MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma

Background

Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.

Methodology/Principal Findings

In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133⁺/CD15⁺ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1^+/- p53^-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.

Conclusions/Significance

Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.     

Evaluation of CD98 light chain-LAT1 as a potential marker of cancer stem-like cells in glioblastoma

ObjectiveGlioma stem cells (GSCs) are a minority population of glioma cells that regarded as the cause of tumor formation and recurrence. Identifying new molecular strategies targeting GSCs must be urgently developed to treat glioblastoma. In this study, one of CD98 light chain-L type amino acid transporter 1 (LAT1) was found as a potential GSC marker. LAT1 served as EAA transporter has been shown to be closely related with tumor invasion, metastasis, angiogenesis, and radiosensitivity.MethodsLAT1⁺ and LAT1^? glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation arrays, and in vitro limiting dilution experiments were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of β-tubulinIII, GFAP, and LAT1 were detected by Western blot. Nude mouse models were constructed to observe tumor formation and metastasis in nude mice.ResultsLAT1⁺ glioma cells were testified a small percentage of all cells and selected as the subsequent sorting marker. LAT1⁺ cells were separated from U87 and U251 cells could express high level of stem cell markers, and possessed GSC properties including self-renewal ability and multi-directional differentiation potential. But LAT1^? cells did not have these characteristics. In addition, LAT1⁺ cells were able to generate tumors in vivo, tumor size of LAT1⁺ cells formed were much bigger than that of LAT1^? cells.ConclusionOur study, including molecular, cell, vitro and vivo experiments, has shown that LAT1⁺ cells possess GSC properties, and present for the first time that LAT1 can be used as a new marker for GSCs screening.     

miR-363-3p induces EMT via the Wnt/β-catenin pathway in glioma cells by targeting CELF2

In our previous study, we reported that CELF2 has a tumour-suppressive function in glioma. Here, we performed additional experiments to elucidate better its role in cancer. The expression profile of CELF2 was analysed by the GEPIA database, and Kaplan–Meier curves were used to evaluate the overall survival rates. Four different online databases were used to predict miRNAs targeting CELF2, and the luciferase assay was performed to identify the binding site. The biological effects of miR-363-3p and CELF2 were also investigated in vitro using MTT, Transwell, and flow cytometry assays. Western blotting, qPCR, and TOP/FOP flash dual-luciferase assays were performed to investigate the impact of miR-363-3p and CELF2 on epithelial-to-mesenchymal transition (EMT) and the Wnt/β-catenin pathway. The effect of miR-363-3p was also tested in vivo using a xenograft mouse model. We observed an abnormal expression pattern of CELF2 in glioma cells, and higher CELF2 expression correlated with better prognosis. We identified miR-363-3p as an upstream regulator of CELF2 and confirmed its direct binding to the 3′-untranslated region of CELF2. Cell function experiments showed that miR-363-3p affected multiple aspects of glioma cells. Suppressing miR-363-3p expression inhibited glioma cell proliferation and invasion, as well as promoted cell death via attenuating EMT and blocking the Wnt/β-catenin pathway. These effects could be abolished by the downregulation of CELF2. Treatment with ASO-miR-363-3p decreased tumour size and weight in nude mice. In conclusion, miR-363-3p induced the EMT, which resulted in increased migration and invasion and reduced apoptosis in glioma cell lines, via the Wnt/β-catenin pathway by targeting CELF2.     

Overexpression of FOXD2‐AS1 enhances proliferation and impairs differentiation of glioma stem cells by activating the NOTCH pathway via TAF‐1

Emerging data have highlighted the importance of long noncoding RNAs (lncRNAs) in exerting critical biological functions and roles in different forms of brain cancer, including gliomas. In this study, we sought to investigate the role of lncRNA FOXD2 adjacent opposite strand RNA 1 (FOXD2‐AS1) in glioma cells. First, we used sphere formation assay and flow cytometry to select U251 glioma stem cells (GSCs). Then, we quantified the expression of lncRNA FOXD2‐AS1, TATA‐box binding protein associated factor 1 (TAF‐1) and NOTCH1 in glioma tissues and GSCs, as well as the expression of GSC stem markers, OCT4, SOX2, Nanog, Nestin and CD133 in GSCs. Colony formation assay, sphere formation assay, and flow cytometry were used to evaluate GSC stemness. Next, the correlations among lncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were investigated. LncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were found to be elevated in glioma tissues and GSCs, and silencing lncRNA FOXD2‐AS1 inhibited stemness and proliferation, while promoting apoptosis and differentiation of GSCs. LncRNA FOXD2‐AS1 overexpression also led to increased NOTCH1 by recruiting TAF‐1 to the NOTCH1 promoter region, thereby promoting stemness and proliferation, while impairing cell apoptosis and differentiation. Mechanistically, lncRNA FOXD2‐AS1 elevation promoted glioma in vivo by activating the NOTCH signalling pathway via TAF‐1 upregulation. Taken together, the key findings of our investigation support the proposition that downregulation of lncRNA FOXD2‐AS1 presents a viable and novel molecular candidate for improving glioma treatment.     

The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.     

siRNA targeting <Emphasis Type="Italic">Notch</Emphasis>-<Emphasis Type="Italic">1</Emphasis> decreases glioma stem cell proliferation and tumor growth

Glioblastoma multiforme (GBM), the most common brain tumor in adults, is neurologically destructive and has a dismal response to virtually all therapeutic modalities. One phenomenon that can contribute to this complexity is the presence of a relatively small subset of glioma stem cells (GSCs) within the tumor and the activation of pathways that control cellular differentiation. The Notch signaling pathway, which is responsible for maintaining a balance between cell proliferation and apoptosis, is believed to be deregulated in cancer stem cells (CSCs), leading to tumor growth through the generation or expansion of CSCs. In this study, Notch-1 small interfering RNA (siRNA) was used to silence Notch-1 gene expression in GSCs. An MTT assay demonstrated inhibitory effects on the proliferation of GSCs in vitro. Real-time PCR showed that Notch-1 expression levels were markedly decreased in GSCs transfected with Notch-1 siRNA in vitro. Notch-1 silenced GSCs engrafted on Balb/c nude mice showed a significantly greater reduction in oncogenicity than the control group (P < 0.05). Furthermore, direct intratumoral injections of Notch-1-siRNA/PEI significantly delayed the growth of pre-established tumors in nude mice (P < 0.05). These results suggest that siRNA-mediated silencing of the Notch-1 gene may represent a novel target for gene therapy of GBM.     

LOXL1‐AS1 communicating with TIAR modulates vasculogenic mimicry in glioma via regulation of the miR‐374b‐5p/MMP14 axis

At present, growing evidence indicates that long non‐coding RNAs (lncRNAs) participate in the progression of glioma. The function of LOXL1‐AS1 in vasculogenic mimicry (VM) in glioma remains unclear. First, the expressions of TIAR, the lncRNA LOXL1‐AS1, miR‐374b‐5p and MMP14 were examined by qRT‐PCR and Western blot in both, glioma tissues and glioma cell lines. Proliferation, migration, invasion and tube formation assays were conducted to evaluate the roles of TIAR, LOXL1‐AS1, miR‐374b‐5p and MMP14 in malignant cellular behaviours in glioma cells. A nude mouse xenograft model and dual staining for CD34 and PAS were used to assess whether VM was affected by TIAR, LOXL1‐AS1 or miR‐374b‐5p in vivo. In this study, low levels of TIAR and high levels of LOXL1‐AS1 were found in glioma cells and tissues. TIAR downregulated the expression of LOXL1‐AS1 by destabilizing it. LOXL1‐AS1 acted like a miRNA sponge towards miR‐374b‐5p so that downregulation of the former greatly inhibited cell proliferation, migration, invasion and VM. Additionally, miR‐374b‐5p overexpression repressed malignant biological behaviours and VM in glioma by modifying MMP14. In summary, we demonstrated that TIAR combined with LOXL1‐AS1 modulates VM in glioma via the miR‐374b‐5p/MMP14 axis, revealing novel targets for glioma therapy.     

MicroRNA-107 Inhibits U87 Glioma Stem Cells Growth and Invasion

Glioma stem cells (GSCs) are thought to be critical for resistance to radiotherapy and chemotherapy and for tumor recurrence after surgery in glioma patients. Identification of new therapeutic strategies that can target GSCs may thus be critical for improving patient survival. MicroRNAs (miRNAs) are small non-coding RNAs that function as tumor suppressors or oncogenes. In this study, we confirmed that miR-107 was down-regulated in GSCs. To investigate the role of miR-107 in tumorigenesis of GSCs, a lentiviral vector over-expressing miR-107 in U87GSCs was constructed. We found that over-expression of miR-107 suppressed proliferation and down-regulated Notch2 protein and stem cell marker (CD133 and Nestin) expression in U87GSCs. Furthermore, enhanced miR-107 expression significantly inhibited U87GSC invasion and reduced matrix metalloproteinase-12 expression. miR-107 also suppressed U87GSCs xenograft growth in vivo. These findings suggest that miR-107 is involved in U87GSCs growth and invasion and may provide a potential therapeutic target for glioma treatment.     

Hypoxic remodelling of Ca2+ signalling in proliferating human arterial smooth muscle

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma. Xue-Ping Zhang, Gang Zheng and Lian Zou are contributed equally to this study.     

Astragaloside IV suppresses transforming growth factor-β1-induced epithelial–mesenchymal transition through inhibition of Wnt/β-catenin pathway in glioma U251 cells

ABSTRACT

Astragaloside IV (AS#IV) has previously demonstrated antitumoractivity. We investigated the effect and mechanisms of AS#IV in relation to epithelial–mesenchymal transition (EMT), viainterference with the Wnt/β-catenin signaling pathway in gliomaU251 cells. Induction of glioma U251 cells by transforming growthfactor (TGF)#β1 activated EMT, including switching E#cadherin toN-cadherin and altering the expression of Wnt/β-catenin signalingpathway components such as vimentin, β-catenin, and cyclin-D1.AS-IV inhibited the viability, invasion, and migration of TGF-β1-induced glioma U251 cells. AS-IV also interfered with the TGF#β1-induced Wnt/β-catenin signaling pathway in glioma U251 cells.These findings indicate that AS#IV prohibits TGF#β1-induced EMTby disrupting the Wnt/β-catenin pathway in glioma U251 cells. AS#IV may thus be a potential candidate agent for treating glioma andother central nervous system tumors.     

miR-195通过下调IGF-1R的表达抑制胶质母细胞瘤的增殖和迁移

目的:探讨miR-195对胶质母细胞瘤(Glioblastoma,GBM)增殖和迁移的影响,并阐明其分子调控机制。方法:采用qRT-PCR检测不同级别胶质瘤中miR-195的表达。将miR-195转染至胶质瘤U251细胞后,应用qRT-PCR验证转染效率,MTT及划痕实验检测U251细胞的增殖及迁移能力的改变,qRT-PCR及Western blot检测胰岛素样生长因子1受体(Insulin-like growth factor 1receptor, IGF-1R)的mRNA和蛋白表达;利用质粒转染过表达miR-195后,同时过表达IGF-1R,再应用MTT及划痕实验检测U251细胞的增殖及迁移能力的变化。结果:随着胶质瘤级别的增加,miR-195的表达逐渐降低,各级别胶质瘤中miR-195的表达差异有统计学意义(P0.05)。体外转染miR-195至U251细胞24、48、72 h后,转染组细胞活力和迁移能力均较对照组显著降低(P0.05),细胞中IGF-1R的mRNA和蛋白的表达也明显减少(P0.05);通过转染IGF-1R过表达质粒可显著逆转miR-195过表达对U251细胞增殖及迁移的抑制作用。结论:miR-195可能通过下调IGF-1R的表达,进而抑制胶质母细胞瘤的增殖和迁移。     

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye

Purpose

By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs).

Methods

C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45⁺ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition.

Results

DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45⁺ cell infiltration in LGs. Likewise, CD45⁺ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice.

Conclusions

Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.     

Topoisomerase I Inhibitors,Shikonin and Topotecan,Inhibit Growth and Induce Apoptosis of Glioma Cells and Glioma Stem Cells

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.     

Effects of miR-19b Overexpression on Proliferation, Differentiation, Apoptosis and Wnt/β-Catenin Signaling Pathway in P19 Cell Model of Cardiac Differentiation In Vitro

MicroRNA (miR)-19b is part of the miR-17–92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/β-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/β-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and β-catenin expressions were decreased, while that of GSK3β was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/β-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.     

LRRC4 inhibits the proliferation of human glioma cells by modulating the expression of STMN1 and microtubule polymerization

LRRC4 is a tumor suppressor of glioma, and it is epigenetically inactivated commonly in glioma. Our previous study has shown that induction of LRRC4 expression inhibits the proliferation of glioma cells. However, little is known about the mechanisms underlying the action of LRRC4 in glioma cells. We employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI -TOF/TOF-MS/MS to identify 11 differentially expressed proteins, including the significantly down-regulated STMN1 expression in the LRRC4-expressing U251 glioma cells. The levels of STMN1 expression appeared to be positively associated with the pathogenic degrees of human glioma. Furthermore, induction of LRRC4 over-expression inhibited the STMN1 expression and U251 cell proliferation in vitro, and the glioma growth in vivo. In addition, induction of LRRC4 or knockdown of STMN1 expression induced cell cycle arrest in U251 cells, which was associated with modulating the p21, cyclin D1, and cyclin B expression, and the ERK phosphorylation, and inhibiting the CDK5 and cdc2 kinase activities, but increasing the microtubulin polymerization in U251 cells. LRRC4, at least partially by down-regulating the STMN1expression, acts as a major glioma suppressor, induces cell cycle arrest and modulates the dynamic process of microtubulin, leading to the inhibition of glioma cell proliferation and growth. Potentially, modulation of LRRC4 or STMN1 expression may be useful for design of new therapies for the intervention of glioma.     

Notch1 expression and ligand interactions in progenitor cells of the mouse olfactory epithelium

Despite the relatively simplified organization of the olfactory epithelium (OE), our understanding of the factors that regulate its cellular diversity is limited. Genetic and localization studies suggest that Notch signaling may be important in this process. We characterize here a population of Notch1 ⁺ olfactory basal cells in embryonic mice that coordinately express both the Notch effector Hes5 and the glycosyltransferase Lfng. These cells are distinct from Mash1 ⁺ neuronal precursors, but give rise to sensory neurons, suggesting that Notch1 signals may in part function to maintain a neurogenic progenitor pool. Furthermore, Lfng ⁺ cells also generate a population of cells in the migratory mass that appear to be ensheathing glial precursors, indicating potential multipotency in these progenitors. The Notch ligand Dll4 is expressed by basal OE cells that are interspersed with Notch1 ⁺ progenitors during later OE neurogenesis. In contrast, mice deficient in Dll1 exhibit a smaller OE and a loss of Hes5 expression, indicating an earlier function in olfactory progenitor cell development. Taken together, these results further support a role for Notch signaling in the regulation of olfactory neurogenesis and cell diversity.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

MMP-2 siRNA inhibits radiation-enhanced invasiveness in glioma cells

Background

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.

Methodology/Principal Findings

We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.

Conclusion/Significance

MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.     

Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells

Background

Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest.

Methods

The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student’s t-test was used to compare differences between treated groups and their controls.

Results

We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice.

Coclusions

In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.     

The cancer stem cell niche(s): The crosstalk between glioma stem cells and their microenvironment

Background

The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized microenvironments (niches) within tumors. These niches represent the hallmarks of malignant gliomas (vascular proliferations, hypoxia/necrosis) and bear analogy to the microenvironments in which physiological stem cells in the brain are found.

Scope of the review

Here we review the progress that has been made towards uncovering the function of the perivascular and the hypoxic niche and the molecular pathways that control the properties of GSCs within them. We propose models of how the different niches and GSC pools in them interact with each other.

Major conclusions

GSCs are not merely passive residents of their niches, but actively contribute to the shaping of the niches through a complex crosstalk with different components of the microenvironment. For example, GSCs play a dominant role in promoting new blood vessel formation through a variety of mechanisms, including the hypoxia dependent stimulation of angiogenesis, recruitment of endothelial progenitor cells and direct transdifferentiation into endothelial cells. Recent work has also revealed that GSCs can recruit and modulate the function of various immune cells to suppress anti-tumor immune responses and to foster tumor-promoting inflammation, which in turn could support the maintenance of GSCs.

General significance

These findings underscore the central role of the GSC microenvironment in driving glioma progression making the GSC niche a prime therapeutic target for the design of therapies aimed at eradicating GSCs.This article is part of a Special Issue entitled Biochemistry of Stem Cells.