DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响。方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异;MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度;BSP实验检测正常胶质细胞株HEB与胶质瘤细胞株A172、U87、U251中miR-133a基因的甲基化水平;去甲基化试剂AZA与胶质瘤细胞株A172、U87、U251共培养72 h后,RT-PCR检测上述3株胶质瘤细胞中的miR-133a表达变化;MTT及Annexin V-FITC凋亡实验分别检测AZA处理后的3株胶质瘤细胞的增殖与凋亡能力变化。结果:RT-PCR实验表明与正常对照脑组织相比,胶质瘤组织中miR-133a表达显著下降;MSP实验结果显示,与正常对照脑组织相比,胶质瘤组织中miR-133a基因的甲基化水平明显增高;BSP实验结果显示与正常对照胶质细胞株HEB相比,胶质瘤细胞A172、U87、U251中miR-133a基因的甲基化水平显著增加;RT-PCR实验显示与去甲基化试剂AZA共培养72 h后,胶质瘤细胞株A172、U87、U251细胞中miR-133a的表达显著增加。MTT与Annexin V-FITC凋亡实验结果表明,去甲基化试剂AZA处理后,胶质瘤细胞A172、U87、U251的增殖能力明显下降,细胞的凋亡发生率显著增加。结论:胶质瘤细胞中DNA甲基化通过调控miR-133a的表达而沉默后者发挥抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的功能。     

和厚朴酚对组织因子途径抑制物 2诱导神经胶质瘤细胞凋亡的实验研究#br#

目的探讨和厚朴酚对组织因子途径抑制物 2（TFPI 2）表达的影响,以及和厚朴酚和TFPI 2过表达对人胶质瘤U251细胞凋亡的影响。 方法体外培养胶质瘤U87、U251、LN18、LN229、A172细胞系,荧光定量PCR（QPCR）检测不同细胞系中TFPI 2的表达。不同浓度（0、10、20、30、40和50 μmol／L）和厚朴酚干预U251细胞,QPCR和Western blotting检测TFPI 2 mRNA和蛋白的表达。采用腺病毒载体pGSadeno TFPI 2过表达U251细胞内TFPI 2的表达,流式细胞术及caspase 3试剂盒检测细胞凋亡率和caspase 3活性。 结果在选取的5种不同胶质瘤细胞株中,U251和A172细胞内TFPI 2 mRNA明显下降（P＜005）。和厚朴酚呈浓度依赖性增加U251细胞内TFPI 2 mRNA水平;其中50 μmol／L和厚朴酚干预24 h后, TFPI 2 mRNA表达水平升高最明显（P＜005）。腺病毒感染U251细胞后TFPI 2表达水平明显增加（P＜005）。过表达TFPI 2和50 μmol／L和厚朴酚干预均可增加U251细胞凋亡水平和caspase 3的活性（P＜005）。 结论和厚朴酚处理可增加U251细胞内TFPI 2的表达;和厚朴酚联合TFPI 2过表达显著诱导胶质瘤细胞凋亡。     

和厚朴酚对组织因子途径抑制物 2诱导神经胶质瘤细胞凋亡的实验研究#br#

目的探讨和厚朴酚对组织因子途径抑制物 2（TFPI 2）表达的影响,以及和厚朴酚和TFPI 2过表达对人胶质瘤U251细胞凋亡的影响。 方法体外培养胶质瘤U87、U251、LN18、LN229、A172细胞系,荧光定量PCR（QPCR）检测不同细胞系中TFPI 2的表达。不同浓度（0、10、20、30、40和50 μmol／L）和厚朴酚干预U251细胞,QPCR和Western blotting检测TFPI 2 mRNA和蛋白的表达。采用腺病毒载体pGSadeno TFPI 2过表达U251细胞内TFPI 2的表达,流式细胞术及caspase 3试剂盒检测细胞凋亡率和caspase 3活性。 结果在选取的5种不同胶质瘤细胞株中,U251和A172细胞内TFPI 2 mRNA明显下降（P＜005）。和厚朴酚呈浓度依赖性增加U251细胞内TFPI 2 mRNA水平;其中50 μmol／L和厚朴酚干预24 h后, TFPI 2 mRNA表达水平升高最明显（P＜005）。腺病毒感染U251细胞后TFPI 2表达水平明显增加（P＜005）。过表达TFPI 2和50 μmol／L和厚朴酚干预均可增加U251细胞凋亡水平和caspase 3的活性（P＜005）。 结论和厚朴酚处理可增加U251细胞内TFPI 2的表达;和厚朴酚联合TFPI 2过表达显著诱导胶质瘤细胞凋亡。     

MPPa-PDT抑制骨肉瘤 MG-63细胞侵袭和迁移

目的:探讨焦脱镁叶绿酸-a甲酯介导的光动力疗法(methyl ester pyropheophorbide-a mediated photodynamic thera-PY,MPPa-PDT)对人骨肉瘤MG-63细胞迁移及侵袭的影响及其可能机制.方法:采用CCK-8检测经MPPa-PDT处理后不同时间点的人骨肉瘤MG-63细胞增殖活性;划痕实验及Transwell实验检测细胞迁移、侵袭能力;Western blotting检测E-钙黏蛋白(E-cad)和基质金属蛋白酶(matrix metalloproteinase,MMP)-2、-9蛋白的表达水平.结果:MPPa-PDT处理MG-63细胞12、24及48 h后MPPa-PDT组细胞增殖能力明显较对照组、MPPa组和LED组细胞降低(均P＜0.05),且MPPa-PDT组细胞的划痕愈合能力、迁移活力及侵袭能力也较这3组细胞显著下降(均P ＜0.05);MPPa-PDT组细胞E-cad的表达显著高于其他3组,MMP-2、MMP-9的表达则明显低于其他3组(均P＜0.05).结论:MPPa-PDT抑制人骨肉瘤MG-63细胞的侵袭和迁移能力;上调E-cad的表达,下调MMP-2、-9的表达可能是其作用机制之一.     

脑胶质瘤细胞系对细小病毒MVM和H1易感性的研究

[目的]测定人胶质瘤细胞系A172,U87MG,U138,U373,和鼠胶质瘤细胞系GL261与MT539对细小病毒MVM和H1的易感性,并观察野生型病毒在肿瘤细胞内部繁殖情况.[方法]体外培养肿瘤细胞,分别用不同剂量的MVM和H1病毒感染GL261,MT539和A172,U87MG,U138,U373细胞1h,继续培养5d,每天进行活细胞计数.空斑形成试验测定感染病毒(MOI=0.1)5d后细胞内部及细胞培养上清中的病毒总量.[结果]与未感染细胞(对照)比较,U138,U373,GL261细胞在MOI 2和5的病毒剂量感染后活细胞数目明显减少,U87细胞数基本维持不变,A172和MT539细胞数轻微增加.MOI=0.1的病毒剂量对细胞的作用与对照没有差异.空斑形成试验结果表明U373和GL261在MOI=0.1的病毒感染5d后细胞内和培养上清中总病毒量略高于初始病毒量,其它细胞的总病毒量均低于初始病毒量.[结论]不同胶质瘤对细小病毒的易感性不同,U138,U373,GL261细胞对细小病毒H1或MVM易感,U87细胞次之,A172和MT539细胞不敏感.U373和GL261细胞有弱的产生子病毒的能力.     

抗人DR5单克隆抗体诱导Ｕ343细胞凋亡研究

目的:探讨抗肿瘤坏死因子相关凋亡配体的死亡受体DR5单克隆抗体对神经胶质瘤细胞株U343的杀伤作用及机制.方法:采用流式细胞仪从蛋白质水平定量地检测DR5在U343的表达;用RT-PCR从mRNA水平检测DR5的表达;免疫细胞化学法明确DR5在U343的分布情况.通过MTT法、琼脂糖凝胶电泳、流式细胞仪DNA倍体分析,观察抗人DR5单克隆抗体对U343细胞增殖的抑制活性及其诱导凋亡效应.结果:神经胶质瘤细胞株U343表达死亡受体DR5,DR5分布于细胞内细胞核的周围.抗人DR5单克隆抗体3μg/ml作用4 h可明显杀伤U343细胞,其机制与诱导凋亡有关.结论:抗人DR5单克隆抗体可以诱导神经胶质瘤细胞株U343凋亡,以死亡受体为靶点的抗体制剂为肿瘤治疗提供新的途径.     

沉默lncRNA GIHCG通过上调miR-146a-3p增加胶质瘤细胞放射敏感性

目的 探讨lncRNA GIHCG对胶质瘤细胞放射敏感性的影响及作用机制。方法 qRT-PCR实验检测人脑正常胶质细胞HEB和胶质瘤细胞系U251、A172、SHG139、U87中GIHCG和miR-146a-3p表达水平。以U251和SHG139细胞为研究对象,沉默GIHCG表达或过表达miR-146a-3p后,MTT检测细胞增殖,流式细胞仪检测细胞凋亡,克隆形成实验检测细胞放射敏感性,蛋白印迹法检测CDK1、CyclinD1、Bcl-2和Bax蛋白表达水平。生物信息学软件预测GIHCG与miR-146a-3p存在结合位点,双荧光素酶报告基因实验和qRT-PCR实验验证GIHCG与miR-146a-3p的靶向关系。结果 与HEB细胞比较,胶质瘤U87、U251、A172和SHG139细胞中GIHCG表达升高(P<0.05),miR-146a-3p表达降低(P<0.05)。沉默GIHCG表达或过表达miR-146a-3p,U251和SHG139细胞存活率、存活分数、CDK1、CyclinD1和Bcl-2蛋白表达降低(P<0.05),凋亡率和Bax蛋白表达升高(P<0.05)。GIHCG在U251和SHG139细胞中靶向负调控miR-146a-3p表达,抑制miR-146a-3p表达逆转了沉默GIHCG对胶质瘤细胞增殖、凋亡及放射敏感性的影响。结论 沉默GIHCG表达可促进miR-146a-3p表达,从而增强胶质瘤细胞的放射敏感性。     

沉默lncRNA GIHCG通过上调miR-146a-3p增加胶质瘤细胞放射敏感性

目的 探讨lncRNA GIHCG对胶质瘤细胞放射敏感性的影响及作用机制。方法 qRT-PCR实验检测人脑正常胶质细胞HEB和胶质瘤细胞系U251、A172、SHG139、U87中GIHCG和miR-146a-3p表达水平。以U251和SHG139细胞为研究对象,沉默GIHCG表达或过表达miR-146a-3p后,MTT检测细胞增殖,流式细胞仪检测细胞凋亡,克隆形成实验检测细胞放射敏感性,蛋白印迹法检测CDK1、CyclinD1、Bcl-2和Bax蛋白表达水平。生物信息学软件预测GIHCG与miR-146a-3p存在结合位点,双荧光素酶报告基因实验和qRT-PCR实验验证GIHCG与miR-146a-3p的靶向关系。结果 与HEB细胞比较,胶质瘤U87、U251、A172和SHG139细胞中GIHCG表达升高(P<0.05),miR-146a-3p表达降低(P<0.05)。沉默GIHCG表达或过表达miR-146a-3p,U251和SHG139细胞存活率、存活分数、CDK1、CyclinD1和Bcl-2蛋白表达降低(P<0.05),凋亡率和Bax蛋白表达升高(P<0.05)。GIHCG在U251和SHG139细胞中靶向负调控miR-146a-3p表达,抑制miR-146a-3p表达逆转了沉默GIHCG对胶质瘤细胞增殖、凋亡及放射敏感性的影响。结论 沉默GIHCG表达可促进miR-146a-3p表达,从而增强胶质瘤细胞的放射敏感性。     

STIP1在脑胶质瘤组织中的表达及对胶质瘤细胞株增殖、凋亡、侵袭的影响

目的:检测磷酸化应激诱导蛋白1（stress-induced phosphoprotein 1,STIP1）在胶质瘤组织和正常脑组织中的表达差异,探讨STIP1对胶质瘤细胞株U87、U251细胞增殖、凋亡及侵袭能力的影响。方法:运用实时定量聚合酶链式反应(qRT-PCR)、免疫组化及蛋白印迹检测胶质瘤组织和正常脑组织中STIP1的表达;小干扰RNA（STIP1-siRNA）转染U87、U251细胞株后,使用噻唑蓝(MTT)法、流式细胞仪和Transwell小室观察细胞的增殖、凋亡和侵袭能力。结果:STIP1在胶质瘤Ⅳ级组织中的表达明显高于正常脑组织,但在胶质瘤Ⅱ、Ⅲ级中的表达与正常脑组织无明显差异。U87、U251细胞株转染STIP1-siRNA后,细胞增殖和侵袭能力明显受到抑制,而细胞凋亡率明显上升。结论:STIP1在胶质母细胞瘤组织中高表达,下调STIP1可以抑制胶质瘤细胞株U87和U251的增殖,阻滞细胞周期,促进其细胞凋亡,并可抑制其侵袭力。     

银杏叶提取物通过调控NF-κB信号通路抑制神经胶质瘤U87细胞增殖和侵袭

[目的]评价银杏叶提取物(EGb761)对神经胶质瘤U87细胞的生长与侵袭的影响,并进一步分析EGb761对U87细胞作用的分子机制。[方法]体外培养人神经胶质瘤U87细胞,给予不同浓度(0、100、200、400mg/L)银杏叶提取物干预(24h、48h、72h)后,采用CCK-8技术检测EGb761对U87细胞增殖的影响。Transwell小室和流式细胞凋亡技术分析EGb761对U87细胞侵袭和凋亡的影响;Western blot检测EGb761处理后U87细胞内NF-κB与cyclin D1、 iNOS和COX-2蛋白的表达变化。[结果]银杏叶提取物可有效抑制神经胶质瘤U87细胞的增殖和侵袭,促进细胞凋亡;Western blot提示EGb761干预的U87细胞内NF-κB与cyclin D1、 iNOS、COX-2蛋白的表达均明显下调。[结论] EGb761抑制神经胶质瘤U87细胞的生长增殖和侵袭能力,其可能是通过NF-κB信号通路而发挥抗肿瘤作用的。     

Isolation and characterization of cancer stem like cells in human glioblastoma cell lines

To identify and compare the features of stem like cells in human glioblastoma cell lines U251, U87MG, A172 with primary cultured glioblastoma stem cells, the ratio of CD133⁺ cells, the ability of tumor sphere formation, and self-renewing capacity of U251, U87MG, A172 cells in serum free medium plus EGF, bFGF and B27 supplement were detected. The results suggested that there might be more cancer stem like cells in U251 cells compared with others. CD133⁺ cells enriched in SP cells and in U251 cells cultured with the serum free medium. They expressed the neural stem cell markers CD133 and Nestin, but lacked of neuronal and astrocyte marker MAP2, β-III tubulin and GFAP. They could apparently generate both neurons and glial cells after serum retrieved in vitro. Gli1, Bmi1, Notch2 and PTEN were also found expressed highly in them. Moreover, CD133⁺ cells were more resistant to hypoxia, irradiations and some chemotherapeutics than CD133⁻cells. So we suggested that glioblastoma stem like cells were existed in CD133⁺ cells in U251 cell line with characteristics of self-renew and generation of an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may be exploited in the development of novel cancer therapeutic drugs.     

Adaphostin cytoxicity in glioblastoma cells is ROS-dependent and is accompanied by upregulation of heme oxygenase-1

Purpose To delineate a role for reactive oxygen species (ROS) induction in adaphostin-induced apoptosis in glioblastoma cells. Methods Three glioblastoma cell lines with different sensitivities to adaphostin were characterized for sensitivity to an oxidant, tert-butyl hydroperoxide. The degree and duration of the ROS levels was assessed in the three cell lines after adaphostin exposure. Antioxidant protein levels were evaluated by Western blotting. Results Of the three glioblastoma cell lines, the U87 cells were least sensitive to adaphostin. These cells were also least sensitive to tert-butyl hydroperoxide, indicating that sensitivity to a direct oxidant stress mirrors the cells’ adaphostin sensitivities. In addition, the antioxidant N-acetylcysteine, (NAC) was protective against adaphostin-induced apoptosis. Direct measurement of intracellular peroxides showed a transient increase in the two less sensitive cell lines (U87 and LN18) which diminishes by 24 h. In contrast, U251 cells, which are most sensitive to adaphostin, display a sustained increase in the ROS levels. After the initial increase in intracellular peroxides, the heat shock protein and antioxidant heme oxygenase-1 (HO-1) was upregulated. Levels of other antioxidant proteins, such as catalase and thioredoxin, however, were not altered by adaphostin in glioblastoma cell lines. NAC attenuated HO-1 upregulation, confirming the time course analysis. Conclusions These results suggest a primary role for ROS in adaphostin-induced apoptosis in glioblastoma. Our data indicate that the duration of intracellular ROS levels is a key factor in mediating sensitivity to adaphostin. Furthermore, upregulation of HO-1 is a novel molecular marker of adaphostin’s action. The kinetics with which adaphostin upregulates HO-1 correlates with sensitivity to the drug. Taken together, our data indicate that a cell’s ability to cope with ROS dictates sensitivity to adaphostin and conceivably other chemotherapies that cause redox perturbations.     

The tyrosine kinase inhibitor imatinib mesylate enhances the efficacy of photodynamic therapy by inhibiting ABCG2.

PURPOSE: The ATP-binding cassette protein ABCG2 (breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) and, thus, may confer resistance to this treatment modality. Tyrosine kinase inhibitors (TKI) can block the function of ABCG2. Therefore, we tested the effects of the TKI imatinib mesylate (Gleevec) on photosensitizer accumulation and in vitro and in vivo PDT efficacy. EXPERIMENTAL DESIGN: Energy-dependent photosensitizer efflux and imatinib mesylate's effects on intracellular accumulation of clinically used second- and first-generation photosensitizers were studied by flow cytometry in murine and human cells with and without ABCG2 expression. Effects of ABCG2 inhibition on PDT were examined in vitro using cell viability assays and in vivo measuring photosensitizer accumulation and time to regrowth in a RIF-1 tumor model. RESULTS: Energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX (PpIX) synthesized from 5-aminolevulenic acid, and the benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) was shown in ABCG2+ cell lines, but the first-generation multimeric photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were minimally transported. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2+ cells, but not in ABCG2- cells, and enhanced PDT efficacy both in vitro and in vivo. CONCLUSIONS: Second-generation clinical photosensitizers are transported out of cells by ABCG2, and this effect can be abrogated by coadministration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other ABCG2 transport inhibitors may enhance efficacy and selectivity of clinical PDT.     

Deactivation of cisplatin-resistant human lung/ovary cancer cells with pyropheophorbide-α methyl ester-photodynamic therapy

The aim of this study was to determine whether photodynamic therapy (PDT) alone or combined with cisplatin (DDP), can deactivate cisplatin-resistant cancer cells. Human cancer cell lines A549 and SKOV3, and chemoresistant sublines A549/DDP and SKOV3/DDP, were subjected to PDT, DDP, or PDT combined with DDP. Cell viability and apoptosis were analyzed, and then intracellular reactive oxygen species (ROS) and proteins related to apoptosis were determined. PDT caused cell death, and PDT combined with DDP led to the highest percentage of dead cells in 4 cell lines; similar results were detected in ROS; a quantification evaluation manifested that the combined effect was addition. DDP increased the percentage of apoptotic cells, and the ROS level in A549 and SKOV3 cells, which was not observed in A549/DDP and SKOV3/DDP cells. Western blot revealed an increase of caspase 3 and Bax, and a decrease of Bcl-2, demonstrating the occurrence of apoptosis. The data suggest that PDT can efficiently deactivate resistant cells and enhance the action of DDP against resistant cancer cells.     

Cellular responses to chlorin-based photosensitizer DH-II-24 under darkness in human gastric adenocarcinoma AGS cells

We investigated cellular responses to chlorin-based photosensitizer DH-II-24 under darkness in human gastric adenocarcinoma AGS cells. Cells were loaded with 0.5-10 μg/mL DH-II-24 for 12 h, and intracellular reactive oxygen species (ROS) and intracellular Ca(2+) levels, in situ tissue transglutaminase (tTGase) activity, cell viability, cell morphology and cell cycle were examined. DH-II-24 treatment had no effect on intracellular ROS production or cell morphology, and did not induce cell detachment at any concentrations tested. In addition, cell viability and cell cycle progression were not altered by the photosensitizer. However, DH-II-24 treatment elevated the basal level of intracellular Ca(2+) in a dose-dependent manner and inhibited tTGase activity without affecting tTGase expression levels. Furthermore, DH-II-24 inhibited lysophosphatidic acid-induced activation of tTGase in a dose-dependent manner. In contrast, photodynamic therapy (PDT) with 1 μg/mL DH-II-24 significantly elevated intracellular ROS and in situ tTGase activity in parallel with a rapid and large increase in intracellular Ca(2+) levels. DH-II-24-mediated PDT decreased cell viability and induced cell detachment. These results demonstrate that DH-II-24 treatment alone under darkness induced different cellular responses to DH-II-24-mediated PDT.     

Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1^R132H protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1^WT and IDH1^R132H were generated, as well as U87MG cell lines overexpressing IDH2^WT and IDH2^R172K. In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1^R132H cells, U373MG-IDH1^R132H cells, and U87MG-IDH2^R172K cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1^R132H cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1^R132H cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1^R132H and IDH2^R172K mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.     

Miconazole induces autophagic death in glioblastoma cells via reactive oxygen species-mediated endoplasmic reticulum stress

Miconazole is an antifungal agent that is used for the treatment of superficial mycosis. However, recent studies have indicated that miconazole also exhibits potent anticancer effects in various types of cancer via the activation of apoptosis. The main aim of the present study was to observe the effect of miconazole on autophagic cell death of cancer cells. Cytotoxicity was measured by viable cell counting after miconazole treatment in glioblastoma cell lines (U343MG, U87MG and U251MG). Induction of autophagy was analyzed by examining microtubule-associated protein light chain 3 (LC3)-II expression levels using western blotting and by detecting GFP-LC3 translocation using a fluorescence microscope. Intracellular ROS production was measured using a fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. It was found that miconazole induced autophagic cell death in the U251MG glioblastoma cell line via the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress response. An association between miconazole-induced ROS production and autophagy was also identified; in particular, pretreatment of the cells with a ROS scavenger resulted in a reduction in the levels of LC3-II. Miconazole-induced ER stress was associated with increases in binding immunoglobulin protein (BiP), inositol-requiring enzyme 1α (IRE1α) and CHOP expression, and phospho-eIF2α levels. The inhibition of ER stress via treatment with 4-phenylbutyric acid or BiP knockdown reduced miconazole-induced autophagy and cell death. These findings suggest that miconazole induces autophagic cell death by inducing an ROS-dependent ER stress response in U251MG glioma cancer cells and provide new insights into the potential antiproliferative effects of miconazole.     

Reduction of nitroxides and radioprotective ability in glioblastoma cells

Electron spin resonance (ESR) analyses were performed to clarify whether glioblastoma cells scavenge hydroxyl radicals (·OH) generated by x-ray irradiation. The rate of bioreduction of nitroxides by three human glioblastoma cells was also evaluated by the same technique and compared with their x-ray sensitivity. Aerated culture media containing 200 mM of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with or without U87MG cells were irradiated with x-rays at a dose of 20 Gy. ESR was measured immediately after each irradiation. Continuous changes of the ESR spectra of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) were analyzed in cell suspensions of TK1, U87MG, and A172 at a concentration of 1.0×10⁷ cells/ml containing 5 μM Tempol. As a result, the signal of DMPO-OH in the U87MG cell suspension decayed faster than that in the control culture media without cells, and the rate of bioreduction of Tempol in each glioblastoma cell suspension was correlated with the x-ray sensitivity defined from the colony-forming assay in those cell lines. It was indicated that the resistance of glioblastoma cells to ionizing radiation could be closely related to their ability to scavenge radical species generated by ionizing radiation.     

Nogo-A/NgR signaling regulates stemness in cancer stem-like cells derived from U87MG glioblastoma cells

Neurite outgrowth inhibitor A (Nogo-A), a member of the reticulon 4 family, is an axon regeneration inhibitor that is negatively associated with the malignancy of oligodendroglial tumors. It has been suggested that the Nogo-A/Nogo Receptor (NgR) pathway plays a promoting effect in regulating cancer stem-like cells (CSCs) derived from glioblastoma, indicating that Nogo-A could exert different roles in CSCs than those in parental cancer cells. In the present study, CSCs were generated from the human Uppsala 87 malignant glioma (U87MG) cell line. These U87MG-CSCs were characterized by the upregulation of CD44 and CD133, which are two markers of stemness. The expression levels of Nogo-A and the differentiation of U87MG-CSCs were investigated. In addition, the proliferation, invasion and colony formation U87MG-CSCs were examined. Using culture in serum-containing medium, U87MG-CSCs were differentiated into neuron-like cells specifically expressing MAP2, β-III-tubulin and nestin. Nogo-A was upregulated in U87MG-CSCs compared with parental cells. Knockdown of Nogo-A and inhibition of the Nogo-A/NgR signaling pathway in U87MG-CSCs markedly decreased cell viability, cell cycle entry, invasion and tumor formation, indicating that Nogo-A could regulate U87MG-CSC function. Moreover, Nogo-A was involved in intracellular ATP synthesis and scavenging of accumulated reactive oxygen species. Nogo-A/NgR pathway exerted protective effects against hypoxia-induced non-apoptotic and apoptotic cell death. These results suggest that Nogo-A plays an important role in regulating U87MG-CSCs via the Nogo-A/NgR signaling pathway. Nogo-A may also different roles in U87MG-CSCs compared with their parental cells.