Decision-making abilities in patients with frontal low-grade glioma

Nucleolin is a multifunctional protein whose expression often correlates with increased cellular proliferation. While the expression of nucleolin is often elevated in numerous cancers, its expression in normal human brain and in astrocytomas has not been previously reported. Using paraffin-embedded sections from normal adult autopsy specimens and glioma resection specimens, we demonstrate that nucleolin expression is limited in the normal human brain specifically to mature neurons, ependymal cells, and granular cells of the dentate gyrus. While astrocytes in the normal human brain do not express nucleolin at significant levels, glioblastoma cell lines and primary human astrocytoma cells exhibit considerable nucleolin expression. Reduction of nucleolin expression through siRNA-mediated knockdown in the U87MG glioblastoma cell line caused a dramatic decrease in cell proliferation and induced cell cycle arrest in vitro. Moreover, conditional siRNA knockdown of nucleolin expression in U87MG intracranial xenografts in nude mice caused dramatic reduction in tumor size. Taken together, these results implicate nucleolin in the regulation of human astrocytoma proliferation in vitro and tumorigenicity in vivo and suggest that nucleolin may represent a potential novel therapeutic target for astrocytomas.     

SRC抑制胶质母细胞瘤的增殖、迁移、侵袭和干性维持的分子机制

目的 研究SRC在胶质母细胞瘤发生发展中的作用,并初步探讨可能的分子机制。方法  采用生物信息学的方法分析SRC在胶质母细胞瘤中的表达变化;利用shRNA下调胶质瘤母细胞系U87MG中SRC的表达,通过RT-PCR和免疫印迹法验证其抑制效率,并筛选出稳定干涉的细胞株;采用WST-1法、划痕愈合实验和Transwell迁移实验检测SRC shRNA干涉后细胞增殖、迁移和侵袭能力的变化;利用干细胞培养液筛选出SRC shRNA稳定干涉的胶质瘤干细胞,观察SRC shRNA对肿瘤干细胞干性的影响;利用细胞免疫荧光法观察干性基因SOX2的表达变化。结果 在胶质母细胞瘤标本中SRC的表达水平高于对照组,筛选到两条有效的SRC shRNA序列;通过shRNA下调SRC的表达后可以显著抑制胶质瘤母细胞U87MG的增殖、迁移、侵袭和肿瘤干细胞干性维持,并且可以明显抑制SOX2的表达。结论 SRC通过调控胶质母细胞瘤的增殖、迁移、侵袭和干性维持影响其发生发展,其对干性维持的作用可能是通过影响SOX2的表达实现的。     

Inhibition of Nodal suppresses angiogenesis and growth of human gliomas

Angiogenesis is the hallmark of malignant gliomas positively correlated with the vascular endothelial growth factor (VEGF) expression. We previously reported that expression levels of Nodal, a member of transforming growth factor-β super family, correlate with the malignant invasive behavior of human glioma cells. In this study, we show that knockdown of Nodal suppresses glioma angiogenesis by inhibition of VEGF. In human primary glioma specimens, expression of Nodal positively correlates with WHO glioma tumor grades and expression of VEGF in the corresponding glioma specimens. In human U87MG glioma cells, knockdown of endogenous Nodal by RNA interference (RNAi) significantly decreases colony formation and secretion of VEGF. In vivo, cellular depletion of Nodal in U87MG inhibited brain glioma growth and prolonged the survival of mice with U87MG/shNodal glioma compared with controls. Inhibition of Nodal suppressed tumor vessel growth in U87MG gliomas. Using Nodal inhibitor (SB431542), silencing Nodal, or overexpressing Nodal in the U87MG, GBM8401, and GBM glioma cells, our further experiments revealed that Nodal-induced VEGF expression might, at least in part, mediate through the ERK1/2-HIF-1α-mediated signaling pathway. Taken together, our data revealed that alteration of Nodal expression in glioma cells resulted in changes to VEGF secretion, and subsequent colony formation, in vivo tumor growth, and angiogenesis, all of which are consistent with the regulation of VEGF through the ERK1/2-HIF-1α-mediated signaling, suggesting that Nodal may serve as a potential therapeutic target for the treatment of human gliomas.     

RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.     

Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth,invasion, and epithelial-to-mesenchymal transition

Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas.     

Chemokine receptor CXCR3 promotes growth of glioma

Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3(+), which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.     

Delayed initiation of radiotherapy for glioblastoma: how important is it to push to the front (or the back) of the line?

The most effective chemotherapeutic for glioblastoma (GBM) is the DNA alkylating agent temozolomide (TMZ). In a recent study by Hegi et al. benefit from TMZ was significantly associated with methylation of the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene; however, the correlation was imperfect. Some patients with methylated tumors were short survivors and others with unmethylated tumors were long survivors. These exceptions have raised the possibility that TMZ response might be influenced by non-MGMT mechanisms. The effect of p53 status on response to TMZ was explored in traditional glioma cell lines (U87MG, U251MG, U343MG, U373MG, SF767, LN443 and LNZ308) and brain tumor initiating cells (BTICs—BT012, BT025, BT042, BT048, BT060 and BT069) in two ways: (1) inhibition of p53 by RNAi and (2) sensitivity in relation to intrinsic p53 status, either wild-type or mutant. Traditional glioma cell lines that did not express a functional p53 were significantly more sensitive to TMZ than cell lines with functionally intact wild-type p53 expression. Altered p53 expression or function had only minor effects on TMZ sensitivity in BTICs and tended to decrease sensitivity to TMZ. RNAi specific for p53 had little effect on sensitivity in p53 null glioma cells. Absence of a functional p53 increases TMZ sensitivity in traditional glioma cell lines, an effect that is independent of MGMT status, and not seen in BTICs. P53 status may influence response to TMZ in differentiated cells in a GBM with a negligible affect on its initiating cells.     

Selective osteopontin knockdown exerts anti‐tumoral activity in a human glioblastoma model

Osteopontin (OPN), a member of the SIBLING (Small Integrin‐Binding LIgand N‐linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti‐tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87‐MG glioma cells transfected with specific anti‐OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio‐allantoic membrane (CAM). OPN‐deficient U87‐MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t‐test, p < 0.05). Accordingly, the amount of proliferating cells in OPN‐deficient tumors showed a six‐fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor‐associated angiogenesis. In vitro, OPN‐silenced U87‐MG and U373‐MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion‐related protein an attractive target for glioma therapy.     

Inhibitory Effect of Benzyl Isothiocyanate on Proliferation in vitro of Human Glioma Cells

Malignant glioma, also known as brain cancer, is the most common intracranial tumor, having an extremelyhigh mortality and recurrence rate. The survival rate of the affected patients is very low and treatment is difficult.Hence, growth inhibition of glioma has become a hot topic in the study of brain cancer treatment. Among thevarious isothiocyanate compounds, it has been confirmed that benzyl isothiocyanate (BITC) can inhibit thegrowth of a variety of tumors, including leukemia, glioma and lung cancer, both inside and outside the body.This study explored inhibitory effects of BITC on human glioma U87MG cells, as well as potential mechanisms.It was found that BITC could inhibit proliferation, induce apoptosis and arrest cell cycling of U87MG cells. Inaddition, it inhibited the expression of SOD and GSH, and caused oxidative stress to tumor cells. Therefore, itis believed that BITC can inhibit the growth of U87MG cells outside the body. Its mechanism may be related tothe fact that BITC can cause oxidative stress to tumor cells.     

The antitumor effects of Angelica sinensis on malignant brain tumors in vitro and in vivo.

PURPOSE: In this study, we have examined the antitumor effects of chloroform extract of Angelica sinensis (AS-C), a traditional Chinese medicine, on glioblastoma multiforme (GBM) brain tumors in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro, GBM cells were treated with AS-C, and the cell proliferation, changes in distributions of cell cycle, and apoptosis were determined. In vivo, human DBTRG-05MG and rat RG2 GBM tumor cells were injected s.c. or i.c. and were treated with AS-C. Effects on tumor growth were determined by tumor volume, magnetic resonance imaging, survival, and histology analysis. RESULTS: The AS-C displays potency in suppressing growth of malignant brain tumor cells without cytotoxicity to fibroblasts. Growth suppression of malignant brain tumor cells by AS-C results from cell cycle arrest and apoptosis. AS-C can up-regulate expression of cdk inhibitors, including p21, to decrease phosphorylation of Rb proteins resulting in cell arrest at the G0-G1 phase for DBTRG-05MG and RG2 cells. The apoptosis-associated proteins are dramatically increased and activated in DBTRG-05MG cells and RG2 cells by AS-C but RG2 cells without p53 protein expression. In vitro results showed AS-C triggered both p53-dependent and p53-independent pathways for apoptosis. In in vivo studies, AS-C not only can suppress growths of malignant brain tumors of rat and human origin but also shrink the volumes of in situ GBM, significantly prolonging survivals. CONCLUSIONS: The in vitro and in vivo anticancer effects of AS-C indicate that it has sufficient potential to warrant further investigation and development as a new anti-brain tumor agent.     

ZMYND11在多形型胶质母细胞瘤中的表达及意义

目的 探讨ZMYND11在多形性胶质母细胞瘤（GBM）中的表达及意义。方法 收集河北医科大学第二医院GBM患者术中肿瘤标本20例（肿瘤组）,重度脑外伤患者正常脑组织标本20例（对照组）,对上述标本进行Western blot及qRT-PCR实验,检测并比较两组ZMYND11的表达;利用ZMYND11过表达的慢病毒转染GBM的细胞系U87细胞使其ZMYND11过表达,通过CCK、Transwell及流式细胞分析检测ZMYND11对U87细胞在增殖、侵袭及凋亡方面的作用;将ZMYND11过表达的U87细胞接种至裸鼠内进行体内试验。结果 肿瘤组中ZMYND11的表达量明显低于对照组（P<0.001）; ZMYND11过表达可明显抑制U87细胞的增殖及侵袭并促进其凋亡,体内实验显示ZMYND11可明显抑制肿瘤的生长。结论 ZMYND11可抑制GBM的发生与发展。     

Fas-induced expression of chemokines in human glioma cells: involvement of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase

Fas transduces not only apoptotic signals through various pathways but also angiogenic and proinflammatory responses in vivo. Human glioma cells express Fas although sensitivity to Fas-mediated cell death is variable, suggesting that Fas may have functions other than apoptosis in these cells. In this study, we addressed alternative functions of Fas expressed on human gliomas by Fas ligation in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG, and the in vivo expression of Fas and chemokines in human glioblastoma multiforme (GBM). Herein, we demonstrate that: (a) stimulation with agonistic anti-Fas monoclonal antibody CH-11 and human recombinant soluble Fas ligand induces expression of the CC chemokine MCP-1 and the CXC chemokine interleukin-8 by human glioma cell lines at the mRNA and protein levels in a dose- and time-dependent manner; (b) selective pharmacological inhibitors of MEK1 (U0126 and PD98059) and p38 mitogen-activated protein kinase (MAPK) (SB202190) suppress Fas-mediated chemokine expression in a dose-dependent manner; (c) Fas ligation on human glioma cells leads to activation of both extracellular signal-regulated kinases ERK1/ERK2 and p38 MAPK; and (d) GBM samples express higher levels of Fas compared with normal control brain, which correlates with increased interleukin 8 expression. These findings indicate that Fas ligation on human glioma cells leads to the selective induction of chemokine expression, which involves the ERK1/ERK2 and p38 MAPK signaling pathways. Therefore, the Fas-Fas ligand system in human brain tumors may be involved not only in apoptotic processes but also in the provocation of angiogenic and proinflammatory responses.     

敲减lncRNA RP1-261G23.7抑制胶质瘤细胞增殖和迁移及其机制探讨北大核心

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)RP1-261G23.7对人胶质瘤细胞增殖及迁移的影响及可能的调控机制。方法:采用GEPIA数据库分析RP1-261G23.7在胶质瘤组织中的相对表达。采用qRT-PCR检测RP1-261G23.7在4种胶质瘤细胞系(U87MG、SNB-19、U251、LN382)中的相对表达。采用Lipofectamine3000向胶质瘤细胞U87MG中单独转染si-RP1-261G23.7质粒(si-RP1-261G23.7组)和si-NC对照质粒(si-NC组)。采用CCK-8实验和细胞划痕实验检测转染后U87MG细胞的增殖及迁移能力。采用生物信息学、qRT-PCR和双荧光素酶报告基因实验研究RP1-261G23.7和miR-525-5p表达的关系。Western blotting检测NF-κB信号通路蛋白的表达。结果:与正常组织相比,RP1-261G23.7在胶质瘤组织中表达上调(P<0.01)。与人脑星型胶质正常细胞系(HEB)相比,RP1-261G23.7在四种胶质瘤细胞系中表达均上调(P<0.01),U87MG细胞中RP1-261G23.7相对表达量最高(P<0.01)。与si-NC组相比,敲减RP1-261G23.7显著抑制了U87MG细胞的增殖(P<0.05)和划痕愈合(P<0.01)。RP1-261G23.7能够直接互补结合miR-525-5p(P<0.01)。与si-NC组相比,敲减RP1-261G23.7表达显著促进了U87MG细胞中miR-525-5p的表达(P<0.01),NF-κB信号通路蛋白表达显著下降(P<0.01)。结论:胶质瘤组织和细胞系中RP1-261G23.7表达明显上调,敲减RP1-261G23.7通过促进miR-525-5p表达、干扰NF-κB信号通路活化,抑制胶质瘤U87MG细胞的增殖和迁移,可能为胶质瘤的靶向治疗开辟新的路径。     

Glioblastoma-secreted factors induce IGFBP7 and angiogenesis by modulating Smad-2-dependent TGF-beta signaling

Insulin-like growth factor-binding protein 7 (IGFBP7) is a selective biomarker of glioblastoma (GBM) vessels, strongly expressed in tumor endothelial cells and vascular basement membrane. IGFBP7 gene regulation and its potential role in tumor angiogenesis remain unclear. Mechanisms of IGFBP7 induction and its angiogenic capacity were examined in human brain endothelial cells (HBECs) exposed to tumor-like conditions. HBEC treated with GBM cell (U87MG)-conditioned media (-CM) exhibited fourfold upregulation of IGFBP7 mRNA and protein compared to control cells. IGFBP7 gene regulation in HBEC was methylation independent. U87MG-CM analysed by enzyme-linked immunosorbent assay contained approximately 5 pM transforming growth factor (TGF)-beta1, a concentration sufficient to stimulate IGFBP7 in HBEC to similar levels as U87MG-CM. Both pan-TGF-beta-neutralizing antibody (1D11) and the TGF-beta1 receptor (activin receptor-like kinase 5, ALK5) antagonist, SB431542, blocked U87MG-CM-induced IGFBP7 expression in HBEC, indicating that TGF-beta1 is an important tumor-secreted effector capable of IGFBP7 induction in endothelial cells. HBEC exposed to either U87MG-CM or IGFBP7 protein exhibited increased capillary-like tube (CLT) formation in Matrigel. Both TGF-beta1- and U87MG-CM-induced Smad-2 phosphorylation and U87MG-CM-induced CLT formation in HBEC were inhibited by the ALK5 antagonist, SB431542. These data suggest that proangiogenic IGFBP7 may be induced in brain endothelial cells by TGF-betas secreted by GBM, most likely through TGF-beta1/ALK5/Smad-2 pathway.     

Glutamate Promotes Cell Growth by EGFR Signaling on U-87MG Human Glioblastoma Cell Line

Accumulating evidences suggest that glutamate plays a key role in the proliferation and invasion of malignant glioblastoma (GBM) tumors. It has been shown that GBM cells release and exploit glutamate for proliferation and invasion through AMPA glutamate receptors. Additionally, amplification of the epidermal growth factor receptor (EGFR) gene occurs in 40–50% of GBM. Since, PI3K/Akt is considered one of the main intracellular pathways involved in EGFR activation, AKT functions could trigger EGFR signaling. Thus, we investigated whether EGFR-phospho-Akt pathway is involved on the glutamate inducing U-87MG human GBM cell line proliferation. For these purpose, we treated the U-87MG cell line with 5 to 200 mM of glutamate and assessed the number of viable cells by trypan blue dye exclusion test. An increase in cell number (50%) was found at 5 mM glutamate, while the addition of DNQX (500 μM), an antagonist of AMPA receptor, inhibited the effect of glutamate on the U87-MG cells proliferation. Also, at 5 mM glutamate we observed an increase on the EGFR and phospho-Akt contents evaluated by immunohistochemistry. Moreover, U-87MG cells treated with glutamate exhibited an increase about 2 times in the EGFR mRNA expression. While, in the presence of the anti-EGFR gefitinib (50 μM) or the PI3K inhibitor wortmannin (5 μM), the U-87MG proliferation was restored to control levels. Together, our data suggest that glutamate signaling mediated by AMPA receptor induces U-87MG human GBM cell line proliferation via EGFR-phospho-Akt pathway.     

AKIP1 promotes glioblastoma viability,mobility and chemoradiation resistance via regulating CXCL1 and CXCL8 mediated NF-κB and AKT pathways

This study aimed to investigate the interaction of A-kinase-interacting protein 1 (AKIP1) with C-X-C motif chemokine ligand (CXCL)1, CXCL2, CXCL8, and their effects on regulating glioblastoma multiforme (GBM) malignant behaviors. AKIP1 expression was modified by pcDNA and pGPH1 vectors in U-87 MG and U-251 MG cells. Subsequently, multiple compensative experiments were conducted via adding CXCL1, CXCL2 and CXCL8 in the pGPH1-AKIP1 (AKIP1 knockdown) transfected U-87 MG and U-251 MG cells, respectively. Furthermore, AKIP1, CXCL1/2/8 expressions in 10 GBM and 10 low-grade glioma (LGG) tumor samples were detected. AKIP1 was elevated in various GBM cell lines compared to normal human astrocytes. AKIP1 overexpression promoted U-87 MG and U-251 MG cell proliferation and invasion while inhibited apoptosis; and it enhanced chemoresistance to temozolomide (but not cisplatin) and radiation resistance; then AKIP1 knockdown showed the opposite effects. Meanwhile, AKIP1 positively regulated CXCL1/2/8, NF-κB pathway, AKT pathway and PD-L1 expression. Further multiple compensative experiments uncovered that CXCL1 and CXCL8 promoted proliferation, invasion, chemoradiation resistance, NF-κB pathway, AKT pathway and PD-L1 expression in U-87 MG and U-251 MG cells, also in pGPH1-AKIP1 (AKIP1 knockdown) transfected U-87 MG and U-251 MG cells; although CXCL2 exhibited similar treads, but its effect was much weaker. Besides, NF-κB pathway inhibitor and AKT pathway inhibitor attenuated the effect of CXCL1&CXCL8 on promoting GBM cell malignant behaviors. Clinically AKIP1 and CXCL1/8 were elevated in GBM compared to LGG tumor samples, and they were inter-correlated. AKIP1 promotes GBM viability, mobility and chemoradiation resistance via regulating CXCL1 and CXCL8 mediated NF-κB and AKT pathways.