Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.     

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.     

骨桥蛋白RNA干扰对人U251胶质瘤细胞在裸鼠体内生长的影响

研究下调骨桥蛋白(osteopontin,OPN)对人U251胶质瘤细胞在裸鼠体内生长的影响并探讨其对胶质瘤生长、侵袭的可能机制.应用RNA干扰技术,将OPN基因的慢病毒干扰载体LV-OPNshRNA感染U251细胞.将对照和试验组U251细胞分别接种裸鼠,建立裸鼠荷瘤模型.3周后测量肿瘤的体积、瘤重并做肿瘤组织病理切片分析;利用RT-PCR和免疫印迹法检测OPN、尿激酶型纤维蛋白酶原激活物(uPA)、基质金属蛋白酶(MMP-2、MMP-9)的mRNA和蛋白表达;免疫组化法检测肿瘤组织微血管密度和血管内皮生长因子（VEGF）表达情况. 经OPN的RNA干扰后,能显著降低肿瘤组织OPN mRNA水平及蛋白表达,有效抑制肿瘤细胞生长及侵袭能力, 肿瘤体积及重量的减小有统计学意义(P<0.05).感染组uPA、MMP-2和MMP-9的mRNA和蛋白表达明显减少, 肿瘤组织的MVD值和VEGF的表达均显著降低.上述结果表明,抑制OPN的表达能明显抑制人U251胶质瘤细胞在裸鼠体内的生长和侵袭,OPN可能通过激活uPA、MMP-2和MMP-9等蛋白酶降解细胞外基质和促进肿瘤血管生成,参与胶质瘤的生长.     

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。     

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．     

Glioma-specific cation conductance regulates migration and cell cycle progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

NDRG2对胶质瘤U87-MG细胞组蛋白乙酰化的影响及机制研究

目的:探讨NDRG2对胶质瘤U87-MG细胞组蛋白乙酰化的影响,从代谢组学角度明确其抑癌机制,为胶质瘤治疗提供新思路。方法:利用慢病毒介导的外源性NDRG2基因在胶质瘤U87-MG细胞株中过表达,并采用MTT检测其对胶质瘤U87-MG细胞增殖的影响,采用Western blot技术研究其对胶质瘤U87-MG细胞组蛋白乙酰化及AKT-ACLY通路磷酸化状态的影响,并使用酶联反应检测胞内乙酰辅酶A的水平。结果:NDRG2在胶质瘤U87-MG细胞中外源过表达可降低AKT及下游分子ACLY的磷酸化水平,减少胞内乙酰辅酶A的合成,抑制组蛋白乙酰化。结论:NDRG2可能通过抑制AKT通路,减少组蛋白乙酰化,进而抑制胶质瘤U87-MG细胞增殖。     

Knockdown of SLC34A2 inhibits cell proliferation,metastasis, and elevates chemosensitivity in glioma

Solute carrier 34 A2 (SLC34A2) is a member of SLC34 family that is a group of phosphate transporters. SLC34A2 has been reported to play critical roles in tumorigenesis and progression. However, the researches about the biological roles of SLC34A2 in glioma have not yet been reported. In this study, we analyzed the expression patterns of SLC34A2 in clinical glioma tumor tissues and cell lines. The results demonstrated that SLC34A2 was generally overexpressed in both glioma tissues and cell lines. To further investigate the roles of SLC34A2 in glioma, lentivirus containing specific SLC34A2 short hairpin RNA (sh-SLC34A2) was used to infect glioma cell lines U251 and U87 for the knockdown of SLC34A2. The following studies proved that SLC34A2 knockdown exhibited suppressive effects on cell proliferation and migration/invasion. SLC34A2 knockdown also inhibited epithelial-mesenchymal transition (EMT) phenotype, as evidenced by the increased E-cadherin expression, and the decreased N-cadherin and fibronectin expressions. Besides, knockdown of SLC34A2 enhanced the temozolomide (TMZ) sensitivity of U251 and U87 cells. In vivo tumorigenicity assay demonstrated that SLC34A2 knockdown inhibited tumor growth. Moreover, SLC34A2 knockdown suppressed the activation of epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway in U87 cells. GW2974 (EGFR inhibitor) increased SLC34A2 knockdown-inhibited cell proliferation, migration/invasion, as well as enhanced SLC34A2 knockdown-increased the TMZ sensitivity of glioma cells. These findings suggested that SLC34A2 might be a new potential therapeutic target for the therapy of glioma patients.     

Expression of ABC-1 transporter is elevated in human glioma cells under irradiation and temozolomide treatment

Summary. Objective: Chemo-therapeutic treatment of glioma patients has minor success. Little is known about mechanisms of a pronounced resistance of gliomas towards actual therapies, yet. ABC-1 belongs to the group of transporters known to be involved in the export of hydrophobic substances and vascular regulation. This study investigates an effect of both temozolomide (TMZ) treatment and/or irradiation on the expression of the ABC-1 transporter in human U87-MG glioma cells.Material and methods: In parallel experiments U87-MG cells underwent either irradiation (RT), chemo-treatment (CT) using TMZ, and combined chemo/radiation-treatment (CT/RT). After each treatment the cells were incubated either 2 or 24 hours at 37°C and counted before protein analysis using Western-Blot technique.Results and conclusions: An exponential growth of cellular density was observed for both untreated and irradiated cells being, however, about 2-times slower in irradiated compared to untreated cells. In contrast the density increase of chemo-treated cells as well as that of cells, which underwent the combined CT/RT treatment was of linear nature. ABC-1 expression was detected in untreated as well as treated cells. Increasing cell density and all kinds of treatment resulted in a considerably enhanced ABC-1 expression. CT treatment resulted in highly up-regulated ABC-1 expression especially in non-confluent cultures compared to untreated cells. Irradiation had a comparable or even higher inducible effect on the ABC-1 expression rates depending, however, on cell density. The highest expression rates were observed in cultures with high cellular density 2 hours after application of the combined treatment. Strong up-regulation of ABC-1 expression under both irradiation and chemo-treatment might be a clue to multidrug and irradiation cross-resistance mechanisms of malignant glioma cells converting the ABC-1 transporter into an attractive pharmacological target for a clinical breakthrough in the therapy of malignant gliomas.     

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.     

Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP)

Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G_q-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.     

Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression

Background

Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/Principal Findings

A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/Significance

Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.     

MicroRNA-383 inhibits anchorage-independent growth and induces cell cycle arrest of glioma cells by targeting CCND1

In recent years, microRNAs (miRNAs) have been proved to be closely related to the tumorigenesis and progression. An increasing number of researches have shown that microRNAs function as oncogenes or tumor suppressor genes in human malignant tumors. This study aims to explore the effects of microRNA-383 (miR-383) on malignant biological function of human gliomas. We detected the expression of miR-383 in glioma tissues and normal brain tissues by quantitative real-time PCR. Anchorage-independent growth assays, and flow cytometry were used to evaluate the functions of miR-383 that involves in cell growth and cell cycle. Western blotting assay was used to examine protein expression levels of Cyclin D1 (CCND1), a cell cycle-associated oncogene which has a predicted binding site of miR-383 within its 3′-untranslated region (3′-UTR), and luciferase activity assay was used to evaluate the 3′-UTR activity of CCND1. In this study, we found that miR-383 expression level was lower in gliomas than normal brain tissues. Overexpression of miR-383 in U251 and U87 cells showed a significant inhibitory effect on cell growth, which accompanied with cell cycle G0/G1 arrest as well as downregulation of CCND1 expression. Moreover, CCND1 was verified to be one of the direct targets of miR-383. In summary, this study suggested that miR-383 plays the role of tumor suppressor by targeting CCND1 in glioma cells, and may be useful for developing a new therapeutic strategy for gliomas.     

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。     

Overexpression of Hsc70 promotes proliferation,migration, and invasion of human glioma cells

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.     

p27Kip1 expression by contact inhibition as a prognostic index of human glioma

The clinical manifestations of human glioma are known to be diverse, ranging from aggressive growth and invasion to apparent dormancy; however, the molecular mechanism underlying this diversity has been largely unexplored. In the present study, we characterized four human glioma cell lines, T98G, A172, U251, and NAC6, each of which has distinct growth properties. A172 and U251 cells continue to grow after confluency, whereas the growth of T98G and NAC6 cells is contact inhibited. Northern and western blot analyses revealed that at high cell density, the expression of p27Kip1 cyclin-dependent kinase inhibitor was dramatically enhanced at both the RNA and the protein levels in T98G and NAC6 cells but not in A172 or U251. These facts together with the finding that overexpression of p27Kip1 caused G1 arrest in A172 and T98G cells suggest that the induction of p27Kip1 represents an important determinant of growth at high cell density. Immunohistochemical analyses of 42 primary gliomas revealed an inverse correlation between the level of p27 protein and the Ki-67 proliferative index. Kaplan-Meier plots demonstrated that a low level of p27 in tumors is associated with decreased overall survival. Thus, disrupted regulation of p27 expression at high cell density may play an important role in determining the clinical behavior of human gliomas as well as the prognosis for glioma patients.