Neuregulin-1 enhances survival of human astrocytic glioma cells

Malignant astrocytic gliomas, referred to as astrocytomas, represent the most commonly diagnosed adult primary brain tumor. These tumors are characterized by unrelenting growth that is often resistant to chemotherapy and radiation therapy. Tumor expansion into the healthy surrounding brain tissue produces severe and often fatal consequences. In this study, we examine the potential for the neuregulin-1/erbB receptor signaling cascade to contribute to this process by modulating glioma cell growth. Using antibodies specific for the erbB receptors, we demonstrate the expression patterns for the erbB2, erbB3, and erbB4 receptors in human glioma biopsy samples. We then verify receptor expression in a panel of human glioma cell lines. Next, we investigate the status of the erbB2 and erbB3 receptors in the human glioma cell lines and find that they are constitutively tyrosine-phosphorylated and heterodimerized. Subsequently, we demonstrate that theses same cell lines express membrane bound and released forms of neuregulins, the erbB receptor ligands, suggesting a possible autocrine or paracrine signaling network. Furthermore, we show that exogenous activation of erbB2 and erbB3 receptors in U251 glioma cells by recombinant Nrg-1beta results in enhanced glioma cell growth under conditions of serum-deprivation. This enhancement is due to an increase in cell survival rather than an increase in cell proliferation and is dependent on the activation of erbB2 and phosphatidylinositol-3 kinase (PI3K). Moreover, Nrg-1beta activates an inhibitor of apoptosis, Akt, implying a possible role for this kinase in mediating Nrg-1beta effects in gliomas. This data suggests that glioma cells may use autocrine or paracrine neuregulin-1/erbB receptor signaling to enhance cell survival under conditions where growth would otherwise be limited.     

Evidence for and against a pivotal role of PI 3-kinase in a neuronal cell survival pathway.

PI 3-kinase has emerged as a key enzyme for regulating neuronal cell survival. However, it has not as yet been demonstrated whether activation of the endogenous pool of the enzyme, that is regulated by the p85 subunit, is sufficient to promote a survival response. It is also not known whether the FGF family of growth factors promote survival via a PI 3-kinase-dependent pathway. We have previously developed a cell permeable p85 binding peptide and shown that it can stimulate a mitogenic response in muscle cells that is dependent on a PI 3-kinase/p70 S6 kinase pathway. In the present study we show that this peptide can rescue cerebellar granule cells from death induced by serum deprivation and that this response is comparable to a growth factor response (FGF2). Experiments with wortmannin, LY294002, and rapamycin suggest that the peptide survival response is dependent on PI 3-kinase activity, but not p70 S6 kinase activity. The peptide response was correlated with a PI 3-kinase-dependent phosphorylation of Akt, an established downstream effector in the PI 3-kinase survival cascade. In contrast to the survival response stimulated by the p85 binding peptide, the response stimulated by FGF2 was not inhibited by wortmannin or LY294002, nor was it associated with phosphorylation of Akt. Thus we can conclude that activation of the endogenous pool of PI 3-kinase that is regulated by p85 is sufficient for cell survival; however, growth factors such as FGF2 can clearly support survival in a PI 3-kinase-independent manner.     

Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells

Phosphoinositol 3-kinase (PI3K) is a downstream effector for multiple ligand-activated receptors and modulates cell responses through activation of its target protein kinase B (Akt). We examined the roles of PI3K-Akt signaling in a primary glial (oligodendrocyte) progenitor cell culture system that is ligand-dependent for cell proliferation, survival, and prevention of differentiation. We demonstrate that PI3K and Akt (Ser-473 phosphorylation) are activated in response to platelet-derived growth factor but not basic fibroblast growth factor-2 (FGF2) and that distinct forms of PI3K are activated in early progenitors and later-maturation pro-oligodendroblasts as identified by their sensitivity to wortmannin. By establishing conditions to examine effects on cell proliferation and survival independently, we demonstrate that PI3K is necessary for a full mitogenic response and that PI3K is also necessary for early progenitor survival. Our results therefore demonstrate that PI3K-Akt signaling independently regulates proliferation and survival, that the form of PI3K is distinct in early progenitors and pro-oligodendroblasts, and that FGF2 does not activate this pathway in either primary glial cell population.     

肉桂醛对神经胶质瘤U251细胞生物学行为的影响

目的 探讨肉桂醛对胶质瘤细胞生物学行为的影响及其分子机制。方法 体外培养胶质瘤细胞系U251细胞,分为对照组、低剂量肉桂醛组（40μmol/L）和高剂量肉桂醛组（80μmol/L）。采用平板克隆和CCK-8试验检测细胞增殖水平;流式细胞术分析细胞凋亡率;Transwell小室实验和细胞划痕试验评估细胞迁移和侵袭水平;免疫印迹法分析凋亡相关蛋白、PI3K/Akt和Wnt/β-catenin通路相关蛋白表达。结果 肉桂醛明显抑制胶质瘤U251细胞增殖、侵袭、迁移能力（P<0.05）,促进细胞凋亡（P<0.05）;抑制Bcl-2、p-PI3K、p-Akt、cycling D、C-Myc、β-catenin表达（P<0.05）,促进Cleaved-Caspase-3、Bax表达（P<0.05）;而且,随剂量增大,肉桂醛的作用明显增强（P<0.05）。结论 肉桂醛抑制胶质瘤细胞增殖、侵袭、迁移,促进细胞凋亡,其机制可能与下调PI3K/Akt和Wnt/β-catenin信号通路有关。     

ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells

The neuroprotective effects of neuregulin (NRG), a polypeptide growth factor, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death and oxidative stress in PC12-ErbB4 cells were investigated. Treatment of PC12-ErbB4 cells with MPP+induced cell death that was markedly attenuated by NRG. The PI3K/PKB/Akt and Ras/MapK signaling pathways probably mediate the survival effect of NRG. NRG induces prolonged activation of PKB/Akt and Erk. Moreover, inhibition of the PI3K and MEK activities prevented the NRG-induced survival effect. Over-expression of constitutively active PI3K or H-Ras (12V) inhibited MPP+-mediated cell death. In addition, MPP+-mediated reactive oxygen species (ROS) elevation was also inhibited by NRG. The effect of NRG on ROS levels was blocked by PI3K and MEK inhibitors, indicating that both signaling pathways can regulate the toxic ROS levels induced by MPP+. Taken together, these results indicate that in PC12-ErbB4 cells, the NRG-induced neuroprotective effect from MPP+treatment, requires PI3K/PKB/Akt and Ras/MapK signaling networks. These authors contributed equally to this work.     

隔蛋白7对胶质瘤细胞系U251细胞周期的影响

目的 检测SEVT7对胶质瘤细胞系U251的细胞周期、增殖、侵袭以及凋亡的影响.方法 流式细胞术分析转染SEPT7对细胞周期的影响,Western blot检测细胞周期因子表达变化,MTT法和Annexin V法评价SEFT7对U251细胞的增殖活性和凋亡的影响,Martrigel三维立体培养分析转染SEPT7后U251细胞侵袭能力的变化.结果 SEPT7使U251细胞G0/G1期阻滞,S期细胞比例(SPF)降低,增殖活性和侵袭能力明显受到抑制,并可诱发细胞凋亡.结论 SEPT7抑制U251细胞侵袭、增殖,促进凋亡.结果 提示,SEPT7是对胶质瘤具有抑制作用的基因.     

Phosphatidylinositol 3-kinase and Akt protein kinase mediate IGF-I- and prosaptide-induced survival in Schwann cells.

Withdrawal of trophic factors necessary for Schwann cell survival regulates Schwann cell number during development and after nerve injury. In the present study, we identified signaling pathways involved in Schwann cell survival by prosaposin, prosaptides (peptides incorporating the neurotrophic sequence of prosaposin), and insulinlike growth factor-I (IGF-I). When postnatal Schwann cells were placed in low serum medium, cells underwent abrupt shrinkage, condensation of nuclei occurred, and smooth rounded apoptotic bodies appeared. Dose-response studies of cell death, measured by lactate dehydrogenase (LDH) release, demonstrated that both prosaptide TX14(A) and IGF-I dose dependently reduced cell death in primary Schwann cells. Histone-associated DNA fragmentation enzyme-linked immunosorbent assay, showed a 10- and 14-fold increase in apoptosis after 4 and 24 hr in low serum medium, respectively, that was reduced by prosaposin, TX14(A), or IGF-I. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, blocked the survival effects of both TX14(A) and IGF-I. In contrast, only TX14(A) anti-apoptotic activity was blocked by the MEK inhbitor, PD98059, although TX14(A) and IGF-I are potent activators of extracellular regulated kinases in Schwann cells. Phosphorylation of the PI3K signaling target, Akt, was measured; TX14(A) and IGF-I increased Akt activity by 12-fold and 22-fold, respectively, that was inhibited by LY294002. These findings indicate that prosaposin and IGF-I use the PI3K/Akt pathway to induce survival of Schwann cells.     

PIAS3过表达抑制人脑胶质瘤U251细胞生长

目的 探讨PIAS3 过表达对人脑胶质瘤U251 细胞生长的作用及其可能的机制.方法 通过实时荧光定量PCR 检测PIAS3 在正常脑细胞和脑胶质瘤U251 细胞中的表达情况.转染PIAS3 过表达载体,提高U251 细胞中PIAS3 表达水平,通过噻唑蓝(MTT)试验检测细胞增殖情况.Western Blot 验证PIAS3 表达与增殖相关基因PI3K 及Akt 间关系.结果 PIAS3 在正常脑组织中相对高表达,而在人脑胶质瘤U251 细胞中表达明显下降(P ＜0.01).体外转染PIAS3 过表达载体能明显抑制U251 细胞生长,并且有效抑制PI3K 活性及p-Akt 表达,但对总的Akt 无明显影响.结论 胶质瘤U251 细胞中异常低表达的PIAS3 发挥着重要的增殖促进作用,过表达PIAS3 可通过抑制PI3K/Akt 通路有效抑制U251 细胞增殖.     

Insulin-like growth factor-I decreased etoposide-induced apoptosis in glioma cells by increasing bcl-2 expression and decreasing CPP32 activity

AIMS: In a variety of tumors, the susceptibility of the tumor cells to apoptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Gliomas are resistant to most chemotherapeutic agents, and its mechanism is not known in detail. In an attempt to understand the mechanism of chemo-resistance, we investigated the roles of insulin-like growth factor-I (IGF-I), IGF-I receptors (IGF-IR), and their relationship with the apoptotic response of two glioma cell lines to etoposide, a chemotherapeutic agent for malignant gliomas. METHODS: Two human glioma cell lines, U-87MG and KNS-42, were used. Etoposide-induced cell growth inhibition was quantified using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), colorimetric assay. Hoechst 33258 staining, DNA fragmentation assay, and western blot were used for the evaluation of apoptosis. ApoAlert caspase assay was used for measuring the activity of caspase-3 (CPP32) and interleukin-1 beta -converting enzyme (ICE) protease. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines. RESULTS: Etoposide inhibited the growth of U-87MG and KNS-42 cells in a concentration-dependent manner. Etoposide increased the expression of wild-type p53, activated CPP32 (but not ICE) activity, and induced apoptosis in these cells. IGF-I prevented etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32. IGF-IR antisense enhanced the apoptotic effect of etoposide. CONCLUSIONS: IGF-I decreased etoposide-induced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The antisense of IGF-IR increased etoposide-induced apoptosis. The anti-apoptotic effect of IGF-I and IGF-IR might be related to the chemo-resistance of glioma to chemotherapeutic agents.     

Activation of NMDA receptor of glutamate influences MMP-2 activity and proliferation of glioma cells

Glioblastoma multiforme (GBM) is the most common malignant glioma, which has high proliferative rate and an extremely invasive phenotype. Major limitations in the effective treatment of malignant gliomas are the proliferation and infiltration into the surrounding brain tissue. Although studies have shown that various stimuli promote glioma cell proliferation and invasion, the underlying mechanisms remain largely unknown. Glioma cells secrete significant amount of glutamate into surrounding tissue and intracellular signaling is thought to be initiated upon glutamate-induced modulation of the ion channels in GBM cells. The objective of the study was to investigate the effect of activation of NMDA (N-methyl-d-aspartate) receptors of glutamate on gelatinase subfamily MMPs and on proliferation of glioma cells. U251MG and U87MG cell lines were maintained in Dulbecco’s Modified Eagle’s Medium. Proliferation assay was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was investigated by gelatin zymography assay. We demonstrate that activated NMDA receptors (NMDAR) increased the activity of MMP-2 only in U251MG glioma cells at concentrations of 100 and 200 μM and increased the proliferation of both U87MG and U251MG glioma cells at concentrations of 50, 100, 150 and 200 μM. Inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly inhibited the effect of activation of NMDAR on MMP-2 activity and on proliferation. We conclude that NMDA receptor activation has role in activity of MMP-2 and proliferation of glioma cells.     

Gas1 inhibits cell proliferation and induces apoptosis of human primary gliomas in the absence of Shh

Growth arrest specific1 (Gas1) is a protein expressed during development and when cells arrest their growth. The potential of Gas1 as an adjuvant in the treatment of cancer, and its role as a tumor suppressor have also been proposed. In this work we are addressing the molecular mechanisms by which Gas1 induces cell arrest and apoptosis of cancer cells, using primary cultures of human gliomas as a model. We had previously demonstrated the structural relationship between Gas1 and the α receptors for the Glial-cell line-Derived Neurotrophic Factor (GDNF) family of ligands, and showed that Gas1 acts by inhibiting the intracellular signaling induced by GDNF. There are also reports indicating that Gas1 positively cooperates with Sonic Hedgehog (Shh) during embryonic development and in this paper we analyzed the potential interactions between Gas1 and Shh. We show that human gliomas do not express Shh, whereas GDNF and the molecular components necessary to transduce its signaling are present in human gliomas. Furthermore, the over-expression of Gas1 induces cell arrest, apoptosis and prevents the activation of Akt, a crucial mediator of survival and cellular proliferation pathways. In the present work, we present evidence demonstrating that Gas1 exerts its effects inhibiting cell growth and inducing apoptosis of glioma cells in the absence of Shh.     

反义hTERT抑制恶性胶质瘤生长的体内外研究

目的 探讨腺病毒介导的反义hTERT在体内外对恶性胶质瘤细胞生长的影响.方法 构建含有hTERT反义序列的腺病毒载体在体内外转染恶性胶质瘤细胞系U251,检测肿瘤细胞生长情况、细胞周期变化、端粒酶活性、hTERT蛋白表达等.结果 腺病毒介导的hTERT反义治疗在体外明显抑制肫瘤细胞生长,增殖减慢,凋亡增多,细胞较多聚集在G1/G0期,转染后第6天细胞生存率为46.9%,端粒酶活性和hTERT蛋白表达都明显下降,在体内实验中肿瘤生长明显减慢.结论 腺病毒介导的反义hTERT在体内外能明显抑制恶性胶质瘤细胞的生长.     

STIP1对胶质瘤U251细胞增殖、侵袭和凋亡的影响

目的 探讨下调磷酸化应激诱导蛋白1（STIP1）表达对胶质瘤U251细胞增殖、侵袭和凋亡的影响,及其对JAK2/STAT3信号通路的调控作用。方法 免疫印迹法检测体外培养的正常胶质细胞（SVG）和人胶质瘤细胞（U251、U87和U37）STIP1蛋白表达水平。NC-siRNA或STIP1-siRNA质粒转染U251细胞,CCK-8法检测U251细胞增殖;Transwell实验检测U251细胞侵袭能力;流式细胞术检测U251细胞凋亡率;免疫印迹法检测JAK2/STAT3信号通路蛋白表达水平。结果 与正常胶质细胞SVG比较,胶质瘤细胞U251、U87和U373的STIP1蛋白表达水平均明显增高（P<0.05）。与NC-siRNA组比较,STIP1-siRNA组STIP1蛋白表达水平、细胞增殖活力和细胞侵袭力明显明显降低（P<0.05）,细胞凋亡率明显增高（P<0.05）,而且,p-JAK2和p-STAT3蛋白表达水平明显降低（P<0.05）。结论 STIP1在胶质瘤细胞中呈高表达,抑制STIP1表达可以抑制胶质瘤细胞的增殖和侵袭、促进凋亡,机制可能与抑制JAK2/STAT3信号通路有关。     

RNA干扰敲低Wnt2的表达抑制U251细胞生长的体内外研究

目的 在体内外研究转染靶向Wnt2的siRNA对U251细胞的抑制效果.方法 向U251细胞转染靶向Wnt2的siRNA后,免疫印记检测转染后Wnt2及相关蛋白表达,MTT检测细胞增殖,annexin标记细胞凋亡,流式细胞仪检测细胞周期分布,鼠尾胶3D生长实验检测细胞侵袭能力的变化.建立裸鼠胶质瘤皮下动物模型,瘤内注射Wnt2 siRNA观察肿瘤生长情况.结果 转染Wnt2siRNA后,U251细胞的Wnt2、frizzled2、磷酸化GSK-3β、β-catenin等表达降低,细胞增殖受抑,凋亡率升高,细胞阻滞于G0/G1期.体内研究发现转染靶向Wnt2的siRNA后,裸鼠皮下肿瘤的生长速度缓慢,相应的蛋白表达降低.结论 转染靶向Wnt2的siRNA可有效敲低该基因在U251细胞内的表达,从而抑制了胶质瘤细胞生长,具有潜在的治疗前景.     

Targeting malignant glioma survival signalling to improve clinical outcomes.

Malignant gliomas are common and aggressive brain tumours in adults. Current treatments for glioblastoma multiforme result in a poor median survival of less than 12 months. The blood-brain barrier restricts the delivery of many chemotherapies to the central nervous system, contributing to the failure of treatment. PI3K/Akt and Ras/MAPK pathways have been identified as important oncogenic pathways in these tumours. The PI3K/Akt pathway mediates cell survival and growth, whereas the Ras/MAPK pathway signals cell differentiation, proliferation and anti-apoptosis. Modern targeted therapies include antibodies to circulating growth factors and cell surface receptors, as well as inhibitors of receptor tyrosine kinases and specific intracellular signalling proteins. Monotherapy with most targeted therapies produces only modest efficacy. Better results are achieved in combination with cytotoxic chemotherapies. Future therapeutics should focus on combination therapy with small lipophilic molecules.     

RNAi下调PIK3 CB表达抑制U251胶质瘤细胞生长的体内外研究

目的 探讨应用RNAi技术靶向磷酸肌醇酯-3-激酶β催化亚单位(PIK3CB)抑制恶性胶质瘤细胞系U251的PIK3CB表达后在体内外对U251细胞生长抑制作用.方法 将短发夹RNA(shRNA)表达载体psiRNA-PIK3CB进行脂质体介导的U251人脑恶性胶质瘤细胞系表达,检测细胞转染前后的细胞增殖能力和凋亡的变化.应用裸鼠皮下荷瘤模型观察脂质体介导shRNA基因治疗对U251细胞生长抑制作用,对肿瘤组织应用免疫荧光双染色和免疫组化的方法分析结果.结果 靶向PIK3CB的shRNA转染后U251细胞生长受到抑制,细胞周期出现G2/M阻滞,细胞明显凋亡.裸鼠皮下荷瘤模型实验显示psiRNA-PIK3CB显著抑制皮下肿瘤生长(P＜0.01).结论 靶向PIK3CB的shRNA基因治疗可以成为胶质瘤治疗的新策略.     

ITGA4基因在脑胶质瘤中的表达及临床意义

目的 探讨整合素α4(ITGA4)基因在脑胶质瘤中的表达与临床意义。方法 计算机检索CGGA数据库获取mRNAseq-325数据集中325例脑胶质瘤的转录组测序（RNA-seq）数据及临床资料,并用m RNAseq-693数据集中693例脑胶质瘤进行验证。结果 胶质瘤ITGA4基因呈高表达（P<0.05）,与胶质瘤IDH基因型、1p/19q共缺失状态、WHO病理分级有关（P<0.05）,与胶质瘤MGMT启动子甲基化状态无明显关系（P>0.05）。多因素Cox回归分析显示,ITGA4高表达是胶质瘤生存预后不良的独立危险因素（P<0.05）。生存曲线分析显示,ITGA4高表达胶质瘤病人生存期明显缩短（P<0.05）。GO分析和KEGG分析显示,ITGA4基因调控的生物过程包括细胞粘附、血管生成及细胞迁移等,主要调控PI3K/AKT信号通路。结论 我们的结果提示胶质瘤ITGA4呈高表达,与胶质瘤不良生存预后密切相关,主要调控PI3K/AKT信号通路。