suggest that overexpression of TRIM48 reduces the viability of U87 MG and U138 MG and leads to cell cycle arrest (in G0-G1 phase), which is associated with blockade of the ERK1/2 pathway and reduction of Cyclin D1. In contrast, knockdown of TRIM48 resulted in the opposite effects. Interestingly, the inhibitory effect of TRIM48 overexpression on human GBM cell growth and the inactivation of ERK1/2 were significantly alleviated with additional curcumin treatment, while it the promoted the effect of siTRIM48 on human GBM cell growth, and the activation of ERK1/2 was significantly alleviated with additional PD98059 treatment.ConclusionsTRIM48 suppressed the growth of human GBM cell via the prevention of ERK1/2 activation.

High Glucose Promotes Human Glioblastoma Cell Growth by Increasing the Expression and Function of Chemoattractant and Growth Factor Receptors

Diabetes mellitus, characterized by hyperglycemia, is considered as a risk factor of cancers including malignant gliomas. However, the direct effect of high glucose on cancer cell behavior is not clear. We therefore investigated the effect of hyperglycemia on the growth of human glioblastoma (GBM) cells. Our results revealed that high glucose (HG) promoted the proliferation and inhibited the apoptosis of a human GBM cell line U87. Mechanistically, HG upregulated the expression and function of a G-protein coupled chemoattractant receptor (GPCR) formyl peptide receptor 1 (FPR1) and epidermal growth factor receptor (EGFR) on GBM cells, which upon activation by their agonists, promoted cell migration and proliferation. In addition, the invasiveness and the production of VEGF by U87 cells were enhanced under HG conditions, the effects of which were mediated by FPR1 and EGFR agonists. The tumor promoting activity of HG was further substantiated by increased tumorigenicity and growth of xenograft tumors formed by GBM cells in nude mice with induced diabetes mellitus. Thus, our study demonstrates the capacity of HG to promote GBM progression via enhancement of the function of chemoattractant and growth factor receptors.

Repression of Septin9 and Septin2 suppresses tumor growth of human glioblastoma cells

Glioblastoma (GBM) is the most common primary malignancy of the central nervous system (CNS) with <10% 5-year survival rate. The growth and invasion of GBM cells into normal brain make the resection and treatment difficult. A better understanding of the biology of GBM cells is crucial to the targeted therapies for the disease. In this study, we identified Septin9 (SEPT9) and Septin2 (SEPT2) as GBM-related genes through integrated multi-omics analysis across independent transcriptomic and proteomic studies. Further studies revealed that expression of SEPT9 and SEPT2 was elevated in glioma tissues and cell lines (A172, U87-MG). Knockdown of SEPT9 and SEPT2 in A172/U87-MG was able to inhibit GBM cell proliferation and arrest cell cycle progression in the S phase in a synergistic mechanism. Moreover, suppression of SEPT9 and SEPT2 decreased the GBM cell invasive capability and significantly impaired the growth of glioma xenografts in nude mice. Furthermore, the decrease in GBM cell growth caused by SEPT9 and SEPT2 RNAi appears to involve two parallel signaling pathway including the p53/p21 axis and MEK/ERK activation. Together, our integration of multi-omics analysis has revealed previously unrecognized synergistic role of SEPT9 and SEPT2 in GBM, and provided novel insights into the targeted therapy of GBM.

Sinomenine Hydrochloride Inhibits Human Glioblastoma Cell Growth through Reactive Oxygen Species Generation and Autophagy-Lysosome Pathway Activation: An In Vitro and In Vivo Study.

Glioblastoma is the most common malignant primary brain tumor, and it is one of the causes of cancer fatality in both adult and pediatric populations. Patients with glioblastoma require chemotherapy after surgical resection and radiotherapy. Therefore, chemotherapy constitutes a viable approach for the eradication of glioblastoma cells. In this study, the anti-tumor activity of sinomenine hydrochloride (SH) was evaluated in U87 and SF767 cells in vitro and in vivo. The results showed that SH potently inhibited U87 and SF767 cell viability and did not cause caspase-dependent cell death, as demonstrated by the absence of significant early apoptosis and caspase-3 cleavage. Instead, SH activated an autophagy-mediated cell death pathway, as indicated by the accumulated microtubule-associated protein light chain 3B (LC3B)-II, triggered autophagic flux and enhanced cell viability after pretreatment with autophagy inhibitors. SH-mediated autophagy in the two cell lines was implicated in reactive oxygen species (ROS) generation, protein kinase B (Akt)-mammalian target of rapamycin (mTOR) pathway suppression and c-Jun NH2-terminal kinase (JNK) pathway activation. The ROS antioxidant N-acetylcysteine (NAC), the Akt-specific activator insulin-like growth factor-1 (IGF-1) and the JNK-specific inhibitor SP600125 attenuated SH-induced autophagy. Moreover, ROS activated autophagy via the Akt-mTOR and JNK pathways. Additionally, SH treatment may promote lysosome biogenesis through activating transcription factor EB (TFEB). The in vivo study found that SH effectively suppressed glioblastoma growth without exhibiting significant toxicity. In conclusion, our findings reveal a novel mechanism of action of SH in cancer cells via the induction of autophagy through ROS generation and autophagy-lysosome pathway activation; these findings also supply a new potential therapeutic agent for the treatment of human glioblastoma.

Abstract 121: Identification and characterization of calcium channel blockers as inhibitors of YAP/TAZ and glioblastoma cells

Abstract Glioblastomas (GBM) are the most aggressive primary brain tumors. Despite major advances in neuroimaging and neurosurgical techniques over the past decades, the neurosurgical management of GBM patients remains challenging. Median survival of the patients is about 14.6 months. In order to radically alter the clinical course of these tumors, it is important to develop targeted therapies based on identified oncogenic mutations and signaling pathways that drive their development and sustain their maintenance. In a screen for small molecules, which can inhibit the growth of human glioblastoma cells, we identified a set of calcium channel blockers (CCB). We found that these CCB induce cell death through an apoptosis-independent process. To understand the mechanisms of the non-apoptotic cell death induced by these CCB, we dissected cellular signaling which is responsible for the cell death induction. We found that these CCB are able to inhibit YAP and TAZ, two oncogenic effectors of the Hippo tumor suppressor pathway. Importantly, GBM cells expressing the YAP active mutant or overexpressing YAP are less sensitive to these CCB. These results suggested that inhibition of YAP/TAZ is a mechanism by which CCB suppress tumor cell growth. The Hippo pathway is an essential signaling network in regulating tissue homeostasis. In this pathway, the transcription co-activators YAP and TAZ promote a gene expression program favoring cell proliferation, survival and self-renewal. In normal brain, the nuclear accumulation of YAP is largely limited to regions harboring neural progenitor cells. However, increased nuclear accumulation of YAP is found in various brain tumors, especially those more aggressive subsets of gliomas. In addition, high expression of YAP is associated with reduced mean survival in astrocytoma patients. Our results identified a novel approach to target the Hippo pathway for GBM treatment. We will present our work in further characterizing these regulations and therapeutic potentials of these calcium channel blockers in the conference. Citation Format: Wei Li, Zhijun Liu, Jennifer Xavier, Hong-Gang Wang. Identification and characterization of calcium channel blockers as inhibitors of YAP/TAZ and glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 121. doi:10.1158/1538-7445.AM2017-121

Inhibitory mechanisms of timosaponin AⅢ on proliferation and growth of human glioblastoma cells

To explore the inhibitory effect of timosaponin AⅢ on the proliferation of human glioblastoma cell line U87MG and investigate its related mechanism. As compared with the model group, the tumor weight was significantly reduced in timosaponin AⅢ-treated group. Timosaponin AⅢinhibited the proliferation of U87MG cell line in a dose-dependent manner. It up-regulated the gene and protein expression levels of p21, meanwhile inhibited the protein expression levels of β-Catenin, Cyclin D1 and Bcl-2. It also inhibited the translocation of β-Catenin into nucleus, suppressed the phosphorylation expression of ERK, but increased the phosphorylation expression of p38 and JNK. Combined use of JNK inhibitor SP600125 and p38 inhibitor SB203580 could decrease p21 and increase β-Catenin protein expressions. Timosaponin AⅢ inhibited the proliferation of human glioblastoma cell line U87MG partly by intervening MAPK and Wnt/β-Catenin signal pathways.