Human Glioblastoma Multiforme Cells Research Articles

Conjugation with polyamines enhances the antitumor activity of naphthoquinones against human glioblastoma cells

Glioblastoma multiform (GBM) is the most common and devastating type of primary brain tumor, being considered the deadliest of human cancers. In this context, extensive efforts have been undertaken to develop new drugs that exhibit both antiproliferation and antimetastasis effects on GBM. 1,4-Naphthoquinone (1,4-NQ) scaffold has been found in compounds able to inhibit important biological targets associated with cancer, which includes DNA topoisomerase, Hsp90 and monoamine oxidase. Among potential antineoplastic 1,4-NQs is the plant-derived lapachol (2-hydroxy-3-prenyl-1,4-naphthoquinone) that was found to be active against the Walker-256 carcinoma and Yoshida sarcoma. In the present study, we examined the effect of polyamine (PA)-conjugated derivatives of lapachol, nor-lapachol and lawsone on the growth and invasion of the human GBM cells. The conjugation with PA (a spermidine analog) resulted in dose-dependent and time-dependent increase of cytotoxicity of the 1,4-NQs. In addition, in-vitro inhibition of GBM cell invasion by lapachol was increased upon PA conjugation. Previous biochemical experiments indicated that these PA-1,4-NQs are capable of inhibiting DNA human topoisomerase II-α (topo2α), a major enzyme involved in maintaining DNA topology. Herein, we applied molecular docking to investigate the binding of PA-1,4-NQs to the ATPase site of topo2α. The most active molecules preferentially bind at the ATP-binding site of topo2α, which is energetically favored by the conjugation with PA. Taken together, these findings suggested that the PA-1,4-NQ conjugates might represent potential molecules in the development of new drugs in chemotherapy for malignant brain tumors.

The Potential of Neural Stem Cell as Vehicle to Deliver Quercus infectoria Extract to Glioma Cell In Vitro

Glioblastoma multiforme (GBM) is the most malignant subtype of brain cancer. However, current clinical treatments for GBM are limited in effectiveness and often impose additional side effects on patients. Here, we developed targeted anti-cancer therapy (TAT) using neural stem cells (NSC) as delivery agent to transport anti-cancer compounds directly to GBM in vitro. Anti-cancer active compounds: Tannic acid (TA) and gallic acid (GA) were extracted from local medicinal plant - Quercus infectoria (QI) using soxhlet technique with 100% methanol (QI-100%) or 70% methanol (QI-70%) solvent. Concentration of TA and GA measured using HPLC were 72.56 and 43.66 μg/mL in QI-100%, while in QI-70%, the concentrations were 72.41 and 43.31 μg/mL, respectively. Cytotoxicity effects of QI-100% and QI-70% on human GBM cell line (DBTRG-05MG), human NSC line (H9-hNSC) and human normal brain glial cell line (SVG-p12) (as negative control) were determined using MTT assay. Both QI-100% and QI-70% showed anti-proliferative properties against DBTRG-05MG at IC50, but not on H9-hNSC and SVG-p12. Taken together, data indicated that both QI extracts contained TA and GA which exhibit anti-proliferative effect specifically on cancerous cells only. Next, QI-treated H9-hNSC was seeded in a modified Boyden chamber for 12 h to investigate its migration capacity towards DBTRG-05MG. The result showed that H9-hNSC migrated towards DBTRG-05MG with 4-folds higher capacity compared to control. In addition, the migration of QI-100% treated H9-hNSC successfully reduced the number of DBTRG-05MG, indicating the anti-GBM potential of these cells after migration. In conclusion, NSC could be a specific anti-cancer compound delivery agent for GBM, reducing unwanted side effects on patients.

Effects of sequentially applied single and combined temozolomide, hydroxychloroquine and AT101 treatment in a long-term stimulation glioblastoma in vitro model.

Glioblastoma multiforme (GBM) is a poorly curable disease due to its heterogeneity that enables single cells to survive treatment regimen and initiate tumor regrowth. Although some progress in therapy has been achieved in the last years, the efficient treatment of GBMs is still a clinical challenge. Besides the standard therapeutic drug temozolomide (TMZ), quinoline-based antimalarial drugs such as hydroxychloroquine (HCQ) and BH3 mimetics such as AT101 were considered as possible drugs for GBM therapy. We investigated the effects of sequentially applied single and combined TMZ, HCQ and AT101 treatments in a long-term stimulation GBM in vitro model. We performed all investigations in parallel in human astrocytes and two differentially TMZ-responsive human GBM cell lines and adjusted used drug concentrations to known liquor/plasma concentrations in patients. We determined amounts of dead cells and still remaining growth rates and depicted our results in a heatmap-like summary to visualize which sequential long-term treatment schedule seemed to be most promising. We showed that sequential stimulations yielded higher cytotoxicity and better tumor growth control in comparison to single TMZ treatment. This was especially the case for the sequences TMZ/HCQ and TMZ + AT101/AT101 which was as effective as the non-sequential combination TMZ + AT101. Importantly, those affected both less and more TMZ-responsive glioma cell lines, whilst being less harmful for astrocytes in comparison to single TMZ treatment. Sequential treatment with mechanistically different acting drugs might be an option to reduce side effects in long-term treatment, for example in local administration approaches.

MicroRNA‑574 inhibits cell proliferation and invasion in glioblastoma multiforme by directly targeting zinc finger E‑box‑binding homeobox 1.

Accumulated evidence has demonstrated that dysregulation of microRNAs (miRNAs) contributes to tumourigenesis and tumour development of glioblastoma multiforme (GBM). Therefore, miRNAs may be promising candidates in the development of prognosis biomarkers and effective therapeutic targets for patients with GBM. A number of studies have reported that miRNA‑574 (miR‑574) is aberrantly expressed in multiple types of human cancers. However, the expression pattern, biological functions and molecular mechanism of miR‑574 in GBM are yet to be elucidated. Therefore, the present study aimed to determine the expression level and biological functions of miR‑574 in GBM and the underlying molecular mechanisms. In the present study, miR‑574 levels were measured by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and were demonstrated to be significantly downregulated in human GBM tissues and cell lines. Functional experiments indicated that restored expression of miR‑574 using mimics led to the inhibition of the cell proliferation and invasion of GBM cells, as determined by Cell Counting kit‑8 and Matrigel invasion assays, respectively. In addition, bioinformatics analysis predicted that zinc finger E‑box‑binding homeobox1 (ZEB1) may be a target of miR‑574. Subsequent RT‑qPCR, western blot analysis and luciferase reporter assays confirmed that ZEB1 was a direct target of miR‑574 in GBM. Additionally, ZEB1 was demonstrated to be upregulated and inversely correlated with miR‑574 expression in clinical GBM tissues. Rescue experiments demonstrated that overexpression of ZEB1 attenuated the inhibitory effects of miR‑574 on the proliferation and invasion of GBM cells. Overall, the results of the present study highlighted the potential tumour inhibitory roles of miR‑574 in GBM, thereby indicating that miR‑574 may be a novel and efficient therapeutic target for the treatment of patients with GBM.

Enhanced radiosensitization of human glioblastoma multiforme cells with phosphorylated peptides derived from Gli2

Glioma-Associated Oncogene Family Zinc Finger 2 (Gli2) seems to be the major nuclear effector of Sonic Hedgehog (SHH) signaling to regulate self-renewal and tumorigenic potential of Glioblastoma multiforme (GBM) cells. Three phosphorylated peptides derived from Gli2 were synthesized and combined with cell-penetrating peptide Tat-(47–57) (AYGRKKRRQRRR). Western Blot was applied to detect the phosphorylation level of Gli2 and cell division protein kinase 6 (CDK6) luciferase reporter was utilized to detect the transcriptional activator function of Gli2. Clonogenic survival assay and apoptosis assay were used to testify the radiosensitization effect. The mixed three phosphorylated peptides derived from Gli2 increased the phosphorylation level of Gli2 and decreased Gli2 transcriptional activator activity significantly than the individually used peptide. The mixed three phosphorylated peptides showed greater radiation-sensitizing effects in GBM cells in clonogenic and survival assay compared with control peptide. We present here a novel rational strategy for developing phosphorylated peptides derived from Gli2 to decrease Gli2 transcriptional activator activity and such administration could radiosensitize GBM.

Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft Models for Glioma Stem Cell Targeted Drug Screening.

Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin, and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold, and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, Temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo, and the same can be used as an effective platform for screening drugs against glioma stem cells.

Regulation of Integrated Stress Response Sensitizes U87MG Glioblastoma Cells to Temozolomide Through the Mitochondrial Apoptosis Pathway.

Glioblastomas are the most frequently diagnosed and worst primary malignancy of the central nervous system, with very poor prognosis. The first-line antiglioma drug temozolomide shows decreasing therapeutic efficacy as treatment progresses. As the integrated stress response (ISR) may be a resistance factor and severe stress might transform the protective effect of the ISR into a damage effect, pharmacological regulation of ISR may be an effective way to sensitize glioma to temozolomide. The aim of the present study was to investigate the mechanisms of the ISR in regulating the therapeutic effect of temozolomide in the human glioblastoma multiforme cell line U87MG. Cultured U87MG cells were treated with temozolomide and PCR array was used to screen key factors in the response to treatment. Cells were co-treated with temozolomide and the eIF2α phosphatase inhibitor salubrinal, and cell apoptosis was measured. Combination treatment with temozolomide and salubrinal had a synergistic effect on cell viability. Salubrinal could upregulate the expression of ATF4, a key factor in the ISR, and enhance temozolomide-induced apoptosis. ATF4 transcriptionally regulated expression of the BH3-ONLY protein NOXA, thus inducing mitochondrial apoptosis. These findings suggest that ISR and ATF4 are involved in the death crosstalk between the endoplasmic reticulum and mitochondria and might be a potential target to enhance the therapeutic effect of temozolomide in patients with glioblastoma multiforme. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Effect of Gynura procumbens Extracts on Anti-Proliferative Activity and its Associated Morphological Changes of Human Glioblastoma multiforme Cell Line (U-87)

Effect of Gynura procumbens Extracts on Anti-Proliferative Activity and its Associated Morphological Changes of Human Glioblastoma multiforme Cell Line (U-87)

In vivo detection of acute intracellular acidification in glioblastoma multiforme following a single dose of cariporide.

Glioblastoma is an aggressive brain cancer that is very difficult to treat. Clinically, it is important to be able to distinguish aggressive from non-aggressive brain tumors. Previous studies have shown that some drugs can induce a rapid change in intracellular pH that could help to identify aggressive cancer. The sodium proton exchanger (NHE1) plays a significant role in maintaining pH balance in the tumor microenvironment. Cariporide is a sodium proton exchange inhibitor that is well tolerated by humans in cardiac applications. We hypothesized that cariporide could selectively acidify brain tumors. The purpose of this study was to determine whether amine/amide concentration-independent detection (AACID) chemical exchange saturation transfer (CEST) MRI measurement of tumor pHi could detect acidification after cariporide injection. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after administration of cariporide (dose: 6mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle injection (DMSO + PBS). Repeated measures t test was used to examine changes in tumor and contralateral tissue regions of interest. Two hours after cariporide injection, there was a significant 0.12 ± 0.03 increase in tumor AACID value corresponding to a 0.48 decrease in pHi and no change in AACID value in contralateral tissue. A small but significant increase of 0.04 ± 0.017 in tumor AACID value was also observed following vehicle injection. This study demonstrates that acute CEST MRI contrast changes, indicative of intracellular acidification, after administration of cariporide could help localize glioblastoma.

Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells.

Replication of mitochondrial DNA is strictly regulated during differentiation and development allowing each cell type to acquire its required mtDNA copy number to meet its specific needs for energy. Undifferentiated cells establish the mtDNA set point, which provides low numbers of mtDNA copy but sufficient template for replication once cells commit to specific lineages. However, cancer cells, such as those from the human glioblastoma multiforme cell line, HSR-GBM1, cannot complete differentiation as they fail to enforce the mtDNA set point and are trapped in a ‘pseudo-differentiated’ state. Global DNA methylation is likely to be a major contributing factor, as DNA demethylation treatments promote differentiation of HSR-GBM1 cells. To determine the relationship between DNA methylation and mtDNA copy number in cancer cells, we applied whole genome MeDIP-Seq and RNA-Seq to HSR-GBM1 cells and following their treatment with the DNA demethylation agents 5-azacytidine and vitamin C. We identified key methylated regions modulated by the DNA demethylation agents that also induced synchronous changes to mtDNA copy number and nuclear gene expression. Our findings highlight the control exerted by DNA methylation on the expression of key genes, the regulation of mtDNA copy number and establishment of the mtDNA set point, which collectively contribute to tumorigenesis.

Serum amyloid A1 in combination with integrin αVβ3 increases glioblastoma cells mobility and progression.

Glioblastoma multiforme (GBM) is a highly malignant type of brain tumor found in humans. GBM cells reproduce quickly, and the median survival time for patients after therapy is approximately 1 year with a high relapse rate. Current therapies and diagnostic tools for GBM are limited; therefore, we searched for a more favorable therapeutic target or marker protein for both therapy and diagnosis. We used mass spectrometry (MS) analysis to identify GBM‐associated marker proteins from human plasma and GBM cell cultures. Additional plasma and 52 brain tissues obtained from patients with gliomas were used to validate the association rate of serum amyloid A1 (SAA1) in different grades of gliomas and its distribution in tumors. Microarray database analysis further validated the coefficient of SAA1 levels in gliomas. The cellular mechanisms of SAA1 in GBM proliferation and infiltration were investigated in vitro. We analyzed the correlation between SAA1 and patients' medication requirement to demonstrate the clinical effects of SAA1 in GBM. SAA1 was identified from MS analysis, and its level was revealed to be correlated with the disease grade, clinical severity, and survival rate of patients with gliomas. In vitro cultures, including GBM cells and normal astrocytes, revealed that SAA1 promotes cell migration and invasion through integrin αVβ3 to activate the Erk signaling pathway. Magnetic resonance imaging and tumor region‐specific microarray analysis identified a correlation between SAA1 and GBM cell infiltration in patients. In summary, our results demonstrate that SAA1 in combination with integrin αV and β3 can serve as an indicator of high glioblastoma risk. We also identified the cellular mechanisms of SAA1 contributing to GBM progression, which can serve as the basis for future GBM therapy.

Duocarmycin SA, a potent antitumor antibiotic, sensitizes glioblastoma cells to proton radiation

New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for GBM. DNA alkylating agents such as temozolomide (TMZ) are known to increase the radiosensitivity of GBM to photon radiation. TMZ is a fairly impotent agent, while duocarmycin SA (DSA) is an extremely potent cytotoxic agent capable of inducing a sequence-selective alkylation of duplex DNA. Here, the effects of sub-nM concentrations of DSA on the radiosensitivity of a human GBM cell line (U-138) to proton irradiation were examined. Radiation sensitivity was determined by viability, apoptosis, necrosis and clonogenic assays. DSA concentrations as low as 0.001 nM significantly sensitized U-138 cells to proton irradiation. DSA demonstrates synergistic cytotoxicity against GBM cells treated with proton radiation in vitro, which may represent a novel therapeutic alternative for the treatment of GBM.

Delivery of DNA encoding RNA Therapeutics to Alter the Expression of Oncogenic Transcripts in Glioblastoma

Glioblastoma multiforme (GBM) is the most common central nervous system (CNS) malignancy. The median survival is only 14 months. It is often characterized by the activation of one or more tyrosine kinase receptors, particularly epidermal growth factor receptor (EGFR). This receptor is dysregulated in about 60% of GBM tumors. EGFR amplification, over‐expression and constitutive activation leads to uncontrolled growth and proliferation of GBM. Although a great deal is known about the aberrant biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. Although anti‐sense RNAs and small interfering RNAs can be used to target and silence gene expression, exogenously expressed RNAs are susceptible to extracellular and intracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we take advantage of a natural noncoding RNA gene architecture and the miRNA expression pathway along with an anti‐sense targeted approach to alter EGFR expression. In addition, we make use of a polycistronic delivery system to express RNAs targeting splicing and alternative poly‐A signal/G‐rich elements of the EGFR transcript. DNA delivery vectors were transfected into human GBM cell lines. Results show that our vectors were expressed at high levels with subsequent reduction in full‐length EGFR mRNA expression and concomitant activation of alternative therapeutic isoforms. Current strategies include using the polycistronic delivery mechanism to target additional oncogenic transcripts and adapting to a mouse model of GBM.Support or Funding InformationBristol‐Myers Squibb, Johnson & Johnson, and the Independent College Fund of New JerseyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fatty Acids Rich Extract From Clerodendrum volubile Suppresses Cell Migration; Abates Oxidative Stress; and Regulates Cell Cycle Progression in Glioblastoma Multiforme (U87 MG) Cells.

Glioblastoma multiforme (GBM) is a malignant primary type of brain cancer with high proliferation and metastasis rates due to involvement of the microglial cell. It is resistant against available chemotherapy. Many strategic protocols have been developed but prognosis and patient life has not improved substantially. In this study, the anti-metastatic and antioxidant effect of fatty acids from Clerodendrum volubile leaves were investigated in U87-MG (Human Glioblastoma Multiforme) cell lines. The extracted fatty acids were incubated with U87-MG cells for 48 h. The anti-proliferative effect was determined by MTT assay, while apoptosis and cell cycle were analyzed with BD FACSCalibur. The transwell assay protocol was utilized in the analysis of cell migration and invasion. The treated cell lines were also assessed for reduced glutathione (GSH) level, catalase, superoxide dismutase (SOD) and lipid peroxidation. The fatty acid extract showed significant inhibitory activity on cell proliferation and cell cycle progression, mitigated oxidative stress, and suppressed migration and invasion in U-87 MG cell lines. These results give credence to the therapeutic potential of this plant against cancer, especially GBM.

PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor due to the lack of effective therapeutic drugs. Cancer therapy targeting programmed cell death protein 1 (PD-1) or programmed death ligand-1 (PD-L1) is of revolutionary. However, the role of intrinsic PD-L1, which determines immune-therapy outcomes, remains largely unclear. Here we demonstrated an oncogenic role of PD-L1 via binding and activating Ras in GBM cells. RNA-sequencing transcriptome data revealed that PD-L1 significantly altered gene expression enriched in cell growth/migration/invasion pathways in human GBM cells. PD-L1 overexpression and knockout or knockdown demonstrated that PD-L1 promoted GBM cell proliferation and migration in vitro and in vivo. Mechanistically, PD-L1 prominently activated epithelial mesenchymal transition (EMT) process in a MEK/Erk- but not PI3K/Akt-dependent manner. Further, we identified intracellular interactions of PD-L1 and H-Ras, which led to Ras/Erk/EMT activation. Finally, we demonstrated that PD-L1 overexpression promoted while knockdown abolished GBM development and invasion in orthotopic GBM models of rodents. Taken together, we found that intracellular PD-L1 confers GBM cell malignancy and aggressiveness via binding Ras and activating the downstream Erk-EMT signaling. Thus, these results shed important insights in improving efficacy of immune therapy for GBM as well as other malignant tumors.

Treatment with 5-azacitidine delay growth of glioblastoma xenografts: a potential new treatment approach for glioblastomas.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults. The epigenetically active ribonucleoside analog 5-azacitidine is a new therapy option that changes tumor cell chromatin, which is frequently modified by methylation and deacetylation in malignant gliomas. In vitro, we analyzed cell viability, cell apoptosis, and migration of human GBM cells. In vivo, we established subcutaneous and intracerebral GBM mouse models originating from U87MG, U373MG, and primary GBM cells as well as one patient-derived xenograft. Xenografts were treated with 5-azacitidine as well as valproic acid, bevacizumab, temozolomide, and phosphate buffered saline. The tumor sizes and Ki67 proliferation indices were determined. Glioma angiogenesis was examined immunohistochemically by expression analysis of endothelial cells (CD31) and pericytes (PDGFRβ). In vitro, 5-azacitidine treatment significantly reduced human glioblastoma cell viability, increased cellular apoptosis, and reduced cellular migration. In vivo, 5-azacitidine significantly reduced growth in two intracerebral GBM models. Notably, this was also shown for a xenograft established from a patient surgery sample; whereas, epigenetically acting valproic acid did not show any growth reduction. Highly vascularized tumors responded to treatment, whereas low-vascularized xenografts showed no response. Furthermore, intracerebral glioblastomas treated with 5-azacitidine showed a clearly visible reduction of tumor angiogenesis and lower numbers of endothelial cells and tumor vessel pericytes. Our data show significant growth inhibition as well as antiangiogenic effects in intracerebral as well as patient-derived GBM xenografts. This encourages to investigate in detail the multifactorial effects of 5-azacitidine on glioblastomas.

Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells

Background: Glioblastoma multiforme (GBM) is the most aggressive form of malignant glioma and is also known as grade IV astrocytoma. This might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal. Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Until now, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells. Methods: Umbilical cord-derived MSCs (UCSCs) were grown on serum-free alphaMEM for 24 hours to prepare the UCSC-CM. Human GBM T98G cells were treated with UCSC-CM for 24 hours. Following this treatment, expression of pluripotency markers SOX2, OCT4 and NANOG genes was analyzed using quantitative RT-PCR. Results: SOX2 and OCT mRNA expression was 4.7-fold (p=0.02) and 1.3-fold (p=0.03) higher in CM-treated cells compared to the control. However, there was no change in NANOG mRNA expression. This might be due to there being others factors regulating NANOG mRNA expression. Conclusions: UCSC-CM could affect the expression of SOX2 and OCT4 in human glioblastoma multiforme T98G cells. Further research is needed to elucidate the mechanism by which pluripotency markers are expressed when induced by the UCSC secretome.

NKG2D ligands in glioma stem-like cells: expression in situ and in vitro.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Tumor stem cells have a major influence on tumor malignancy, and immunological escape mechanisms, involving the Natural Killer Group 2, member D (NKG2D) receptor-ligand-system, are key elements in tumor immuno-surveillance. We analyzed the expression profile and localization of NKG2D ligands (NKG2DL) and embryonic and neural stem cell markers in solid human GBM and stem-like cells isolated from glioma cell lines by qRT-PCR and immunohistochemistry, including quantitative analysis. We also evaluated the effect of Temozolomide (TMZ), the standard chemotherapeutic agent used in GBM therapy, on NKG2DL expression. NKG2DL-positive cells were mostly found scattered and isolated, were detectable in glial fibrillary acidic protein (GFAP)-positive tumor regions and partly in the penumbra of tumor vessels. NKG2DL were found in a distinct tumor stem-like cell subpopulation and were broadly costained with each other. Quantitative analysis revealed, that dependent on the individual NKG2DL investigated, cell portions costained with different stem cell markers varied between small (Musashi-1) and high (KLf-4) amounts. However, a costaining of NKG2DL with CD3γ, typically found in T cells, was also observable, whereas CD11b as a marker for tumor micoglia cells was only rarely costained with NKG2DL. Stem-like cells derived from the glioma cell lines T98G and U251MG showed a distinct expression pattern of NKG2DL and stem cell markers, which seemed to be balanced in a cell line-specific way. With differentiation, T98G displayed less NKG2DL, whereas in U251MG, only expression of most stem cell markers decreased. In addition, stimulation with TMZ led to a significant upregulation of NKG2DL in stem-like cells of both lines. As stem-like glioma cells tend to show a higher expression of NKG2DL than more differentiated tumor cells and TMZ treatment supports upregulation of NKG2DL, the NKG2D system might play an important role in tumor stem cell survival and in GBM therapy.

Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking

Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Pyr3 Induces Apoptosis and Inhibits Migration in Human Glioblastoma Cells

Background/Aims: Glioblastoma, also known as glioblastoma multiforme (GBM), is a fast-growing type of tumor that is the most aggressive brain malignancy in adults. According to GEO profile analysis, patients with high transient receptor potential canonical 3 (TRPC3) expression have poor survival rates. The aim of this study is to evaluate the effects of Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a selective TRPC3 channel blocker, on the proliferation and migration of human glioblastoma cells. Methods: We first analyzed the TRPC3 mRNA expression in Gene Expression Omnibus (GEO) database. Then, TRPC3 protein expression was analyzed by Western blotting in three human GBM cell lines. The survival rate was measured by sulforhodamine B. JC1 staining was used to analyze the mitochondria membrane potential by flow cytometric analysis. Besides, the migration and invasion were evaluated by wound healing and Transwell assays. Annexin V and 7-aminoactinomycin D staining was used to monitor the apoptosis by flow cytometric analysis. The expression of apoptotic-related and migration-related proteins after Pyr3 treatment was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of Pyr3 in the in vivo study. Results: Basis on the results of bioinformatics study, glioma patients with higher TRPC3 expression had a shorter survival time than those with lower TRPC3 expression. GBM cell proliferation was decreased by Pyr3 treatment. The migration and invasion abilities of glioma cells were also inhibited via focal adhesion kinase and myosin light chain dephosphorization after Pyr3 treatment. Moreover, Pyr3 induced caspase-dependent apoptosis and mitochondria membrane potential imbalance in the GBM cells. In a xenograft animal model, Pyr3 in combination with temozolomide (TMZ) inhibited GBM tumor growth. Conclusion: Pyr3 inhibited GBM tumor growth in vitro and in vivo. Pyr3-TMZ combination therapy could be used to treat glioblastoma in the future.