榄香烯抑制Raf/MEK/ERK信号通路诱导人源胶质瘤U87细胞凋亡

目的探讨莪术提取物榄香烯体外诱导胶质瘤细胞凋亡的机制。方法采用流式细胞术、West-ern印迹等方法,分别检测不同浓度榄香烯对人源U87胶质瘤细胞的凋亡诱导及其对U87细胞Raf-1、ERK、癌基因Bcl-2蛋白质表达的影响。结果榄香烯对人源U87胶质瘤细胞具有明显的凋亡诱导作用,该增殖抑制效应呈时间依赖性。榄香烯可明显下调U87细胞的磷酸化Raf-1、ERK、Bcl-2表达。结论体外榄香烯对胶质瘤细胞具有明显的凋亡诱导作用(呈时间依赖性),抑制Raf/MEK/ERK信号通路,从而下调其下游信号癌基因Bcl-2的表达,最终启动凋亡程序可能是榄香烯诱导U87细胞凋亡的机制。     

GMF-β与MEK信号通路cross-talk介导榄香烯的抗胶质瘤作用

目的探讨神经胶质成熟因子-β(Glia Maturation Factor-β,GMF-β)在榄香烯致人U87MG胶质瘤细胞增殖抑制中的作用。方法以不同浓度榄香烯作用于人U87MG胶质瘤细胞,噻唑蓝(Methyl Thiazolyl Tet-razolium,MTT)法检测细胞活性。免疫沉淀联合Western blot法检测经榄香烯处理的U87MG细胞中总GMF-β及磷酸化GMF-β(p-GMF-β)蛋白的表达水平。通过RNA干扰技术使GMF-β的表达沉默,然后以MTT法检测榄香烯的抗胶质瘤增殖能力。Western blot法检测榄香烯对丝裂原活化蛋白激酶激酶3/6(Mitogen-activated Protein Ki-nase Kinase 3/6,MEK3/6)的激活作用是否因GMF-β的沉默而受到影响。结果榄香烯显著抑制了人U87MG细胞的增殖,并可使GMF-β、MEK3和MEK6的磷酸化水平上调。沉默GMF-β的表达导致MEK3和MEK6的磷酸化水平下调,榄香烯的抗胶质瘤细胞增殖作用减弱。结论 GMF-β与MEK信号通路的交互作用(cross-talk)介导了榄香烯的抗人胶质瘤细胞增殖作用。     

榄香烯抑制人脑胶质瘤U251细胞增殖和PCNA基因表达的研究

目的研究榄香烯(elemene)抑制人脑胶质瘤U251细胞增殖和抑制增殖细胞核抗原(PCNA)基因表达作用。方法应用四甲基偶氮唑蓝法(MTT)测定不同浓度榄香烯对人脑胶质瘤U251细胞的抑制作用,同时观察时间—效应关系,并求出半数致死浓度(IC50);免疫组化法检测IC50浓度榄香烯作用0、24和48h的U251细胞中PCNA蛋白表达差异;RT-PCR检测不同浓度或不同作用时间的榄香烯抑制PCNA基因的表达。结果榄香烯对U251细胞株增殖具有抑制作用,呈剂量和作用时间的依赖性,IC50为0.062g·L-1。榄香烯对U251细胞内PCNA蛋白表达有明显的抑制作用;RT-PCR分别证实榄香烯抑制PCNA基因的表达,其PCNA基因mRNA表达值随药物浓度增加或作用时间延长增加而不断下降,呈剂量和作用时间的依赖性。结论榄香烯能有效下调PCNA基因表达,从而抑制人脑胶质瘤U251细胞增殖,对抗人脑胶质瘤具有广阔的前景。     

榄香烯阻碍大鼠胶质瘤C6细胞ERK信号通路中Hsp90/Raf-1分子复合体的形成

目的探讨莪术提取物榄香烯抑制胶质瘤细胞体外增殖的机制。方法采用细胞计数、免疫共沉淀、Western印迹等方法,分别检测不同浓度、不同作用时间榄香烯对C6胶质瘤细胞的增殖影响、对C6细胞中与Raf-1结合形成分子复合体的Hsp90水平的影响、对C6细胞Hsp90、Raf-1、ERK蛋白质表达的影响。结果榄香烯对大鼠C6胶质瘤细胞具有明显的抑制增殖作用,该增殖抑制效应呈剂量、时间依赖性。榄香烯可明显下调C6细胞中与Raf-1结合的Hsp90水平及C6细胞的磷酸化Raf-1、ERK表达,但不影响总Hsp90的表达水平。结论榄香烯能有效抑制胶质瘤细胞的体外增殖,破坏Hsp90的分子伴侣功能,阻碍Hsp90/Raf-1复合体的形成,使Raf-1不能被活化,最终下调胶质瘤细胞赖以生存的Raf/MEK/ERK通路,这可能是榄香烯抑制C6细胞增殖的机制。     

异硫氰酸苄酯抑制人胶质瘤U87MG细胞体外侵袭和诱导凋亡的作用和机制

目的 研究异硫氰酸苄酯对人胶质瘤U87MG细胞体外侵袭和凋亡的影响,并初步探讨其作用机制。方法 采用MTS实验考察异硫氰酸苄酯对肿瘤细胞体外增殖的抑制作用;2,5 μmol·L^-1异硫氰酸苄酯作用24 h后,采用Transwell实验、黏附实验和划痕实验观察异硫氰酸苄酯对人胶质瘤细胞侵袭、黏附和迁移能力的影响;应用Real-time-PCR和Western blot法检测相应浓度异硫氰酸酯处理后人胶质瘤U87MG细胞中MMP-2、MMP-9、CD44、Survivin、Bcl-2、Net1、RohA、caspase-3、caspase-8和p-AKT的表达变化;应用报告基因技术检测NF-κB转录活性的变化;采用ELISA法测定胞内8-OH-dG含量;10,20 μmol·L^-1异硫氰酸酯作用24 h后,采用流式细胞术观察其对细胞凋亡的作用。结果 异硫氰酸苄酯可显著抑制人胶质瘤U87MG细胞体外增殖;与0 μmol·L^-1组相比,2,5 μmol·L^-1异硫氰酸苄酯对人胶质瘤细胞U87MG侵袭、黏附和迁移能力有明显的抑制作用;不同浓度异硫氰酸苄酯处理后,肿瘤细胞MMP-2、MMP-9、CD44、Survivin、Bcl-2、NET1和RhoA的mRNA和蛋白表达、AKT磷酸化水平和NF-κB转录活性明显下调,caspase-3和caspase-8表达以及8-OH-dG含量显著上调;10,20 μmol·L^-1异硫氰酸苄酯可显著诱导细胞凋亡。结论 异硫氰酸苄酯抑制人胶质瘤细胞U87MG的侵袭能力,诱导细胞凋亡,其机制可能与抑制AKT/NF-κB信号转导途径,进而调节侵袭和凋亡相关基因表达有关。     

TIM-3在人胶质瘤中的表达及其对人胶质瘤细胞U251凋亡与侵袭的作用研究

目的 探讨T细胞免疫球蛋白黏蛋白3（TIM-3）在人胶质瘤中的表达及其对胶质瘤细胞U251凋亡与侵袭的影响。方法 收集2018年3月—2019年3月在我院进行手术切除的30例胶质瘤患者术后的瘤组织和瘤旁正常组织,免疫组化检测胶质瘤组织中TIM-3的表达情况。将胶质瘤U251细胞分为正常组、对照组、实验组：正常组细胞常规培养,不做任何处理;对照组和实验组细胞分别转染TIM-3-siRNA-NC和TIM-3-siRNA。采用实时荧光定量聚合酶链反应（RT-PCR）检测各组细胞中TIM-3 mRNA的表达,四甲基偶氮唑盐（MTT）实验检测各组细胞的增殖活力,流式细胞术检测各组细胞的凋亡率,Transwell小室检测各组细胞的侵袭能力。结果 TIM-3的表达主要定位在胞质中。TIM-3在Ⅰ级和Ⅱ级胶质瘤组织中的阳性表达率为（48.37±4.68）%,在Ⅲ级和Ⅳ级胶质瘤组织中的阳性表达率为（87.94±6.41）%,与正常组织中的阳性表达率［（12.34±4.92）%］相比,差异均有统计学意义（t=24.87, P<0.05）。与正常组细胞相比,实验组细胞中TIM-3的表达水平、细胞的增殖和侵袭能力均明显下降,凋亡率明显上升,差异均有统计学意义（P<0.05）。结论 TIM-3在胶质瘤中呈高表达,沉默其表达能明显抑制胶质瘤细胞的增殖和侵袭,并促进其凋亡。     

二氢杨梅素对胶质瘤细胞株U87增值凋亡及PI3K/Akt通路的影响

摘 要 目的：探讨二氢杨梅素（DMY）对胶质瘤细胞株U87增殖、凋亡、自噬及磷脂酰肌醇 3激酶/丝 苏氨酸蛋白激酶（PI3K/Akt）信号通路的影响。 方法: 体外培养胶质瘤U87细胞,U87细胞分为4组：空白对照组、DMY低（25 μmol·L-1）、中（50 μmol·L-1）、高（100 μmol·L-1）剂量组。采用MTT法检测U87细胞增殖情况,Hoechst染色法检测U87细胞凋亡,透射电镜下观察自噬体形成,蛋白印迹（WB）法检测凋亡蛋白B细胞淋巴瘤 2（Bcl 2）及其相关蛋白（Bax）、裂解的半胱氨酸天冬氨酸蛋白酶 3（cleaved caspase 3）、自噬相关蛋白Beclin 1、微管相关蛋白1轻链3（LC3）、P62及PI3K/Akt信号通路相关蛋白PI3K及其磷酸化激酶（p PI3K）、Akt及其磷酸化蛋白（p Akt）表达。 结果: 与空白对照组相比,DMY处理后U87细胞存活率、P62、Bcl 2、p PI3K及p Akt蛋白表达量均显著降低（P＜0.05）,且高剂量组显著低于低、中剂量组（P＜0.05）;与空白对照组相比,DMY处理后U87细胞凋亡率、自噬体数量、自噬泡/胞质总面积、LC3 Ⅱ/LC3 Ⅰ比值、LC3 Ⅱ、Beclin 1、Bax及cleaved caspase 3蛋白表达量均显著升高（P＜0.05）,且高剂量组显著高于低、中剂量组（P＜0.05）。 结论: 二氢杨梅素可有效抑制胶质瘤U87细胞增殖,并可诱导自噬发生促使细胞凋亡,其作用机制可能与PI3K/Akt信号通路有关。     

榄香烯乳联合更昔洛韦对C6脑肿瘤干细胞侵袭能力及TIMP-1 mRNA表达的影响

目的:研究榄香烯乳联合更昔洛韦对C6脑肿瘤干细胞(BTSCs)侵袭能力及组织金属蛋白酶抑制物1(TIMP-1)mRNA表达的影响。方法:从大鼠C6恶性胶质瘤细胞株中分离培养传代3次的细胞,采用免疫细胞化学法检测其中干细胞标志物CD133、Nestin蛋白表达情况。将20只大鼠随机分为空白对照组(生理盐水)、更昔洛韦组(ig,216 mg/kg)、榄香烯乳组(尾iv,36 mg/kg)和联用组(ig更昔洛韦216 mg/kg+尾iv榄香烯乳36 mg/kg),每组5只,每天给药1次,连续给药10 d。于末次给药后2 h采集各组大鼠血液并分离血清,分别与C6 BTSCs在体外培养基中共同培养24 h。采用Boyden侵袭试验检测C6 BTSCs的细胞侵袭能力,实时荧光定量聚合酶链式反应(RT-PCR)法测定C6 BTSCs的TIMP-1 mRNA表达情况。结果:传代3次后的细胞中CD133、Nestin蛋白有明显表达,表明其为BTSCs。与空白对照组比较,更昔洛韦组、榄香烯乳组和联用组C6 BTSCs的细胞侵袭率均明显下降、TIMP-1 mRNA表达均明显增强,差异有统计学意义(P<0.05);与更昔洛韦组和榄香烯乳组比较,联用组C6 BTSCs的细胞侵袭率明显下降、TIMP-1 mRNA表达明显增强,差异有统计学意义(P<0.05)。结论:榄香烯乳联合更昔洛韦可明显抑制C6 BTSCs的细胞侵袭,其作用机制可能与促进TIMP-1的生成有关,且联用效果强于单用。     

ERK1/2信号通路在青蒿琥酯抑制PDGF-BB诱导HSC细胞增殖中的作用

目的:观察青蒿琥酯(Artesunate,Art)对血小板衍生生长因子-BB(platelet derived growthfactor-BB,PDGF-BB)刺激大鼠肝星状细胞(he-patic stellate cell,HSC)增殖,磷酸化细胞外信号调节蛋白激酶1/2(phosphorylated extracellularsignal regulated kinase 1/2,p-ERK1/2)表达的影响,探讨Art抗肝纤维化的分子机制。方法:实验分为对照组、PDGF-BB组、PDGF-BB+Art组和PDGF-BB+PD98059组。以PDGF-BB诱导HSC增殖,再分别加入不同浓度的Art和ERK1/2抑制剂PD98059。CCK-8法检测各组HSC细胞的增殖情况,ELISA法测定细胞上清液中Ⅰ型胶原的含量,免疫印迹(Western blot-ting)法检测各组细胞ERK1/2的表达及活化情况。结果:PDGF-BB可诱导HSC细胞增殖,p-ERK1/2表达增高,而PDGF-BB+Art组、PDGF-BB+PD98059组HSCⅠ型胶原的含量及p-ERK1/2活性均降低。结论:ERK1/2参与了PDGF-BB诱导HSC细胞增殖的作用,Art抗PDGF-BB所诱导的HSC细胞增殖作用与其抑制ERK1/2的活化有关。     

黄连素通过诱导G0/G1或G2/M期阻滞抑制前列腺癌细胞株PC-3的增殖作用分析

目的 观察黄连素在体外对前列腺癌细胞株PC-3的抑制及凋亡的诱导,并进一步探讨其作用机制.方法 采用MTT法检测不同浓度黄连素对前列腺癌细胞株PC-3的增殖抑制作用,采用流式细胞术(FCM)检测细胞的凋亡,通过PI染色法结合流式细胞仪检测黄连素对前列腺癌细胞株PC-3细胞周期的影响;蛋白免疫印迹(WB)方法检测黄连素作...     

Geldanamycin induces G2 arrest in U87MG glioblastoma cells through downregulation of Cdc2 and cyclin B1

Cell cycle progression requires precise expression and activation of several cyclins and cyclin-dependent kinases. Geldanamycin (GA) affects cell cycle progression in various kinds of cells. We analyzed GA-induced cell cycle regulation in glioblastoma cells. GA-induced G2 or M arrest in glioblastoma cells in a cell line-dependent manner. GA decreased the expression of Cdc2 and cyclin B1 in U87MG cells. And phosphorylated Cdc2 decreased along with Cdc2 in the GA-treated cells. This cell line showed G2 arrest after GA treatment. In contrast, GA failed to down-regulate these cell cycle regulators in U251MG cells. In U251MG cells, the cell cycle was arrested at M phase in addition to G2 by GA. Next, we analyzed the mechanism of the GA-induced regulation of Cdc2 and cyclin B1 in U87MG cells. Cdc2 and cyclin B1 were ubiquitinated by GA. MG132 abrogated the GA-induced decrease of Cdc2 and cyclin B1 indicating that these proteins were degraded by proteasomes. In conclusion, GA controls the stability of Cdc2 and cyclin B1 in glioblastomas cell species-dependently. Cdc2 and cyclin B1 might be responsible for the different responses of glioblastoma cell lines to GA.     

Berberine induces G1 arrest and apoptosis in human glioblastoma T98G cells through mitochondrial/caspases pathway

Berberine is an isoquinoline plant alkaloid with a long history of being used for the treatment of many diseases in Chinese herbal medicine. Berberine has a wide range of biochemical and pharmacological effects, including antitumor activities, but its mechanism of action is not clearly understood. In this study, we investigated that the relationship between the antiproliferative activities of berberine and the apoptotic pathway associated with its molecular mechanism of action in human glioblastoma T98G cells. Berberine treatment of T98G cell lines inhibited cell proliferation and induced cell death in a dose (50-200 microg/ml) dependent manner with an IC50 value of 134 microg/ml, which was associated with an increase in G1 arrest. Western blot analysis showed that the berberine-induced G1 arrest was mediated through the increased expression of P27 and the decreased expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D, and cyclin E proteins. Berberine treatment also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of the Bax/Bcl-2 proteins, the disruption of mitochondrial membrane potential, and the activation of procaspase-9, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Berberine can inhibit T98G cell proliferation by inducing G1 arrest and apoptosis. These results demonstrate that the berberine-induced apoptosis of T98G cells is primarily mediated through the mitochondrial/caspases-dependent pathway.     

Preparation and characterization of teniposide PLGA nanoparticles and their uptake in human glioblastoma U87MG cells

Many studies have demonstrated the uptake mechanisms of various nanoparticle delivery systems with different physicochemical properties in different cells. In this study, we report for the first time the preparation and characterization of teniposide (VM-26) poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and their cellular uptake pathways in human glioblastoma U87MG cells. The nanoparticles prepared with oil-in-water (O/W) single-emulsion solvent evaporation method had a small particle size and spherical shape and provided effective protection against degradation of teniposide in PBS solution. Differential scanning calorimeter (DSC) thermograms concluded that VM-26 was dispersed as amorphous or disordered crystalline phase in the PLGA matrix. A cytotoxicity study revealed that, in a 24h period, blank PLGA NPs had no cytotoxicity, whereas teniposide-loaded PLGA NPs (VM-26-NPs) had U87MG cytotoxicity levels similar to free teniposide. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) images showed the distribution and degradation processes of nanoparticles in cells. An endocytosis inhibition test indicated that clathrin-mediated endocytosis and macropinocytosis were the primary modes of engulfment involved in the internalization of VM-26-NPs. Our findings suggest that PLGA nanoparticles containing a sustained release formula of teniposide may multiplex the therapeutic effect and ultimately degrade in lysosomal within human glioblastoma U87MG cells.     

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.     

Punicalagin induces apoptotic and autophagic ce death in human U87MG glioma cells

Aim： To investigate the effects of punicalagin, a polyphenol isolated from Punica granatum, on human U87MG glioma cells in vitro. Methods： The viability of human U87MG glioma cells was evaluated using MTT assay. Cell cycle was detected with flow cytometry analysis. The levels of Bcl-2, cleaved caspase-9, cleaved poly（ADP-ribose） polymerase （PARP）, phosphor-AMPK and phosphor-p27 at Thr198 were measured using immunoblot analyses. Caspase-3 activity was determined with spectrophotometer. To determine autopha~Lv, LC3 cleavage and punctate patterns were examined. Results： Punicalagin （1-30 pp=VmL） dose-dependently inhibited the cell viability in association with increased cyclin E level and decreased cyclin B and cyclin A levels. The treatment also induced apoptosis as shown by the cleavage of PARP, activation of caspase-9, and increase of caspase-3 activity in the cells. However, pretreatment of the cells with the pan-caspase inhibitor z-DEVD- fmk （50 pmol/L） did not completely prevent the cell death. On the other hand, punicalagin treatment increased LC3-11 cleavage and caused GFP-LC3-11-stained punctate pattern in the cells. Suppressing autopha~, of cells with chloroquine （1-10 pmol/L） dose- dependently alleviated the cell death caused by punicalagin. Punicalagin （1-30 pp=VmL） also increased the levels phosphor-AMPK and phosphor-p27 at Thr198 in the cells, which were correlated with the induction of autophagic cell death. Conclusion： Punicalagin induces human U87MG glioma cell death through both apoptotic and autophagic pathways.     

Anti-proliferative properties of commercial Pelargonium sidoides tincture,with cell-cycle G0/G1 arrest and apoptosis in Jurkat leukaemia cells

Context Pelargonium sidoides DC (Geraniaceae) is an important medicinal plant indigenous to South Africa and Lesotho. Previous studies have shown that root extracts are rich in polyphenolic compounds with antibacterial, antiviral and immunomodulatory activities. Little is known regarding the anticancer properties of Pelargonium sidoides extracts.

Objective This study evaluates the anti-proliferative effects of a Pelargonium sidoides radix mother tincture (PST).

Materials and methods The PST was characterized by LC-MS/MS. Anti-proliferative activity was evaluated in the pre-screen panel of the National Cancer Institute (NCI-H460, MCF-7 and SF-268) and the Jurkat leukaemia cell line at concentrations of 0–150?μg/mL. The effect on cell growth was determined with sulphorhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 72?h. The effect on cell cycle and apoptosis induction in Jurkat cells was determined by flow cytometry with propidium iodide and Annexin V: fluorescein isothiocyanate staining.

Results Dihydroxycoumarin sulphates, gallic acid as well as gallocatechin dimers and trimers were characterized in PST by mass spectrometry. Moderate anti-proliferative effects with GI₅₀ values between 40 and 80?μg/mL were observed in the NCI-pre-screen panel. Strong activity observed with Jurkat cells with a GI₅₀ value of 6.2?μg/mL, significantly better than positive control 5-fluorouracil (GI₅₀ value of 9.7?μg/mL). The PST arrested Jurkat cells at the G₀/G₁ phase of the cell cycle and increased the apoptotic cells from 9% to 21%, while the dead cells increased from 4% to 17%.

Conclusion We present evidence that P. sidoides has cancer cell type-specific anti-proliferative effects and may be a source of novel anticancer molecules.     

Exploring the cell death mechanisms of cytotoxic [1,2,3]triazolylcarborane lead compounds against U87 MG human glioblastoma cells

Moving towards high-grade glioma drug discovery, this study aimed to detect the mechanism of cellular death (apoptosis, necrosis and/or autophagy) induced by three carboranyl-based lead compounds. For that, we performed in U87 MG cells, flow cytometry experiments, as the gold standard technique, as well as confocal microscopy and ¹H-NMR experiments as non-invasive assays. We selected three hybrid leads ( 1–3 ) from the in-house-library and the corresponding parent compounds, and recognized tyrosine kinase inhibitors (lapatinib, sunitinib and erlotinib) to put to the test in these experiments. Flow cytometry with Annexin V-FITC/DAPI staining showed that leads 1 and 3 and lapatinib mainly induced necrosis in U87 MG upon a 24 h treatment at IC₅₀ dose; meanwhile, hybrid 2 , sunitinib and erlotinib seem to induce apoptosis in such cells. In general, confocal microscopy studies were in agreement with flow cytometry observing loss of cell membrane integrity in necrotic cells and features of apoptosis, that is, chromatin condensation, in apoptotic cells. Finally, NMR results showed that glioblastoma cells treated with hybrid 1 , 3 or lapatinib displayed changes in CH₂/CH₃ signal ratio and choline signals that could indicate necrotic cell death mechanism: meanwhile, 2 -, sunitinib- or erlotinib-treated cells showed apoptotic characteristic behaviors. Additionally, carboranyl-hybrid 2 also produced autophagy in U87 MG cells.