Invasion Of Human Glioblastoma Cells Research Articles

TRAIL and Celastrol Combinational Treatment Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells via Targeting Wnt/β-catenin Signaling Pathway.

To investigate the mechanistic basis for the anti-proliferation and anti-invasion effect of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) and celastrol combination treatment (TCCT) in glioblastoma cells. Cell counting kit-8 was used to detect the effects of different concentrations of celastrol (0-16 µmol/L) and TRAIL (0-500 ng/mL) on the cell viability of glioblastoma cells. U87 cells were randomly divided into 4 groups, namely control, TRAIL (TRAIL 100 ng/mL), Cel (celastrol 0.5 µmol/L) and TCCT (TRAIL 100 ng/mL+ celastrol 0.5 µmol/L). Cell proliferation, migration, and invasion were detected by colony formation, wound healing, and Transwell assays, respectively. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to assess the levels of epithelial-mesenchymal transition (EMT) markers (zona occludens, N-cadherin, vimentin, zinc finger E-box-binding homeobox, Slug, and β-catenin). Wnt pathway was activated by lithium chloride (LiCl, 20 mol/L) and the mechanism for action of TCCT was explored. Celastrol and TRAIL synergistically inhibited the proliferation, migration, invasion, and EMT of U87 cells (P<0.01). TCCT up-regulated the expression of GSK-3β and down-regulated the expression of β-catenin and its associated proteins (P<0.05 or P<0.01), including c-Myc, Cyclin-D1, and matrix metalloproteinase (MMP)-2. In addition, LiCl, an activator of the Wnt signaling pathway, restored the inhibitory effects of TCCT on the expression of β-catenin and its downstream genes, as well as the migration and invasion of glioblastoma cells (P<0.05 or P<0.01). Celastrol and TRAIL can synergistically suppress glioblastoma cell migration, invasion, and EMT, potentially through inhibition of Wnt/β-catenin pathway. This underlies a novel mechanism of action for TCCT as an effective therapy for glioblastoma.

Progesterone and its metabolite allopregnanolone promote invasion of human glioblastoma cells through metalloproteinase‑9 and cSrc kinase.

Glioblastomas are the most aggressive and common primary brain tumors in adults. Glioblastoma cells have a great capacity to migrate and invade the brain parenchyma, often reaching the contralateral hemisphere. Progesterone (P4) and its metabolite, allopregnanolone (3α-THP), promote the migration and invasion of human glioblastoma-derived cells. P4 induces migration in glioblastoma cells by the activation of the proto-oncogene tyrosine-protein kinase Src (cSrc) and focal adhesion kinase (Fak). In breast cancer cells, cSrc and Fak promote invasion by increasing the expression and activation of extracellular matrix metalloproteinases (MMPs). However, the mechanism of action by which P4 and 3a-THP promote invasion in glioblastoma cells remains unclear. The effects of P4 and 3α-THP on the protein expression levels of MMP-2 and -9 and the participation of cSrc in progestin effects in U251 and U87 human glioblastoma-derived cells were evaluated. It was determined by western blotting that the P4 increased the protein expression level of MMP-9 in U251 and U87 cells, and 3α-THP increased the protein expression level of MMP-9 in U87 cells. None of these progestins modified MMP-2 protein expression levels. The increase in MMP-9 expression was reduced when the intracellular progesterone receptor and cSrc expression were blocked with small interfering RNAs. Cell invasion induced by P4 and 3α-THP was also blocked by inhibiting cSrc activity with PP2 or by cSrc gene silencing. These results suggest that P4 and its metabolite 3α-THP induce the invasion of glioblastoma cells by increasing MMP-9 expression through the cSrc kinase family. The results of this study provide information of interest in the context of targeted therapies against molecular pathways involved in glioblastoma invasion.

Platycodin D suppresses proliferation, migration, and invasion of human glioblastoma cells through regulation of Skp2

BackgroundPlatycodin D (PD) is a major bioactive component of Platycodon grandiflorum, a medicinal herb that is widely used in China, and is effective against various human cancers, including glioblastoma multiforme (GBM). S phase kinase-related protein 2 (Skp2) is oncogenic and overexpressed in various human tumors. It is highly expressed in GBM and its expression is correlated with tumor growth, drug resistance and poor prognosis. In this study, we investigated whether inhibition of glioma progression by PD is mediated by decreasing expression of Skp2. MethodsCell Counting Kit-8 (CCK-8) and Transwell assays were used to determine the effects of PD on GBM cell proliferation, migration, and invasion in vitro. mRNA and protein expression were determined by real time polymerase chain reaction (RT-PCR) and western blotting, respectively. The U87 xenograft model was used to verify the anti-glioma effect of PD in vivo. Expression levels of Skp2 protein were analyzed by immunofluorescence staining. ResultsPD suppressed proliferation and motility of GBM cells in vitro. The expression of Skp2 in U87 and U251 cells was significantly reduced by PD. PD mainly decreased the cytoplasmic expression of Skp2 in glioma cells. Skp2 protein expression was downregulated by PD, resulting in upregulation of its downstream targets, p21and p27. The inhibitory effect of PD was enhanced by Skp2 knockdown in GBM cells and reversed in cells with Skp2 overexpression. ConclusionPD suppresses glioma development by regulation of Skp2 in GBM cells.

MiR-373/miR-520s-CD44 Axis Significantly Inhibits the Growth and Invasion of Human Glioblastoma Cells

The expression and regulation of microRNAs (miRNAs) play an important role in glioblastoma (GBM) tumorigenesis, progression and prognosis. Little is known about the role of the miRNA regulatory network of GBM risk-related genes in GBM growth and invasiveness. The UALCAN and Oncomine gene expression dataset were used to explore gene expression profiles in human GBM. The Kaplan-Meier method was performed to evaluate the prognostic values of the GBM-related genes. Multiple bioinformatics databases were analysed to predict the GBM-related genes targeted by miRNAs. A luciferase reporter assay and other molecular cell function experiments were conducted to reveal the mechanisms of interaction between the identified miRNAs and their targets. The CD44 expression is significantly higher in GBM tissues than that in normal tissues, and negatively correlated with survival duration in GBM patients. In normal physiological conditions, CD44 expression is lower in various parts of the central nervous system than in other organ systems. The mRNA encoding CD44 is a direct target of miR-373 and miR-520s, and this finding was verified by molecular biology experiments. We further found that miR-373 and miR-520s expression was negatively associated with CD44 expression in GBM specimens, and that the miR-373 or miR-520s-CD44 interaction network significantly affected the growth and invasiveness of GBM cells. The miR-373 and miR-520s exert their functions by suppressing CD44 expression in GBM cells, and their expression, together with that of CD44, could thus serve as a valuable biomarker of GBM prognosis.

Echinacoside (ECH) suppresses proliferation, migration, and invasion of human glioblastoma cells by inhibiting Skp2-triggered epithelial-mesenchymal transition (EMT)

BackgroundEchinacoside (ECH) is a phenylethanoid extracted from the stems of Cistanches salsa, an herb used in Chinese medicine formulations, and is effective against glioblastoma multiforme (GBM). Epithelial-mesenchymal transition (EMT) is the cornerstone of tumorigenesis and metastasis, and increases the malignant behavior of GBM cells. The S phase kinase-related protein 2 (skp2), an oncoprotein associated with EMT, is highly expressed in GBM and significantly associated with drug resistance, tumor grade and dismal prognosis. The aim of this study was to explore the inhibitory effects of ECH against GBM development and skp2-induced EMT. MethodsCCK-8, EdU incorporation, transwell, colony formation and sphere formation assays were used to determine the effects of ECH on GBM cell viability, proliferation, migration and invasion in vitro. The in vivo anti-glioma effects of ECH were examined using a U87 xenograft model. The expression levels of skp2 protein, EMT-associated markers (vimentin and snail) and stemness markers (Nestin and sox2) were analyzed by immunofluorescence staining and western blotting experiments. ResultsECH suppressed the proliferation, invasiveness and migration of GBM cells in vitro, as well as the growth of U87 xenograft in vivo. In addition, ECH downregulated the skp2 protein, EMT-related markers (vimentin and snail) and stemness markers (sox2 and Nestin). The inhibitory effects of ECH were augmented in the skp2-knockdown GBM cells, and reversed in cells with ectopic expression of skp2. ConclusionECH inhibits glioma development by suppressing skp2-induced EMT of GBM cells.

CD95 gene deletion may reduce clonogenic growth and invasiveness of human glioblastoma cells in a CD95 ligand-independent manner

CD95 (Fas/APO-1) is a multifunctional cell surface receptor with antithetic roles. First described to mediate cell death, interactions of CD95 with its natural ligand, CD95L, have also been described to induce tumor-promoting signaling leading to proliferation, invasion and stem cell maintenance, mainly in cancer cells that are resistant to CD95-mediated apoptosis. While activation of CD95-mediated apoptosis in cancer cells may not be clinically practicable due to toxicity, inhibition of tumor-promoting CD95 signaling holds therapeutic potential. In the present study, we characterized CD95 and CD95L expression in human glioma-initiating cells (GIC), a glioblastoma cell population with stem cell features, and investigated the consequences of CRISPR-Cas9-mediated CD95 or CD95L gene deletion. In vitro, GIC expressed CD95 but not CD95L and were sensitive to CD95-mediated apoptosis. Upon genetic deletion of CD95, GIC acquired resistance to CD95L-induced apoptosis but exhibited inferior clonogenic growth, sphere-forming capacity, and invasiveness compared with control cells, suggesting the existence of CD95L-independent constitutive CD95 signaling with tumor-promoting properties in GIC. In vivo, GIC expressed CD95 and a non-canonical form of CD95L lacking the CD95-binding region. CD95 genetic deletion did not prolong survival in immunocompromised GIC-bearing mice. Altogether, these data indicate that canonical CD95L may not be expressed in human GIC and suggest the existence of a CD95L-independent CD95-signaling pathway that maintains some malignancy traits of GIC. The lack of altered survival of tumor-bearing mice after genetic deletion of CD95 suggests that CD95 signaling is not essential to maintain the growth of human GIC xenografted into the brains of nude mice. The ligand-independent tumor-promoting role of constitutive CD95 in our GIC models in vitro highlights the complexity and challenges associated with targeting CD95 with therapeutic intent.

Progesterone and its Metabolite Allopregnanolone Promote Invasion of Human Glioblastoma Cells Through Metalloproteinase-9 and cSrc Kinase

Progesterone and its Metabolite Allopregnanolone Promote Invasion of Human Glioblastoma Cells Through Metalloproteinase-9 and cSrc Kinase

BIRB796, an Inhibitor of p38 Mitogen-Activated Protein Kinase, Inhibits Proliferation and Invasion in Glioblastoma Cells.

Glioblastoma (GBM) is the most common malignant tumor, and it is characterized by high cellular proliferation and invasion in the central nervous system of adults. Due to its high degree of heterogeneity and mortality, there is no effective therapy for GBM. In our study, we investigated the effect of the p38-MAPK signaling pathway inhibitor BIRB796 on GBM cells. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EDU) staining, and cell cycle distribution analysis were performed, and the results showed that BIRB796 decreased proliferation in U87 and U251 cells. Moreover, wound healing and invasion assays were performed, which showed that BIRB796 inhibited the migration and invasion of human GBM cells. We found that BIRB796 treatment significantly decreased the formation of the cytoskeleton and thus downregulated the movement ability of the cells, as shown by phalloidin staining and vimentin immunofluorescence staining. Real-time polymerase chain reaction showed that the mRNA levels of MMP-2, Vimentin, CyclinD1, and Snail-1 were downregulated. Consistently, the expressions of MMP-2, Vimentin, CyclinD1, and p-p38 were also decreased after BIRB796 treatment. Taken together, all our results demonstrated that BIRB796 could play an antitumor role by inhibiting the proliferation and invasion in GBM cells. Thus, BIRB796 may be used as an adjuvant therapy to improve the therapeutic efficacy of GBM treatment.

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells.

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

Foretinib induces G2/M cell cycle arrest, apoptosis, and invasion in human glioblastoma cells through c-MET inhibition

Glioblastoma multiforme (GBM) is one of the most aggressive human cancers. The c-MET receptor tyrosine kinase (RTK) which is frequently deregulated in GBM is considered as a promising target for GBM treatment. The c-MET plays a key role in cell proliferation, cell cycle progression, invasion, angiogenesis, and metastasis. Here, we investigated the anti-tumour activity of foretinib, a c-MET inhibitor, on three human GBM cells (T98G, U87MG and U251). Anti-proliferative effect of foretinib was determined using MTT, crystal violet staining, and clonogenic assays. PI and Annexin V/PI staining flow cytometry were used to evaluate the effects of foretinib on cell cycle and apoptosis, respectively. Scratch assay, qRT-PCR, western blot, and zymography analyses were applied to elucidate the molecular mechanisms underlying the anti-tumour activity of foretinib. Foretinib treatment reduced phosphorylation of c-MET on T98G and U251 cells, but not in U87MG cells. The highest inhibitory effect was observed in T98G cells (IC50 = 4.66 ± 0.29µM) and the lowest one in U87MG cells (IC50 = 29.99 ± 1.31µM). The results showed that foretinib inhibited the proliferation of GBM cells through a G2/M cell cycle arrest and mitochondrial-mediated apoptosis in association with alternation in expression of the related genes and protein-regulated G2/M phase and apoptosis. Foretinib diminished GBM cell invasion through downregulation of the proteolytic cascade of MMP2, uPA and uPAR and epithelial-mesenchymal transition (EMT)-related genes. A different GBM cell sensitivity pattern was noticeable in all experiments which demonstrated T98G as a sensitive and U87MG as a resistant phenotype to foretinib treatment. The results indicated that foretinib might have the therapeutic potential against human GBM which deserve further investigation.

Norepinephrine inhibits migration and invasion of human glioblastoma cell cultures possibly via MMP-11 inhibition

PurposeGrowing evidence has shown that the stress hormones affect tumor progression. Patients with surgery to remove tumor often have increased norepinephrine during the perioperative period. However, the effect of norepinephrine on the progression of glioblastoma has not yet studied. Therefore, the present study aimed at investigating the effects of norepinephrine on the migration and invasion of the human glioblastoma U87 and U251 cell lines and the mechanism for the effects. MethodsThe U87 and U251 cells were treated with 0, 0.1, 1, 5, 10 or 50 μM norepinephrine. A scratch wound healing assay and a transwell invasion assay were used to investigate cell migration and invasion, respectively. The Human Tumor Metastasis RT2 Profiler PCR Array was used to detect the expression of 84 genes known to be involved in metastasis. ResultsFollowing norepinephrine treatment, the ability of the U87 and U251 cells to migrate and invade was significantly decreased. Human Tumor Metastasis RT2 Profiler PCR Array assay showed that matrix metallopeptidase-11 (MMP-11) was decreased following norepinephrine treatment. The β-adrenergic receptor blocker (AR) propranolol blunted the suppressive effect of norepinephrine on the migration and invasion of U251 cells but did not have such an effect on the invasion of U87 cells. MMP-11 silencing inhibited the migration and invasion of U87 and U251 cells. The Cancer Genome Atlas data showed that patients with higher expression of MMP-11 in the glioblastoma tissues had poorer prognosis. ConclusionOur results indicate that norepinephrine inhibits the migration and invasion of human glioblastoma cells. This effect may be mediated by the decrease of MMP-11. β-AR may be a regulatory factor for this effect in U251 cells.

Galangin (GLN) Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells by Targeting Skp2-Induced Epithelial-Mesenchymal Transition (EMT).

BackgroundGalangin (GLN), a pure natural flavonoid compound found in plants, has been shown to exert anti-cancer effects against multiple cancer types, including glioma. However, its underlying molecular mechanism remains unclear. Epithelial-to-mesenchymal transition (EMT) performs an important function in the genesis and development of cancer. Skp2, a pivotal component of SCFSkp2 E3 ubiquitin ligase, has been shown to function as an oncogene in GBM invasion that contributes to the EMT process. Thus, we explored whether GLN inhibited Skp2-mediated EMT and the mechanism underlying the Skp2 degradation pathway.MethodsCCK-8 assay, wound healing assay and transwell assay were used to examine cell proliferation, migration, and invasion after treatment with or without GLN. RT-PCR and Western blotting analysis were performed to evaluate mRNA and protein expression, respectively. Co-immunoprecipitation was conducted to detect ubiquitinated Skp2 levels in vitro and in vivo after GLN treatment. Bioluminescence imaging was performed to examine the intracranial tumor size of U87 xenograft mice. Microscale thermophoresis (MST) experiment was used to detect interactions between Skp2 and GLN.ResultsGLN suppressed GBM cell growth, migration, and invasion, and also downregulated the expression of Skp2 and mesenchymal markers (Zeb1, N-cadherin, snail, vimentin) in vitro. Moreover, the overexpression of Skp2 in GBM cells decreased the effect of GLN on EMT. Furthermore, we demonstrated that GLN degraded skp2 protein through the ubiquitination proteasome pathway and directly interacted with skp2 protein, as shown through the MST assay.ConclusionThis study is the first to identify Skp2 as a novel target of GLN for the treatment of GBM and report of Skp2 protein degradation in a ubiquitination proteasome pathway. Results from our study indicated the potential of GLN for the treatment of GBM through ubiquitin-mediated degradation of Skp2.

The Bradykinin-BDKRB1 Axis Regulates Aquaporin 4 Gene Expression and Consequential Migration and Invasion of Malignant Glioblastoma Cells via a Ca2+-MEK1-ERK1/2-NF-κB Mechanism.

Glioblastoma multiforme (GBM) is the most common form of brain tumor and is very aggressive. Rapid migration and invasion of glioblastoma cells are two typical features driving malignance of GBM. Bradykinin functionally prompts calcium influx via activation of bradykinin receptor B1/B2 (BDKRB1/2). In this study, we evaluated the roles of bradykinin in migration and invasion of glioblastoma cells and the possible mechanisms. Expressions of aquaporin 4 (AQP4) mRNA and protein were upregulated in human glioblastomas. Furthermore, exposure of human U87 MG glioblastoma cells to bradykinin specifically increased levels of BDKRB1. Successively, bradykinin stimulated influx of calcium, phosphorylation of MEK1 and extracellular signal-regulated kinase (ERK)1/2, translocation and transactivation of nuclear factor-kappaB (NF-κB), and expressions of AQP4 mRNA and protein. Concomitantly, migration and invasion of human glioblastoma cells were elevated by bradykinin. Knocking-down BDKRB1 concurrently decreased AQP4 mRNA expression and cell migration and invasion. The bradykinin-induced effects were further confirmed in murine GL261 glioblastoma cells. Therefore, bradykinin can induce AQP4 expression and subsequent migration and invasion through BDKRB1-mediated calcium influx and subsequent activation of a MEK1-ERK1/2-NF-κB pathway. The bradykinin-BDKRB1 axis and AQP4 could be precise targets for treating GBM patients.

α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.

Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness

Glioblastoma (GBM) is the most aggressive tumor of the brain. NF1, a tumor suppressor gene and RAS-GTPase, is one of the highly mutated genes in GBM. Dysregulated NF1 expression promotes cell invasion, proliferation, and tumorigenesis. Loss of NF1 expression in glioblastoma is associated with increased aggressiveness of the tumor. Here, we show that NF1-loss in patient-derived glioma cells using shRNA increases self-renewal, heightens cell invasion, and promotes mesenchymal subtype and epithelial mesenchymal transition-specific gene expression that enhances tumorigenesis. The neurofibromin protein contains at least four major domains, with the GAP-related domain being the most well-studied. In this study, we report that the leucine-rich domain (LRD) of neurofibromin inhibits invasion of human glioblastoma cells without affecting their proliferation. Moreover, under conditions tested, the NF1-LRD fails to hydrolyze Ras-GTP to Ras-GDP, suggesting that its suppressive function is independent of Ras signaling. We further demonstrate that rare variants within the NF1-LRD domain found in a subset of the patients are pathogenic and reduce NF1-LRD’s invasion suppressive function. Taken together, our results show, for the first time, that NF1-LRD inhibits glioma invasion, and provides evidence of a previously unrecognized function of NF1-LRD in glioma biology.

CD164 regulates proliferation, progression, and invasion of human glioblastoma cells.

Grade IV astrocytoma, also known as glioblastoma multiforme (GBM), is the most common and aggressive intracranial glial tumor. GBM is associated with very poor survival and effective treatments have remained elusive so far. Mounting evidence indicates that CD164 contributes to stemness and tumorigenesis in normal cells and is overexpressed in various tumor types, including glioblastoma. Using tissue microarray immunohistochemistry, we show that there is a significant correlation between CD164 expression and glioma type and grade. Depletion of CD164 expression in human glioblastoma cells with siRNA reduced proliferation, migration, and invasiveness. In parallel, immunoblotting showed that downregulation of CD164 expression decreased Akt activation and modified the expression of autophagy markers by upregulating Beclin-1 and LC3B and downregulating p62. These effects were mimicked by inhibition of Akt with MK2206, which suggests that CD164 induces autophagy via Akt/Beclin-1 signaling. We propose that CD164 may serve as a GBM molecular marker and a potential target in therapeutic strategies aimed to improve outcomes for this devastating brain tumor.

MiR-210 inhibits cell migration and invasion by targeting thebrain-derived neurotrophic factor in glioblastoma.

Recently, there is increasing evidence that microRNAs are related to the development, diagnosis, treatment, and prognosis of glioblastoma. microRNA-210 (miR-210) had been identified in many human cancers, but the specific function of miR-210 remains unclear in glioblastoma. The present study mainly focused on exploring its biological role and potential molecular mechanisms in glioblastoma. We found that miR-210 expression was decreased in glioblastoma, and downregulation of miR-210 was related to worse prognosis in glioblastoma patients. In addition, miR-210 overexpression inhibited the migration and invasion of human glioblastoma cells. At the same time, we found that miR-210 directly targets the brain-derived neurotrophic factor (BDNF) and reduces BDNF expression level. Consistently, BDNF silencing had the same effects as miR-210 overexpression in glioblastoma, and upregulation of BDNF counteracted the inhibitory effect of miR-210 in glioblastoma. In conclusion, miR-210 suppressed the migration and invasion of glioblastoma cells by targeting BDNF.

Baicalin suppresses proliferation, migration, and invasion in human glioblastoma cells via Ca2+-dependent pathway.

ObjectiveBaicalin, a kind of flavonoid extracted from the dry root of Scutellaria, possesses potent anticancer bioactivities in various tumor cell lines. Accumulating evidences show that baicalin induces autophagy and apoptosis to suppress the cancer growth. Moreover, the antineoplastic role of baicalin in human glioblastoma cells remains to be uncovered.MethodsBoth U87 and U251 human glioblastoma cell lines were employed in the present study. Cell viability was tested by Cell Counting Kit-8 and colony-forming assay; Flow cytometry was employed to analyze cell apoptosis, cell cycle, and Ca2+ content. Cell immunofluorescence assays were used for analyzing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), light chain 3 beta (LC3B), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanineiodide (JC-1), and Ca2+ content. The protein levels were tested by Western blot. The SPSS software was used for statistical analysis.ResultsBaicalin suppressed the proliferation, migration, and invasion ability of human glioblastoma cells in a dose-dependent manner. Baicalin induced the loss of mitochondrial membrane potential and led to mitochondrial apoptosis. The maturation of microtubule-associated protein 1A/1B-LC3B indicated the activation of autophagy potentially through PI3K/Akt/mTOR pathway, and inhibition of autophagy by 3-methyladenine decreased the apoptotic cell ratio. Besides, baicalin increased the intercellular Ca2+ content; meanwhile, chelation of free Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited both apoptotic and autophagy. Finally, baicalin suppressed tumor growth in vivo.ConclusionOur observations suggest that baicalin exerts cytotoxic effects on human glioblastoma cells by the autophagy-related apoptosis through Ca2+ movement to the cytosol. Furthermore, baicalin has the potential as a candidate for the treatment of glioblastoma.

Oxymatrine induces cell cycle arrest and apoptosis and suppresses the invasion of human glioblastoma cells through the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3.

Oxymatrine (OM), a natural quinolizidine alkaloid extracted from the traditional Chinese herb Sophora flavescens, has been revealed to produce antitumor activities in various cancer cell lines, including glioblastoma lines, invitro. However, the mechanisms by which OM exerts its antitumor effect against glioma are poorly understood. The aim of this study was to investigate the role of OM in the proliferation, apoptosis and invasion of glioma cells and to reveal the underlying mechanisms. The effects of OM on U251MG cells invitro were determined using a Cell Counting Kit‑8 (CCK‑8) assay, flow cytometric analysis, AnnexinV‑FITC/PI staining, DAPI staining, a terminal deoxynucleotidyl transferase‑mediated dUTP nick end‑labeling (TUNEL) assay, a Transwell assay and western blotting. Our data indicated that OM inhibited proliferation, arrested the cell cycle at the G0/G1phase, decreased the expression levels of G1 cell cycle regulatory proteins (cyclinD1, CDK4 and CDK6), inhibited invasion and induced apoptosis in glioma cells. Additional investigations revealed that the expression levels of p‑STAT3 and key proteins in the EGFR/PI3K/Akt/mTOR signaling pathway, such as p‑EGFR, p‑Akt and p‑mTOR, were markedly decreased after OM treatment, while the total STAT3, EGFR, Akt and mTOR levels were not affected. These findings indicated that the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3 suppression may be a potential mechanism of the OM‑mediated antitumor effect in glioblastoma cells and that EGFR may be a target of OM. Hence, OM may be a promising drug and may offer a novel therapeutic strategy for malignant gliomas in the future.

Activation of membrane progesterone receptor-alpha increases proliferation, migration, and invasion of human glioblastoma cells.

Glioblastoma is the most frequent and aggressive brain tumor due to its high capacity to migrate and invade normal brain tissue. The steroid hormone progesterone (P4) contributes to the progression of glioblastoma by promoting proliferation, migration, and cellular invasion through the activation of its intracellular receptor (PR). However, the use of PR antagonist RU486 partially blocks the effects of P4, suggesting the participation of signaling pathways such as those mediated by membrane receptors to P4 (mPRs). Therefore, this study aimed to investigate the effects of mPRα subtype activation on proliferation, migration, and invasion of human glioblastoma cells. We treated human glioblastoma cell lines U87 and U251 with the specific mPRα agonist Org OD 02-0, and evaluated its effects on cell number, proliferation, migration, and invasion. Additionally, we measured the phosphorylation of the kinases Src and Akt in both cell lines upon Org OD 02-0 treatment. Org OD 02-0 (100 nM) augmented the number of U87 and U251 cells by increasing cell proliferation. The treatment with this agonist also increased U87 and U251 cell migration and invasion. Both proliferation and cell invasion decreased when mPRα expression was silenced. Finally, we observed that Org OD 02-0 increased the content of p-Src and p-Akt in both cell lines. Our data suggest that P4 produces its effects in human glioblastoma progression not only by PR interaction but also through cell signaling pathways activated by mPRα.