沉默ABCE1对神经胶质瘤细胞U251增殖、迁移和凋亡的影响

ABCE1是ATP结合盒蛋白亚家族成员之一,在病毒感染,细胞增殖,抗凋亡,翻译起始和核糖体生物发生等过程中有重要的作用。为了探讨ABCE1对神经胶质瘤细胞U251增殖、迁移和凋亡的作用,本研究通过实时荧光定量PCR和免疫印迹实验检测ABCE1在神经胶质瘤细胞和正常胶质细胞中的mRNA和蛋白质表达水平,结果发现ABCE1在神经胶质瘤细胞U251中的表达高于在正常胶质细胞中的表达。利用siRNA靶向沉默ABCE1后,神经胶质瘤细胞U251中ABCE1 mRNA和蛋白的表达水平均显著减少,细胞的凋亡率显著提高,细胞增殖和迁移明显受到抑制,而且细胞对化疗药物替莫唑胺的敏感性增强。此外,沉默ABCE1后,Bcl-2的mRNA和蛋白质表达水平显著下调,而Bax的mRNA和蛋白质表达水平显著上调。以上研究结果表明,ABCE1与神经胶质瘤细胞的增殖和迁移密切相关,通过siRNA靶向沉默ABCE1基因可显著降低U251细胞的增殖和迁移能力。     

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.     

Bone morphogenetic protein (BMP) type II receptor is required for BMP-mediated growth arrest and differentiation in pulmonary artery smooth muscle cells

Bone morphogenetic protein (BMP) signals regulate the growth and differentiation of diverse lineages. The association of mutations in the BMP type II receptor (BMPRII) with idiopathic pulmonary arterial hypertension suggests an important role of this receptor in vascular remodeling. Pulmonary artery smooth muscle cells lacking BMPRII can transduce BMP signals using ActRIIa (Activin type II receptor). We investigated whether or not BMP signaling via the two receptors leads to differential effects on vascular smooth muscle cells. BMP4, but not BMP7, inhibited platelet-derived growth factor-activated proliferation in wild-type pulmonary artery smooth muscle cells, whereas neither ligand inhibited the growth of BMPRII-deficient cells. Adenoviral gene transfer of BMPRII enabled BMP4, as well as BMP7, to inhibit proliferation in BMPRII-deficient cells. BMP-mediated growth inhibition was also reconstituted by the BMPRII short isoform, lacking the C-terminal domain present in the long form. BMP4, but not BMP7, induced the expression of osteoblast markers in wild-type cells, whereas neither ligand induced these markers in BMPRII-deficient cells. Overexpression of short or long forms of BMPRII in BMPRII-deficient cells enabled BMP4 and BMP7 to induce osteogenic differentiation. Although signaling via BMPRII or ActRIIa transiently activated SMAD1/5/8, only BMPRII signaling led to persistent SMAD1/5/8 activation and sustained increases in Id1 mRNA and protein expression. Pharmacologic blockade of BMP type I receptor function within 24 h after BMP stimulation abrogated differentiation. These data suggest that sustained BMP pathway activation, such as that mediated by BMPRII, is necessary for growth and differentiation control in vascular smooth muscle.     

EphA2对神经胶质瘤细胞系U251的凋亡﹑增殖﹑迁移和侵袭的研究

为了研究EphA2对神经胶质瘤细胞系U251在增殖、凋亡、迁移和侵袭方面所起的作用,用RT-PCR方法检测正常脑组织标本与两种恶性胶质瘤细胞系中EphA2 mRNA表达水平,然后用化学合成的针对EphA2基因的小干扰RNA(siRNA)下调该基因的表达,以检测其在U251中的生物学功能．证实了EphA2基因在正常脑组织标本中的表达水平远低于两种恶性胶质瘤细胞系．把体外化学合成针对EphA2基因的小干扰RNA(siRNA- EphA2)转染入U251细胞后,Western blot, 实时定量 RT-PCR检测到U251细胞中EphA2蛋白及mRNA表达水平都明显降低,并且细胞增殖受到显著抑制,同时出现了明显的细胞凋亡．伤口愈合实验(检测细胞迁移能力),Transwell小室实验(检测细胞侵袭能力)均表明,下调EphA2的表达后,细胞的迁移和侵袭能力较阴性对照组显著减弱．上述结果表明,在神经胶质瘤U251细胞中,EphA2与其恶性增殖及高度侵染性相关,可作为分子治疗的有效靶点．     

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.     

紫草素对人脑胶质瘤U251 细胞增殖和凋亡的影响

目的:观察紫草素对体外培养的人脑胶质瘤U251细胞的增殖和凋亡的影响。方法:应用MTT法和Annexin V-FITC/PI双染流式细胞术分别检测2.5、5、10μM/L的紫草素对U251细胞的体外抑杀作用以及凋亡诱导作用,进一步应用Western blot方法检测紫草素对凋亡相关蛋白Bcl-2及Bax表达水平的影响。结果:紫草素对人U251胶质瘤细胞具有明显的增殖抑制作用,且呈一定的剂量依赖性和时间依赖性。紫草素可明显上调U251细胞Bax的表达,下调Bcl-2的表达,与对照组相比存在显著性差异(P0.05)。结论:紫草素对人胶质瘤U251细胞具有明显的抑制增殖和促进凋亡作用。     

LRRC4 inhibits glioblastoma cell proliferation, migration, and angiogenesis by downregulating pleiotropic cytokine expression and responses

Leucine-rich repeat C4 (LRRC4) has been shown to inhibit glioma cell proliferation, however, little is known about the mechanism(s) underlying the action of LRRC4. Here, we show that two glioblstoma U251 cell clones stably expressing LRRC4 were established. LRRC4 expression significantly inhibited the expression of some cytokines and their receptors determined by microarray and Western blot assays, and dramatically reduced cytokine-induced AP-1, NF-kB, and CyclinD1 activation in glioma cells. Furthermore, LRRC4 expression in glioma cells significantly downregulated spontaneous and cytokine-induced expression of K-RAS and phosphorylation of c-Raf, ERK, AKT, NF-kBp65, p70S6K, and PKC, suggesting that LRRC4 inhibited receptor tyrosine kinase (RTK) signaling pathways. Moreover, treatment with bFGF, IGF1, or IGF2 stimulated LRRC4(-/-), but not the LRRC4(+), glioma cell proliferation, indicating that LRRC4 mitigated cytokine-stimulated proliferation in glioma cells. In addition, treatment of LRRC4(-/-) glioma cells with EGF, IGF2, or PDGF promoted long distance mobilization, but induced little migration in LRRC4(+) glioma cells, suggesting that LRRC4 retarded cytokine-promoted glioma cell migration in vitro. Finally, human vessel endothelial cells (ECV304) treated with VEGF grew, aligned and formed hollow tube-like structures in vitro. In contrast, LRRC4(+) ECV304 treated with VEGF failed to form vessel-tube structures. Collectively, LRRC4 expression inhibited the expression of some growth factors, cytokines and their receptors, and the capacity of glioma cells responding to cytokine stimulation, leading to inhibition of glioma cell proliferation. Conceivably, induction of LRRC4 expression may provide new intervention for human glioma in the clinic.     

Reduced expression of proteolipid protein 2 increases ER stress-induced apoptosis and autophagy in glioblastoma

Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.     

LRRC4 inhibits human glioblastoma cells proliferation, invasion, and proMMP-2 activation by reducing SDF-1 alpha/CXCR4-mediated ERK1/2 and Akt signaling pathways

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. The alpha-chemokine stromal cell-derived factor (SDF)-1 alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation by activating multiple signal transduction pathways. Leucine-rich repeats containing 4 (LRRC4), a putative glioma suppressive gene, inhibits glioblastoma cells tumorigenesis in vivo and cell proliferation and invasion in vitro. We also previously demonstrated that LRRC4 controlled glioblastoma cells proliferation by ERK/AKT/NF-kappa B signaling pathway. In the present study, we demonstrate that CXC chemokine receptor 4 (CXCR4) is expressed in human glioblastoma U251 cell line, and that SDF-1 alpha increases the proliferation, chemotaxis, and invasion in CXCR4+ glioblastoma U251 cells through the activation of ERK1/2 and Akt. The reintroduction of LRRC4 in U251 cells inhibits the expression of CXCR4 and SDF-1 alpha/CXCR4 axis-mediated downstream intracellular pathways such as ERK1/2 and Akt leading to proliferate, chemotactic and invasive effects. Furthermore, we provide evidence for proMMP-2 activation involvement in the SDF-1 alpha/CXCR4 axis-mediated signaling pathway. LRRC4 significantly inhibits proMMP-2 activation by SDF-1 alpha/CXCR4 axis-mediated ERK1/2 and Akt signaling pathway. Collectively, these results suggest a possible important "cross-talk" between LRRC4 and SDF-1 alpha/CXCR4 axis-mediated intracellular pathways that can link signals of cell proliferation, chemotaxis and invasion in glioblastoma, and may represent a new target for development of new therapeutic strategies in glioma.     

PMA and Ionomycin Induce Glioblastoma Cell Death: Activation-Induced Cell-Death-Like Phenomena Occur in Glioma Cells

Phorbol myristate acetate (PMA) and ionomycin (Io) can induce T cell activation and proliferation. Furthermore, they stimulate activation-induced cell death (AICD) in mature lymphocytes via Fas/Fas ligand (FasL) up-regulation. In this study, we explored the influence of PMA/Io treatment on glioblastoma cells, and found that AICD-like phenomena may also occur in glioma. Using the MTT assay and cell counting, we demonstrated that treatment of PMA/Io significantly inhibited the proliferation of glioma cell lines, U87 and U251. TUNEL assays and transmission electron microscopy revealed that PMA/Io markedly induced U87 and U251 cell apoptosis. Propidium iodide staining and flow cytometry showed that treatment with PMA/Io resulted in an arrestment of cell cycle and an increase in cell death. Using real-time PCR and western blot, we found that PMA/Io up-regulated the expression of Fas and FasL at both mRNA and protein level, which confirmed that PMA/Io induced glioma cell death. Specific knockdown of NFAT1 expression by small hairpin RNA greatly reduced the PMA/Io induced cell death and apoptosis by inhibition of FasL expression. Microarray analysis showed that the expression of NFAT1 significantly correlated with the expression of Fas. The coexistence of Fas with NFAT1 in vivo provides the background for AICD-like phenomena to occur in glioma. These findings demonstrate that PMA/Io can induce glioblastoma cell death through the NFAT1-Fas/FasL pathway. Glioma-related AICD-like phenomena may provide a novel avenue for glioma treatment.     

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.     

CFTR activation suppresses glioblastoma cell proliferation,migration and invasion

The aim of this study was to investigate the function of Cystic fibrosis transmembrane conductance regulator (CFTR) in human glioblastoma (GBM) cells. Data dining results of the Human Protein Atlas showed that low CFTR expression was associated with poor prognosis for GBM patients. We found that CFTR protein expression was lower in U87 and U251 GBM cells than that in normal humane astrocyte cells. CFTR activation significantly reduced GBM cell proliferation. In addition, CFTR activation significantly abrogated migration and invasion of GBM cells. Besides, CFTR activator Forskolin treatment markedly reduced MMP-2 protein expression. These effects of CFTR activation were significantly inhibited by CFTR inhibitor CFTRinh-172 pretreatment. Our findings suggested that JAK2/STAT3 signaling was involved in the anti-glioblastoma effects of CFTR activation. Moreover, CFTR overexpression in combination with Forskolin induced a synergistic anti-proliferative response in U87?cells. Overall, our findings demonstrated that CFTR activation suppressed GBM cell proliferation, migration and invasion likely through the inhibition of JAK2/STAT3 signaling.     

Flouride Promotes Viability and Differentiation of Osteoblast-Like Saos-2 Cells Via BMP/Smads Signaling Pathway

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.     

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.     

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

ABSTRACT: BACKGROUND: The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. METHODS: We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4',6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. RESULTS: Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. CONCLUSIONS: Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas.