Deep learning identified glioblastoma subtypes based on internal genomic expression ranks.

BACKGROUND: Glioblastoma (GBM) can be divided into subtypes according to their genomic features, including Proneural (PN), Neural (NE), Classical (CL) and Mesenchymal (ME). However, it is a difficult task to unify various genomic expression profiles which were standardized with various procedures from different studies and to manually classify a given GBM sample into a subtype. METHODS: An algorithm was developed to unify the genomic profiles of GBM samples into a standardized normal distribution (SND), based on their internal expression ranks. Deep neural networks (DNN) and convolutional DNN (CDNN) models were trained on original and SND data. In addition, expanded SND data by combining various The Cancer Genome Atlas (TCGA) datasets were used to improve the robustness and generalization capacity of the CDNN models. RESULTS: The SND data kept unimodal distribution similar to their original data, and also kept the internal expression ranks of all genes for each sample. CDNN models trained on the SND data showed significantly higher accuracy compared to DNN and CDNN models trained on primary expression data. Interestingly, the CDNN models classified the NE subtype with the lowest accuracy in the GBM datasets, expanded datasets and in IDH wide type GBMs, consistent with the recent studies that NE subtype should be excluded. Furthermore, the CDNN models also recognized independent GBM datasets, even with small set of genomic expressions. CONCLUSIONS: The GBM expression profiles can be transformed into unified SND data, which can be used to train CDNN models with high accuracy and generalization capacity. These models suggested NE subtype may be not compatible with the 4 subtypes classification system.

Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo.

BACKGROUND: Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity. RESULTS: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo. CONCLUSIONS: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8-10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Outer membrane protein A (OmpA) as a potential therapeutic target for Acinetobacter baumannii infection.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing serious nosocomial infections, which is considered as the most threatening Gram-negative bacteria (GNB). Outer membrane protein A (OmpA), a major component of outer membrane proteins (OMPs) in GNB, is a key virulence factor which mediates bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance and immunomodulation. The characteristics of OmpA in Escherichia coli (E. coli) have been extensively studied since 1974, but only in recent years researchers started to clarify the functions of OmpA in A. baumannii. In this review, we summarized the structure and functions of OmpA in A. baumannii (AbOmpA), collected novel therapeutic strategies against it for treating A. baumannii infection, and emphasized the feasibility of using AbOmpA as a potential therapeutic target.

Loop-armed DNA tetrahedron nanoparticles for delivering antisense oligos into bacteria.

BACKGROUND: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria. RESULTS: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 µL/µg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure. CONCLUSIONS: This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

Hypoxia Regulated Gene Network in Glioblastoma Has Special Algebraic Topology Structures and Revealed Communications Involving Warburg Effect and Immune Regulation.

Hypoxia regulated genes (HRGs) formed a complex molecular interaction network (MINW), contributing to many aspects of glioblastoma (GBM) tumor biology. However, little is known about the intrinsic structures of the HRGs-MINW, mainly due to a lack of analysis tools to decipher MINWs. By introducing general hyper-geometric distribution, we obtained a statistically reliable gene set of HRGs (SR-HRGs) from several datasets. Next, MINWs were reconstructed from several independent GBM expression datasets. Algebraic topological analysis was performed to quantitatively analyze the amount of equivalence classes of cycles in various dimensions by calculating the Betti numbers. Persistent homology analysis of a filtration of growing networks was further performed to examine robust topological structures in the network by investigating the Betti curves, life length of the cycles. Random networks with the same number of node and edge and degree distribution were produced as controls. As a result, GBM-HRGs-MINWs reconstructed from different datasets exhibited great consistent Betti curves to each other, which were significantly different from that of random networks. Furthermore, HRGs-MINWs reconstructed from normal brain expression datasets exhibited topological structures significantly different from that of GBM-HRGs-MINWs. Analysis of cycles in GBM-HRGs-MINWs revealed genes that had clinical implications, and key parts of the cycles were also identified in reconstructed protein-protein interaction networks. In addition, the cycles are composed by genes involved in the Warburg effect, immune regulation, and angiogenesis. In summary, GBM-HRGs-MINWs contained abundant molecular interacting cycles in different dimensions, which are composed by genes involved in multiple programs essential for the tumorigenesis of GBM, revealing novel interaction diagrams in GBM and providing novel potential therapeutic targets.

Advances in the delivery of antisense oligonucleotides for combating bacterial infectious diseases.

Discovery and development of new antibacterial drugs against multidrug resistant bacterial strains have become more and more urgent. Antisense oligonucleotides (ASOs) show immense potential to control the spread of resistant microbes due to its high specificity of action, little risk to human gene expression, and easy design and synthesis to target any possible gene. However, efficient delivery of ASOs to their action sites with enough concentration remains a major obstacle, which greatly hampers their clinical application. In this study, we reviewed current progress on delivery strategies of ASOs into bacteria, focused on various non-virus gene vectors, including cell penetrating peptides, lipid nanoparticles, bolaamphiphile-based nanoparticles, DNA nanostructures and Vitamin B12. The current review provided comprehensive understanding and novel perspective for the future application of ASOs in combating bacterial infections.

A potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with LED209 targeting QseC receptor to inhibit the virulence genes of gram negative bacteria.

The pandemic of multidrug-resistant Gram negative bacteria (GNB) is a worldwide healthcare concern, and very few antibiotics are being explored to match the clinical challenge. Recently, amino-terminated poly(amidoamine) (PAMAM) dendrimers have shown potential to function as broad antimicrobial agents. However, PAMAM displays a generation dependent cytotoxicity to mammalian cells and low selectivity on bacterial cells, which limits PAMAM to be developed as an antibacterial agent for systemic administration. We conjugated G3 PAMAM with LED209, a specific inhibitor of quorum sensor QseC of GNB, to generate a multifunctional agent PAMAM-LED209. Intriguingly, PAMAM-LED209 showed higher selectivity on GNB and lower cytotoxicity to mammalian cells, yet remained strong antibacterial activity. PAMAM-LED209 also inhibited virulence gene expression of GNB, and did not induce antibiotic-resistance. The present work firstly demonstrated that PAMAM-LED209 conjugate had a highly selective anti-GNB activity and low cytotoxicity, which offered a feasible strategy for combating multidrug-resistant GNB infections. FROM THE CLINICAL EDITOR: This research team demonstrated that a novel PAMAM-LED209 conjugate had highly selective activity against Gram-negative bacteria, coupled with low cytotoxicity, offering a potential strategy for combating multidrug-resistant infections.

LGR5 is a proneural factor and is regulated by OLIG2 in glioma stem-like cells.

The biological functional roles of LGR5 (leucine-rich repeat containing G protein-coupled receptor 5, also known as GPR49), a novel potential marker for stem-like cells in glioblastoma (GSCs), is poorly acknowledged. Here, we demonstrated that LGR5 was detected in glioblastoma tissues and GSCs. Bioinformatics analysis revealed that LGR5 is closely related to neurogenesis and neuronal functions, and preferentially expressed in Proneural subtype of GBMs. Furthermore, LGR5 is regulated by Proneural factor OLIG2, which is important for both neurogenesis and GSC maintenance. Biological experiments in GSC cells validated the bioinformatics analysis results and revealed that LGR5 regulated the tumor sphere formation capacity, an important stem cell property for GSCs. Therefore, LGR5 expression may be functionally correlated with the neurogenic competence, and be regulated by OLIG2 in GSCs.

The Role of Hypoxia and Cancer Stem Cells in Development of Glioblastoma.

Glioblastoma multiform (GBM) is recognized as the most malignant brain tumor with a high level of hypoxia, containing a small population of glioblastoma stem like cells (GSCs). These GSCs have the capacity of self-renewal, proliferation, invasion and recapitulating the parent tumor, and are major causes of radio-and chemoresistance of GBM. Upregulated expression of hypoxia inducible factors (HIFs) in hypoxia fundamentally contributes to maintenance and progression of GSCs. Therefore, we thoroughly reviewed the currently acknowledged roles of hypoxia-associated GSCs in development of GBM. In detail, we recapitulated general features of GBM, especially GSC-related features, and delineated essential responses resulted from interactions between GSC and hypoxia, including hypoxia-induced signatures, genes and pathways, and hypoxia-regulated metabolic alterations. Five hypothesized GSC niches are discussed and integrated into one comprehensive concept: hypoxic peri-arteriolar niche of GSCs. Autophagy, another protective mechanism against chemotherapy, is also closely related to hypoxia and is a potential therapeutic target for GBM. In addition, potential causes of therapeutic resistance (chemo-, radio-, surgical-, immuno-), and chemotherapeutic agents which can improve the therapeutic effects of chemo-, radio-, or immunotherapy are introduced and discussed. At last, as a potential approach to reverse the hypoxic microenvironment in GBM, hyperbaric oxygen therapy (HBOT) might be an adjuvant therapy to chemo-and radiotherapy after surgery. In conclusion, we focus on demonstrating the important role of hypoxia on development of GBM, especially by affecting the function of GSCs. Important advantages have been made to understand the complicated responses induced by hypoxia in GBM. Further exploration of targeting hypoxia and GSCs can help to develop novel therapeutic strategies to improve the survival of GBM patients.

Lemur tyrosine kinase 2 has a tumor-inhibition function in human glioblastoma by regulating the RUNX3/Notch pathway.

Deregulation of lemur tyrosine kinase 2 (LMTK2) is a vital determinant for the onset and progression of malignancies, yet the relationship between LMTK2 and glioblastoma (GBM) is undetermined. This study was carried out to determine the relevance of LMTK2 in GBM. Initiating investigation by assessing The Cancer Genome Atlas (TCGA) data showed LMTK2 mRNA levels were decreased in GBM tissue. Later examination of clinical specimens confirmed low levels of LMTK2 mRNA and protein in GBM tissue. The downregulated level of LMTK2 in patients with GBM was related to poor overall survival. A suppressive function of LMTK2 on the proliferative capability and metastatic potential of GBM cells was demonstrated by overexpressing LMTK2 in GBM cell lines. Moreover, the restoration of LMTK2 augmented the sensitivity of GBM cells to the chemotherapy drug temozolomide. The mechanistic investigation uncovered LMTK2 as a regulator of the runt-related transcription factor 3 (RUNX3)/Notch signaling pathway. The overexpression of LMTK2 increased the expression of RUNX3 while inhibiting the activation of Notch signaling. The silencing of RUNX3 diminished the regulatory role of LMTK2 on Notch signaling. The inhibition of Notch signaling reversed the LMTK2-silencing-elicited protumor effects. Importantly, LMTK2-overexpressed GBM cells displayed weakened tumorigenicity in xenograft models. Our findings illustrate that LMTK2 has a tumor-inhibition function in GBM by constraining Notch signaling via RUNX3. This work indicates the deregulation of the LMTK2-mediated RUNX3/Notch signaling pathway may be a novel molecular mechanism for the malignant transformation of GBMs. This work highlights the interest in LMTK2-related approaches for treating GBM.

Cuproptosis-related gene expression is associated with immune infiltration and CD47/CD24 expression in glioblastoma, and a risk score based on these genes can predict the survival and prognosis of patients.

Introduction: Glioblastoma (GBM) is the most invasive type of glioma, is insensitive to radiotherapy and chemotherapy, and has high proliferation and invasive ability, with a 5-year survival rate of <5%. Cuproptosis-related genes (CRGs) have been successfully used to predict the prognosis of many types of tumors. However, the relationship between cuproptosis and GBM remains unclear. Methods: Here, we sought to identify CRGs in GBM and elucidate their role in the tumor immune microenvironment and prognosis. To that aim, changes in CRGs in The Cancer Genome Atlas (TCGA) transcriptional and Gene Expression Omnibus (GEO) datasets (GEO4290 and GEO15824) were characterized, and the expression patterns of these genes were analyzed. Results: A risk score based on CRG expression characteristics could predict the survival and prognosis of patients with GBM and was significantly associated with immune infiltration levels and the expression of CD47 and CD24, which are immune checkpoints of the "don't eat me "signal. Furthermore, we found that the CDKN2A gene may predict GBM sensitivity and resistance to drugs. Discussion: Our findings suggest that CRGs play a crucial role in GBM outcomes and provide new insights into CRG-related target drugs/molecules for cancer prevention and treatment.

CEBPD is a master transcriptional factor for hypoxia regulated proteins in glioblastoma and augments hypoxia induced invasion through extracellular matrix-integrin mediated EGFR/PI3K pathway.

Hypoxia contributes to the initiation and progression of glioblastoma by regulating a cohort of genes called hypoxia-regulated genes (HRGs) which form a complex molecular interacting network (HRG-MINW). Transcription factors (TFs) often play central roles for MINW. The key TFs for hypoxia induced reactions were explored using proteomic analysis to identify a set of hypoxia-regulated proteins (HRPs) in GBM cells. Next, systematic TF analysis identified CEBPD as a top TF that regulates the greatest number of HRPs and HRGs. Clinical sample and public database analysis revealed that CEBPD is significantly up-regulated in GBM, high levels of CEBPD predict poor prognosis. In addition, CEBPD is highly expressed in hypoxic condition both in GBM tissue and cell lines. For molecular mechanisms, HIF1α and HIF2α can activate the CEBPD promotor. In vitro and in vivo experiments demonstrated that CEBPD knockdown impaired the invasion and growth capacity of GBM cells, especially in hypoxia condition. Next, proteomic analysis identified that CEBPD target proteins are mainly involved in the EGFR/PI3K pathway and extracellular matrix (ECM) functions. WB assays revealed that CEBPD significantly positively regulated EGFR/PI3K pathway. Chromatin immunoprecipitation (ChIP) qPCR/Seq analysis and Luciferase reporter assay demonstrated that CEBPD binds and activates the promotor of a key ECM protein FN1 (fibronectin). In addition, the interactions of FN1 and its integrin receptors are necessary for CEBPD-induced EGFR/PI3K activation by promoting EGFR phosphorylation. Furthermore, GBM sample analysis in the database corroborated that CEBPD is positively correlated with the pathway activities of EGFR/PI3K and HIF1α, especially in highly hypoxic samples. At last, HRPs are also enriched in ECM proteins, indicating that ECM activities are important components of hypoxia induced responses in GBM. In conclusion, CEPBD plays important regulatory roles in the GBM HRG-MINW as a key TF, which activates the EGFR/PI3K pathway through ECM, especially FN1, mediated EGFR phosphorylation.

Biomimetic three-dimensional glioma model printed in vitro for the studies of glioma cells and neurons interactions.

1The interactions between glioma cells and neurons are important for glioma progression but are rarely mimicked and recapitulated in in vitro three-dimensional (3D) models, which may affect the success rate of relevant drug research and development. In this study, an in vitro bioprinted 3D glioma model consisting of an outer hemispherical shell with neurons and an inner hemisphere with glioma cells is proposed to simulate the natural glioma. This model was produced by extrusion-based 3D bioprinting technology. The cells survival rate, morphology, and intercellular Ca2+ concentration studies were carried out up to 5 days of culturing. It was found that neurons could promote the proliferation of glioma cells around them, associate the morphological changes of glioma cells to be neuron-like, and increase the expression of intracellular Ca2+ of glioma cells. Conversely, the presence of glioma cells could maintain the neuronal survival rate and promote the neurite outgrowth. The results indicated that glioma cells and neurons facilitated each other implying a symbiotic pattern established between two types of cells during the early stage of glioma development, which were seldom found in the present artificial glioma models. The proposed bioprinted glioma model can mimic the natural microenvironment of glioma tissue, provide an in-depth understanding of cell-cell interactions, and enable pathological and pharmacological studies of glioma.

Survivin promotes glioma angiogenesis through vascular endothelial growth factor and basic fibroblast growth factor in vitro and in vivo.

Survivin is involved in multiple signaling mechanisms in tumor maintenance, and accumulated studies elucidate that knockdown of survivin in endothelial cells could inhibit angiogenesis; however, the role of survivin in tumor cells to regulate tumor-derived angiogenesis remains largely unclear. In the present study 80 cases of brain glioma were chosen and protein expressions of survivin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in glioma cells were investigated by immunohistochemistry (IHC). Human umbilical vein endothelial cells (HUVEC) were cocultured with human glioma U251 wild-type cells, U251 cells survivin silenced, SHG44 wild-type cells, and SHG44 survivin-overexpressing cells, respectively. The proliferation and migration of HUVEC were evaluated by MTT assay and transwell chamber assay. The microvessels density (MVD) marked by CD31 expression in vascular endothelial cells in glioma xenografts in nude mice was detected by IHC. VEGF, bFGF, and PDGF in the aforementioned cells were detected by quantitive PCR (qPCR), Western blot, ELISA, and IHC in vitro and in vivo. The results showed that VEGF immunoreactivity score (IRS), bFGF IRS, and PDGF IRS were all positively correlated with survivin IRS in gliomas, respectively (P < 0.01). Survivin in human glioma cells could significantly promote the proliferation and migration of HUVEC and increase MVD, which could be contributed to survivin-dependent burst of VEGF and bFGF expression, followed by increase of tumor growth and proliferation. In summary, survivin, through upregulation of VEGF and bFGF, plays an essential role during glioma angiogenesis.

Aberrant expression of N-methylpurine-DNA glycosylase influences patient survival in malignant gliomas.

AIM: To examine the expression of N-methylpurine-DNA glycosylase (MPG) gene and protein in glioma samples with different WHO grades and its association with patients' survival. METHODS: Immunohistochemistry assay, quantitative real-time PCR and Western blot analysis were carried out to investigate the expression of MPG gene and protein in 128 glioma and 10 non-neoplastic brain tissues. RESULTS: MPG gene expression level in glioma tissues was significantly higher than that in non-neoplastic brain tissues (P < 0.001). Immunohistochemistry also showed that MPG protein was over-expressed in glioma tissues, which was consistent with the resutls of Western blot analysis. Additionally, the expression levels of MPG gene and protein both increase from grade I to grade IV glioma according to the results of real-time PCR, immunohistochemistry and western blot analysis. Moreover, the survival rate of MPG-positive patients was significantly lower than that of MPG-negative patients (P < 0.001). We further confirmed that the over-expression of MPG was a significant and independent prognostic indicator in glioma by multivariate analysis (P < 0.001). CONCLUSIONS: Our data showed the over-expression of MPG gene and protein in human gliomas, and also suggested for the first time that MPG be an unfavorable independent prognostic indicator for glioma patients.

Promoting effects of chemical permeation enhancers on insulin permeation across TR146 cell model of buccal epithelium in vitro.

To find potential enhancers for facilitating the buccal delivery of insulin, a TR146 cell-culture model of buccal epithelium, cultured on commercially available insert plates, was used to evaluate the permeability-enhancing effects of several traditional and new types of chemical enhancers, including N-acetyl-L-cysteine (NAC), sodium deoxycholate (SDC), sodium nitroprusside (SNP), reduced glutathione (GSH), glutamine (Gln), chitosan (CS), L-arginine (Arg), 1-dodecylazacycloheptan-2-one (Azone), and soybean lecithin (SPC) (50 and 10 µg/mL respectively). Permeability studies were performed to determine the enhancing effects of these compounds on insulin permeation across the cell-culture model. The enhancing effects of the enhancers were assessed by calculating the apparent permeability coefficients and enhancement ratio. Cytotoxicity of the permeation enhancers at different concentrations was investigated by using the methylthiazolydiphenyl-tetrazolium bromide (MTT) assay. Results showed that 50 µg/mL of NAC, SDC, GSH, CS, Arg, Azone, SPC, SNP, and 10 µg/mL of SNP had a significant enhancing effect on promoting the transport of insulin across the TR146 cell model. MTT assays showed that 50 µg/mL of Gln, Azone, SDC, SNP, Arg, 10 µg/mL SDC, and Arg had obvious toxic effects on TR146 cells. Therefore, NAC, GSH, CS, SPC, and SNP appear to be safe, effective permeability enhancers that promote the transport of insulin across the TR146 cell-culture model of buccal epithelium and may be potential enhancers for buccal delivery of insulin with both low toxicity and high efficiency.

Protein-protein interaction network of E. coli K-12 has significant high-dimensional cavities: new insights from algebraic topological studies.

As a model system, Escherichia coli has been used to study various life processes. A dramatic paradigm shift has occurred in recent years, with the study of single proteins moving toward the study of dynamically interacting proteins, especially protein-protein interaction (PPI) networks. However, despite the importance of PPI networks, little is known about the intrinsic nature of the network structure, especially high-dimensional topological properties. By introducing general hypergeometric distribution, we reconstruct a statistically reliable combined PPI network of E. coli (E. coli-PPI-Network) from several datasets. Unlike traditional graph analysis, algebraic topology was introduced to analyze the topological structures of the E. coli-PPI-Network, including high-dimensional cavities and cycles. Random networks with the same node and edge number (RandomNet) or scale-free networks with the same degree distribution (RandomNet-SameDD) were produced as controls. We discovered that the E. coli-PPI-Network had special algebraic typological structures, exhibiting more high-dimensional cavities and cycles, compared to RandomNets or, importantly, RandomNet-SameDD. Based on these results, we defined degree of involved q-dimensional cycles of proteins (q-DCprotein ) in the network, a novel concept that relies on the integral structure of the network and is different from traditional node degree or hubs. Finally, top proteins ranked by their 1-DCprotein were identified (such as gmhB, rpoA, rplB, rpsF and yfgB). In conclusion, by introducing mathematical and computer technologies, we discovered novel algebraic topological properties of the E. coli-PPI-Network, which has special high-dimensional cavities and cycles, and thereby revealed certain intrinsic rules of information flow underlining bacteria biology.

Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors.

Glioblastoma multiforme (GBM) is the most aggressive and common kind of primary brain tumor in adults, and is thought to be driven by a subpopulation of glioma stem cells (GSCs). GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1α and HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT-PCR and western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by small-interfering RNA (siRNA) inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1α or HIF2α, which has a central role in the hypoxia-induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1α or HIF2α was targeted by siRNA. HIF2α knockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1α knockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs, which is regulated by HIFs released by the hypoxic environment of the tumor.

Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax.

BACKGROUND: Boron neutron capture therapy (BNCT) is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE) of BNCT, γ-ray and reactor neutron irradiation. METHODS: The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR) and γ-rays were obtained from [60Co] γ source of the Fourth Military Medical University (FMMU) in China. Human glioma cells (the U87, U251, and SHG44 cell lines) were irradiated by neutron beams at the XAPR or [60Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [60Co] γ-rays; Group C included cells treated with 8 Gy of [60Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine)-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM). The apoptosis rate was detected by flow cytometer (FCM). The level of Bcl-2 and Bax protein was measured by western blot analysis. RESULTS: Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [60Co] γ-rays (P < 0.01). Nuclear condensation was determined using both a fluorescence technique and electron microscopy in all cell lines treated with BPA-BNCT. Furthermore, the cellular apoptotic rates in Group D and Group E treated with BPA-BNCT were significantly higher than those in Group B and Group C irradiated by [60Co] γ-rays (P < 0.01). The clonogenicity of glioma cells was reduced by BPA-BNCT compared with cells treated in the reactor (Group F, G, H, I), and with the control cells (P < 0.01). Upon BPA-BNCT treatment, the Bax level increased in glioma cells, whereas Bcl-2 expression decreased. CONCLUSIONS: Compared with γ-ray and reactor neutron irradiation, a higher RBE can be achieved upon treatment of glioma cells with BNCT. Glioma cell apoptosis induced by BNCT may be related to activation of Bax and downregulation of Bcl-2.

Maintenance of critical properties of brain tumor stem-like cells after cryopreservation.

It would be very useful to be able to classify brain tumor stem cells (BTSCs) by certain criteria to afford the design of specific or individualized treatment. Here, we studied two BTSC lines with differing biological and molecular features and whose respective features were well preserved after cryopreservation as single cells in SFM or 90% serum with 10% DMSO, a method not previously reported. The resuscitated BTSCs shared properties indistinguishable from their respective parental cells, including tumor sphere forming potentials, growth and differentiation properties, and tumorigenesis in vivo. The two cell lines also had differing molecule profiles, which can be well preserved after cryopreservation, similar to that of their respective primary tumors. Therefore, BTSCs from different patients, that have their own properties, were well retained by the present cryopreservation method, which might be a useful and reliable method for preserving BTSCs for long-term studies, such as classification and specific therapy design.