Survivin-specific small interfering RNAs enhance sensitivity of glioma U-87MG cells to paclitaxel by promoting apoptosis

A survivin siRNA expression vector was transfected into glioma U-87MG cells and these cells were then treated with paclitaxel.The results showed that survivin-specific siRNA combined with paclitaxel treatment synergistically inhibited glioma U-87MG cell proliferation and promoted apoptosis.This treatment also inhibited the expression of the cell cycle regulatory proteins,survivin,cyclinD1,c-Myc and CDK4 and enhanced the sensitivity of U-87MG cells to paclitaxel.     

表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

Monoclonal antibody against human glioblastoma multiforme (U-87MG) immunoprecipitates a protein of molecular mass 38 kDa and inhibits tumor growth in nude mice

A monoclonal antibody 6DS₁ against a human glioblastoma multiforme cell line U-87MG recognizes a tumor-specific, cell surface antigen of human glioblastoma cell lines. Partial cross-reactivity is observed with two human neuroblastoma cell lines, SK-N-SH and SK-N-MC, with little or no reactivity towards a rat glioma cell line C6 or normal human adult and fetal brain tissues. The antibody recognizes an antigen of molecular mass 38 kDa as inferred from Western blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate. The monoclonal antibody 6DS₁ inhibits both the attachment to substratum and growth of U-87MG cells. It strongly cross-reacts with xenotransplants of U-87MG cells and inhibits tumorigenesis (subcutaneous implants of U-87MG cells) in nude mice.     

多聚凝胶介导IGF-IR反义寡核苷酸治疗体内胶质瘤的实验研究

目的探讨反义寡核苷酸阻断IGF-IR基因表达对裸鼠移植胶质瘤的抑制作用。方法首先建立胶质瘤裸鼠移植模型,随机分为4组:空白对照组、多聚凝胶组、正义核酸组和反义核酸组,反义核酸组在瘤内注射多聚凝胶包裹的反义寡核苷酸,其他每组均作相应的瘤内注射处理。利用多聚凝胶缓释反义寡核苷酸的作用,观察肿瘤的抑制情况、肿瘤病理和IGF-IR表达结果。结果反义核酸组用反义核苷酸开始治疗后的第1周、第2周和第3周,肿瘤的生长速度明显减慢,与空白对照组相比,抑制率分别为65.2%、76.38%和69.6%;而多聚凝胶组和正义核酸组肿瘤生长速度与空白对照组相比无明显差异,其抑制率在各时间点均低于10%。肿瘤的组织病理发现多聚凝胶组、正义核酸组如同空白对照组(野生型),肿瘤细胞密集分布,呈明显的恶性增殖表现,而反义核酸组肿瘤细胞稀疏,可见部分细胞呈凋亡改变,细胞染色质浓聚边缘化。在空白对照组(野生型)、多聚凝胶组和正义核酸组IGF-IR蛋白均呈高表达,而反义核酸组IGF-IR的表达明显减弱。结论IGF-IR的反义寡核苷酸对体内胶质瘤的生长具有明显的抑制作用,并能诱导部分肿瘤细胞凋亡。因此,IGF-IR可作为胶质瘤基因治疗的靶。     

Differentiation inducing effects of vesnarinone on human glioma cells

The effects of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone) on the growth of glioma cells were examined in vitro. Vesnarinone at a dose of 100 mug/ml suppressed the growth of four different glioma cell lines, U-251MG, U-373MG, U-87MG and A-172, by approximately 50%, with an elongation of the cytoplasmic process on day 5 of culture. The long-term culture of U-87MG with 10 mug/ml of vesnarinone was continued up to day 34. Although growth suppression was approximately 25% on day 5, it reached over 95% on day 34. An increase in the cyclic adenosine monophosphate content of glioma cells cultured with vesnarinone was observed by enzyme-linked immunosorbent assay (ELISA). The accumulation of glial fibrillary acidic protein was observed to occur with vesnarinone by ELISA. These findings suggest that vesnarinone suppressed the growth and induced differentiation of glioma cells in vitro.     

miR-27b影响β-catenin／Tcf-4通路调控U87胶质瘤细胞生物学行为

目的研究miR-27b在胶质瘤细胞系U87中的表达、功能及作用机制。方法在多个胶质瘤细胞系及胶质瘤标本中检测miR-27b的表达;利用反义miR-27b转染U87,下调rniR-27b的表达,应用流式细胞术和MTT法,评价细胞生长、增殖、凋亡及周期变化：采用荧光素酶法及Western印迹法检测miR-27b作用的初步机制及通路蛋白的变化。结果miR-27b在胶质瘤细胞系及胶质瘤标本中均呈高表达;转染反义miR-27b后,表达降低86％,MTT法显示转染反义miR-27b后细胞生长受到抑制且凋亡细胞增加13％;荧光素酶实验结果显示：miR-27b发挥作用可能与B—catenin／Tcf-4通路相关,Western印迹法显示通路相关蛋白STAT3、c-myc和CyclinD1在转染反义miR-27b后表达下降。结论miR-27b可促进人脑胶质瘤细胞生长、增殖,具有原癌基因的作用,其发挥作用可能与β-catenin／Tcf-4通路密切相关。     

反义IGF-IR基因片段对裸鼠胶质瘤形成的影响

目的 探讨胰岛素样生长因子-I受体(IGF—IR)的反义基因片段对裸鼠胶质瘤的形成及其病理的影响。方法 体外用反义IGF—IR基因片段孵育胶质瘤细胞，并接种裸鼠体内，了解其肿瘤形成能力及其肿瘤组织学和IGF—IR的表达：结果 野生型C6细胞和正义寡核苷酸孵育的C6细胞在BALB／c裸鼠体内形成肿瘤的潜伏期均为4d，而经反义寡核苷酸孵育的C6细胞在裸鼠体内形成肿瘤的潜伏期为11～12d，肿瘤形成的潜伏期明显延长，肿瘤的生长得到抑制。肿瘤形成7d时，研究发现正义核酸组与野生组肿瘤病理组织学是一致的，且IGF—IR均呈高表达，反义核酸组肿瘤细胞变得稀疏，而且可见部分肿瘤细胞核固缩，染色质凝聚边缘化，甚至有细胞出现核碎裂等凋亡征象，IGF—IR表达明显减少。而在肿瘤形成的25d时，反义核酸组胶质瘤的病理表现和IGF—IR蛋白的表达与对照组和正义核酸组无明显差异。结论反义IGF—IR基因片段能抑制裸鼠胶质瘤的形成能力，但逃脱反义IGF—IR基因片段封闭的肿瘤细胞经过体内的增殖可形成肿瘤。     

Activation of CPP32 during apoptosis of neurons and astrocytes

Members of the interleukin-1β-converting enzyme (ICE)/CED-3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED-3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum-free media. We show that specific inhibitors of ICE/CED-3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell-free system. Cell extracts from neurons induced to undergo apoptosis contained ICE/CED-3 protease activity. To determine which member of the ICE/CED-3 family was activated in neurons and astrocytes during apoptosis, we developed a novel affinity-labeling technique that labeled the active site cysteine and identified a 17-kDa subunit of the activated protease. The affinity-labeled 17-kDa protease subunit shares antigenic and molecular mass identity with the processed form of CPP32 on immunoblots, suggesting that CPP32 may be the prinicipal effector in the apoptotic pathway in neurons and astrocytes. In time-course experiments, the activation of CPP32 preceded the detection of PARP cleavage and DNA laddering, suggesting that processing of CPP32 is a very early event in apoptosis of neurons and astrocytes and may be involved in the proteolytic action on specific cellular targets. The affinity-labeling technique developed and used in this report with neural cells allows for the sensitive detection, purification, and identification of ICE/CED-3 proteases that may be activated in other cells types under a variety of conditions, including certain diseased states. J. Neurosci. Res. 48:168–180, 1997. © 1997 Wiley-Liss, Inc.     

Expression of antisense bcl-2 cDNA abolishes tumorigenicity and enhances chemosensitivity of human malignant glioma cells

Bcl-2 is a key antiapoptotic protein, and it confers survival advantages on many types of tumors by inhibiting apoptotic cell death. Malignant gliomas are the most common primary central nervous system tumors, but the role of bcl-2 in these tumors has not been defined. We investigated the impact of bcl-2 on malignant gliomas by suppressing its expression. Antisense human bcl-2 cDNA was transfected into human malignant glioma cells. The effects of bcl-2 protein down-regulation on glioma cell morphology, in vitro tumor growth, and tumorigenicity in nude mice, as well as chemosensitivity to cisplatin, were studied. Expression of antisense bcl-2 cDNA decreased bcl-2 protein by more than sixfold. Antisense bcl-2 stable transfectants (AS-bcl-2) showed profound morphological change and markedly retarded cell growth in vitro. Transplantation of AS-bcl-2 cells resulted in no tumor formation, whereas backbone plasmid transfectant control formed tumors in each mouse transplanted. Expression of antisense bcl-2 in glioma cells resulted in significantly increased cytotoxicity of cisplatin. In conclusion, antisense bcl-2 expression can effectively reduce glioma survival, including retarding in vitro growth, complete loss of tumorigenicity, and significantly enhanced cisplatin cytotoxicity. These results suggest that bcl-2 plays an important role in glioma malignancy and chemoresistance. Development of strategies targeted at bcl-2 has the potential to advance treatment for malignant gliomas.     

IGF-IR反义寡核苷酸对胶质瘤细胞增殖的影响

目的 根据胰岛素样生长因子-受体（IGF-IR）基因序列设计了IGF-IR mRNA起始密码子下游的硫代磷酸型反义寡核苷酸和正义寡核苷酸，研究其对C6胶质瘤细胞增殖作用。方法 实验分为反义核酸组、正义核酸组和空白对照组，经细胞计数、MTT法研究反义核苷酸对胶质瘤细胞生长和增殖的抑制作用。并应用免疫细胞化学方法检测胶质瘤细胞IGF-IR和增殖细胞核抗原（PCNA）蛋白的表达。结果 反义寡核苷酸能有效地降低IGF-IR基因的表达，抑制体外培养的胶质瘤细胞的增殖，其抑制作用24h显效，96h仍有作用，反义寡核苷酸的抑制效果与其作用浓度有关。而正义寡核苷酸对其无抑制作用。同时可见反义寡核苷酸抑制增殖作用中，能明显下调PCNA蛋白的表达。结论 IGF-IR介导的IGF-I-IGF-IR的自（旁）分泌环对胶质瘤生长极其重要，通过IGF-IR反义寡核苷酸治疗能较有效地抑制胶质瘤细胞的增殖。     

Differential control of VEGF synthesis and secretion in human glioma cells by IL-1 and EGF.

VEGF (vascular endothelial growth factor), one of the most potent angiogenic factors, has recently been identified as an inducer of neoangiogenesis in many tumors including gliomas. VEGF itself appears to be regulated through different pathways. Since malignant gliomas frequently show EGF receptor amplification and express IL-1, a pivotal regulatory cytokine involved in angiogenesis, we analyzed interactions between EGF/EGF receptor and IL-1/IL-1 receptor and VEGF in the established glioblastoma cell lines U-87 MG and A-172. Basal VEGF expression was an order of magnitude higher in U-87 MG compared to A-172. IL-1 caused a fast and strong increase of VEGF secretion in U-87 MG which appeared to harbor an intracellular VEGF pool for enhanced exocytosis. The IL-1 receptor antagonist (IL-1-ra) reversed this effect suggesting an IL-1 receptor-associated mechanism. In contrast, VEGF secretion could not be increased by exogenous IL-1 exposure in A-172, which apparently lacked an intracellular VEGF pool for augmented exocytosis. However, IL-1-ra treatment alone caused a significant reduction of basal VEGF secretion in both U-87 MG and A-172. This suggests that baseline secretion of VEGF involves IL-1 receptor activation by endogenously produced IL-1. EGF also stimulated the secretion of VEGF into the cell supernatant. However, this effect, observed in both U-87 MG and A-172, was delayed and only occurred following replenishment of the intracellular VEGF pool. EGF upregulated the amount of VEGF mRNA. In general, the effects of IL-1 and EGF on VEGF were additive, suggesting independent mechanisms. Since IL-1 appears to be involved in VEGF secretion in glial tumors through an autocrine/paracrine mechanism, recombinant human IL-1-ra may evolve as a new agent for anti-angiogenic glioma therapy.     

Physiology of transformed glial cells

Much of our present knowledge of glial cell function stems from studies of glioma cell lines, both rodent (C6, C6 polyploid, and TR33B) and human (1321N1, 138MG, D384, R-111, T67, Tp-301MG, Tp-483MG, Tp-378MG, U-118MG, U-251MG, U-373MG, U-787MG, U-1242MG, and UC-11MG). New methods such as patch clamp and Ca²⁺ imaging have lead to rapid progress the last few years in our knowledge about glial cells, where an unexpected presence and diversity of receptors and ion channels have emerged. Basic mechanisms related to membrane potential and K⁺ transport and the presence of voltage gated ion channels (Na⁺, inwardly rectifying K⁺, Ca²⁺ activated K⁺, Ca²⁺, and Cl^? channels) have been identified. Receptor function and intracellular signaling for glutamate, acetylcholine, histamine, serotonin, cathecolamines, and a large number of neuropeptides (bradykinin, cholecystokinin, endothelin, opioids, and tachykinins) have been characterized. Such studies are facilitated in cell lines which offer a more homogenous material than primary cultures. Although the expression of ion channels and receptors vary considerably between different cell lines and comparative studies are rare, a few differences (compared to astrocytes in primary culture) have been identified which may turn out to be characteristic for glioma cells. Future identification of specific markers for receptors on glial and glioma cells related to cell type and growth properties may have great potential in clinical diagnosis and therapy. © 1995 Wiley-Liss, Inc.     

Protection of adult mouse progenitor cells and human glioma cells by de novo decorin expression in an oxygen- and glucose-deprived cell culture model system.

We employed an in vitro hypoxia cell culture model system and gene transfer technology to examine the effect of the decorin gene on cell survival against oxygen and glucose deprivation (OGD). Ectopic expression of decorin in subventricular zone (SVZ) cells from adult male mouse brain and human glioblastoma U-87 cells kept the cells viable against 24 h of OGD. Fewer than 1% of decorin-synthesizing cells were apoptotic after 12 h of OGD. In contrast, 100% of the control cells were apoptotic even after 4 h of OGD. De novo decorin synthesis in SVZ and U-87 cells induced expression of p21, p27 and Ras, AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma), and phosphorylated AKT. Blocking of phosphoinositide 3-kinase (PI-3K), Ras, and the epidermal growth factor receptor with specific inhibitors had no effect on induction of Ras, p21, and p27 at the messenger RNA level in decorin-synthesizing SVZ and U-87 cells. PI-3K inhibitors significantly increased apoptosis in decorin-expressing cells. Our data indicate that induction of p21, p27, Ras, AKT, and phosphorylated AKT by decorin inhibits apoptosis and protects U-87 and SVZ cells against OGD. Therefore, our data suggest that decorin is a potent trophic factor that protects neuronal progenitor cells and glioma cells from OGD.     

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.     

Role of high molecular weight extracellular matrix proteins in glioma cell migration

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that α3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.     

As2O3对不同p53表型的人胶质瘤细胞系细胞周期蛋白B1、D1表达的影响

目的研究三氧化二砷(As2O3)对两种不同p53表型的人胶质瘤细胞系(U87MG和T98G)细胞周期蛋白B1、D1表达及细胞周期的影响。方法应用激光扫描细胞计数分析仪(LSC),共聚焦显微镜以及Western印迹分析等方法检测As2O3对细胞周期蛋白B1、D1和p53及细胞周期的作用。结果As2O3能使两个胶质瘤细胞系的p53蛋白水平升高,细胞周期蛋白B1表达降低及G2/M期俘获,但仅U87MG细胞的细胞周期蛋白D1表达下降。As2O3还能诱导U87MG细胞凋亡。结论体外低浓度的As2O3能有效地抑制U87MG和T98G细胞增殖,提示As2O3有希望用于神经胶质瘤患者的治疗。