MLN4924对紫杉醇耐药的前列腺癌细胞增殖和凋亡的影响

目的:研究MLN4924对紫杉醇耐药的前列腺癌细胞增殖和凋亡的影响,从而为紫杉醇耐药前列腺癌的临床治疗提供新的理论依据。方法:不同浓度的MLN4924作用紫杉醇耐药的前列腺癌细胞PC3-TxR和DU145-TxR 48和72小时后,采用MTS检测细胞活力。Western blot检测凋亡相关蛋白Caspase-3、CDT1、p27以及EMT标志物水平。结果:MLN4924能够显著抑制PC3-TxR和DU145-TxR细胞的增殖,并且具有时间和浓度的依懒性。Western blot结果显示MLN4924作用后显著增加细胞内凋亡相关蛋白Caspase-3、CDT1和p27的表达。结论:MLN4924能够显著抑制紫杉醇耐药的前列腺癌细胞增殖、促进细胞凋亡,其相关机制为MLN4924可通过上调p27蛋白表达促进紫杉醇耐药的前列腺癌细胞凋亡。     

熊果酸对A549细胞增殖、凋亡的影响及其机制

目的探讨三萜酸类化合物熊果酸(UrsolicAcid,UA)对人肺腺癌A549细胞增殖和凋亡的影响及其分子机制。方法利用噻唑蓝(MTT)比色法观察UA对A549细胞增殖的影响。流式细胞仪检测UA对细胞周期的影响。用Hoechst33258荧光染色观察凋亡细胞核形态变化。利用免疫印迹法(Western-blot法)检测细胞中活化型Caspase-3蛋白及NF-κB表达的变化。结果UA明显抑制A549细胞的生长(P<0.05),并呈时间和浓度依赖。流式细胞仪检测发现UA作用24和48h后A549细胞阻滞在G0/G1期(P均<0.05)。经UA处理后的实验组细胞呈现典型的凋亡核固缩表现,胞核呈致密浓染的颗粒状或块状荧光。胞质检测到活性Caspase-3蛋白的表达增强,并呈时间依赖,同时,NF-κB表达随时间延长而减弱。结论熊果酸在体外对A549细胞具有抗增殖和诱导凋亡的作用。核因子NF-κB参与肺癌细胞的增殖与凋亡调控,UA可通过抑制NF-κB活性,激活Caspase-3,从而诱导细胞的凋亡。     

自噬在吉西他滨诱导肺癌细胞A549凋亡中的作用及其相关机制

  目的  研究自噬对吉西他滨(gemcitabine, GEM)诱导的肺癌细胞A549凋亡的影响并探讨其可能机制。  方法  MDC染色后采用荧光显微镜对自噬进行形态观察; 以CCK8检测3-MA抑制自噬前后经吉西他滨诱导的A549细胞存活率; RT-PCR检测自噬相关基因Beclin-1表达的变化。Western blot分别检测自噬相关性蛋白Beclin-l及凋亡活化蛋白Caspase-3的变化。  结果  吉西他滨可诱导肺癌细胞A549产生自噬, 自噬在基因及蛋白水平表达均增加; 3-MA和吉西他滨联合用药可增强肺癌A549细胞凋亡。  结论  吉西他滨诱导肺癌细胞A549凋亡的过程中自噬可能起到保护作用, 3-MA特异性抑制自噬后可增强吉西他滨诱导的A549细胞凋亡。      

阻断NF-κB信号通路对人肝门部胆管癌细胞QBC939增殖的影响

  目的  研究阻断NF-κB信号通路对人肝门部胆管癌细胞QBC939生长增殖的影响。  方法  常规培养QBC939细胞, 采用PDTC(100μmol/L)阻断NF-κB信号通路, Western blot检测核内P65蛋白水平的表达, CCK-8检测细胞生长增殖抑制率, 流式细胞仪观察细胞周期与凋亡的变化情况。  结果  经PDTC阻断NF-κB信号通路后, 与对照组相比, 细胞核内P65蛋白水平明显下降(P < 0.05)。CCK-8检测显示细胞增殖活性明显受到抑制, 并随时间的延长而愈渐明显, 12、24、36h后细胞增殖抑制率分别为(22.53±0.03)%、(46.54±0.03)%、(58.02±0.03)%, 与对照组相比差异有统计学意义(P < 0.05)。流式细胞仪检测发现实验组G₀/G₁期细胞比例高于对照组, 分别为(68.53±1.72)%、(38.3±1.35)%, 差异有统计学意义(P < 0.05);细胞凋亡率在实验组为(27.68±2.77)%, 对照组仅为(9.27±2.36)%, 差异有统计学意义(P < 0.05)。  结论  阻断NF-κB信号通路诱导人肝门部胆管癌细胞G₀/G₁期阻滞及细胞凋亡, 从而抑制细胞生长与增殖, 提示NF-κB信号通路组成性激活参与人肝门部胆管癌的发生发展, 以NF-κB信号通路作为治疗的靶点可能给人肝门部胆管癌带来新的治疗选择。      

沉默泛素结合酶E2O对人非小细胞肺癌细胞增殖和侵袭的影响

  目的  研究泛素结合酶E2O（ubiquitin-conjugating enzyme E2O,UBE2O）蛋白在非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞增殖、细胞周期、迁移、侵袭中的作用。  方法  通过慢病毒介导的shRNA靶向抑制人NSCLC细胞株A549中UBE2O基因的表达,应用CCK-8法、流式细胞术分别检测UBE2O对A549细胞增殖、细胞周期和凋亡的影响,应用Transwell实验检测下调UBE2O表达后对A549细胞迁移、侵袭能力的影响,应用Western blot检测细胞上皮间质转化（epithelial-mesenchymal transition,EMT）两种标志分子E-cadherin蛋白、N-cadherin蛋白和PI3K-Akt信号通路相关蛋白表达量的变化。  结果  下调UBE2O表达后,A549细胞UBE2O mRNA和蛋白表达均下调（均P < 0.01）,CCK-8实验结果显示沉默UBE2O可以抑制A549的增殖（P < 0.001）,Transwell实验显示下调UBE2O的表达能够抑制A549细胞的迁移、侵袭（均P < 0.001）。Western blot结果表明下调UBE2O的表达可使A549细胞中的E-cadherin蛋白表达水平升高、p-Akt蛋白表达水平下降。  结论  UBE2O蛋白能够促进NSCLC细胞侵袭和迁移,作用机制可能是通过诱导肺癌细胞发生EMT和促进pI3K-Akt信号通路的激活。      

BAY11-7082对前列腺癌细胞内NF-κB及其生物学行为的影响

  目的  探讨BAY11-7082对前列腺癌细胞NF-κB表达及其生物学行为的影响。  方法  应用CCK-8方法、蛋白印迹、NF-κB激活-核转运检测, 细胞增殖抑制实验和集落形成实验方法观察BAY11-7082作用下前列腺癌细胞NF-κB蛋白的表达, 细胞生长等指标的变化。  结果  BAY11-7082对前列腺癌细胞转移有明显的抑制作用。BAY11-7082处理的前列腺癌细胞内NF-κB蛋白表达降低, 减弱NF-κB的激活, 细胞增殖和形成集落的能力下降。  结论  BAY11-7082可抑制前列腺癌细胞NF-κB的激活与表达, 提示NF-κB可作为前列腺癌基因治疗的重要靶点之一。      

MLN4924通过抑制类泛素化通路上调人肺腺癌A549细胞中PD-L1的表达

目的:探究MLN4924对肺腺癌A549细胞中程序性死亡-配体1(PD-L1)表达的影响及其可能的内在机制。方法:体外培养A549细胞,按(0、0.25、0.5、0.75、1、5、10μmol/L)MLN4924分组分别处理24、48、72 h后,CCK-8法检测细胞增殖抑制率,作图并计算各组半数抑制浓度(IC₅₀),流式细胞术检测PD-L1的表达,选择最适时间72 h及其IC₅₀用于后续实验。后续实验按(0、0.469μmol/L)MLN4924分组培养72 h, Western blot法检测FBXW2蛋白、肌段同源盒2(MSX2)蛋白、Y-染色体性别决定基因盒2(SOX2)蛋白、PD-L1蛋白表达,qPCR法测定PD-L1 mRNA表达。结果:MLN4924在24、48、72 h的IC₅₀值分别为(1.976、 0.647、0.469)μmol/L;与对照组相比,实验组A549细胞PD-L1表达均上调,呈时间-浓度依赖性。与对照组相比,实验组A549细胞FBXW2、SOX2蛋白表达降低,MSX2蛋白、PD-L1 m...     

肺腺癌穿膜蛋白 125 通过高甲基化介导 NF-κB 信号通路的激活降低对 地西他滨的敏感性

目的：探讨穿膜蛋白125（transmembrane protein125,TMEM125）在肺腺癌组织与A549细胞中的表达,以及影响细胞的增殖和侵袭能力的分子机制。方法：从癌症基因组图谱（the cancer genome atlas,TCGA）数据库收集肺腺癌数据包,下载临床信息及基因表达谱数据。分析 TMEM125在肺腺癌组织中的表达及其与患者总生存期的相关性。构建 TMEM125过表达 A549细胞株,以CCK-8法、细胞划痕实验检测TMEM125过表达对肿瘤细胞的增殖和迁移能力的影响;流式细胞术检测TMEM125过表达对A549细胞的细胞周期和凋亡的影响。WB检测TMEM125过表达对下游NF-κB信号通路、凋亡蛋白的影响;免疫共沉淀法（co-immunoprecipitation,Co-IP）检 测 TMEM125 与 NF- κB 抑 制 因 子 结 合 Ras 样 2（NF- κB inhibitor interacting Ras-like 2,NKIRAS2）的相互作用。利用TNFα（10 ng/ml）处理TMEM125过表达A549细胞,CKK-8、流式细胞术及WB检测其对细胞增殖、凋亡以及NF-κB信号通路相关蛋白表达的影响。去甲基化试剂地西他滨处理A549细胞,qPCR和WB检测TMEM125基因和蛋白的表达。结果：TMEM125 mRNA在肺腺癌组织中表达水平显著低于正常组织（P<0.001）,启动子甲基化水平显著高于正常组织（P<0.001）,并且低、中表达患者总生存期显著低于高表达患者（P<0.001）。过表达TMEM125抑制了A549细胞的增殖和迁移（P<0.01）,增加细胞 G2/M 期,促进细胞凋亡（P<0.01）;过表达 TMEM125 与 NKIRAS2 相互作用,显著抑制 NF-κB 的活性（P<0.01）;地西他滨处理 A549 细胞可促进 TMEM125 表达并且抑制细胞增殖（P<0.01）。结论：启动子高甲基化水平降低了TMEM125基因表达,导致其抑制NF-κB活性功能和抑制细胞增殖的作用下降,并且降低了细胞对地西他滨的敏感性。     

抗MUC1单克隆抗体C595对人卵巢癌0VCAR-3细胞增殖和凋亡的研究

  目的  探讨抗MUCI单克隆抗体C595在体外抑制高表达MUC1的人卵巢癌OVCAR-3细胞增殖并诱导其凋亡的作用。  方法  免疫荧光标记法和流式细胞术检测单克隆抗体C595标记的MUC1在OVCAR-3细胞表面的表达; MTT法、克隆形成试验检测处理后细胞生长抑制情况, TUNEL法检测细胞凋亡, EIISA检测细胞内细胞色素C和Caspase-3活性的变化。  结果  单克隆抗体C595对人卵巢癌OVCAR-3细胞具有明显的增殖抑制作用, 且呈剂量-时间依赖性, TUNEL结果表明C595(IC₅₀)可以诱导细胞凋亡。ELLSA检测显示治疗后的细胞凋亡与释放细胞色素C和Caspase-3浓度的上升相关。  结论  单克隆抗体C595可以在体外抑制人卵巢癌OvCAR-3细胞增殖并诱导其凋亡, 其机制可能与上调Caspase-3蛋白活性有关。      

异甘草素增加人脑胶质瘤干细胞放射敏感性的实验研究

  目的  研究异甘草素对胶质瘤干细胞（glioma stem cells，GSCs）放射敏感性的影响及作用机制。  方法  无血清培养法提取SHG44人脑GSCs。检测异甘草素和X射线联合运用下GSCs活性和干细胞球形成情况。检测Notch1信号通路、NF-κB和cas-pase-3的表达情况。  结果  采用10 μM异甘草素在8 Gy较高剂量X射线存在的情况下，能够抑制GSCs球的数目和直径（P＜0.05）。20 μM异甘草素杀伤GSCs的作用明显，且在4、8 Gy的X射线下联合杀伤GSCs的能力依次增强（P＜00.05）。异甘草素干作用48 h后Notch1的表达下调（P＜00.05）。4 Gy的X射线照射后，分别于6、24 h，异甘草素干预组P-NF-κB表达逐渐增高（P＜00.05），而cleaved caspase-3的表达在X射线照射后的24 h开始升高（P＜00.05）。  结论  异甘草素能够增加GSCs的放射敏感性，抑制Noch1信号通路，上调NF-κB和caspase-3的表达，参与X射线对GSCs的损伤过程。      

Inhibition of proliferation and induction of apoptosis by trimethoxyl stilbene (TMS) in a lung cancer cell line

Trimethoxyl stilbene (TMS) is a derivative of resveratrol, a compound shown to inhibit development of a variety of tumor types. We aimed to evaluate the effect of TMS on cell proliferation and apoptosis in the A549 non-small cell lung cancer cell line. Growth inhibition rate and colony formation was measured and apoptosis was determined with Hoechst 33258 staining. Protein expression levels of caspase-3, STAT3, STAT5b, JAK2, NF-κB, and IκB were examined by Western blotting. Furthermore, localization of NF-κB protein was also explored. TMS inhibited proliferation (IC50 8.6 μmol/L) and induced apoptosis of the cells in a concentration-dependent manner., also inducing apoptosis accompanied by up-regulated expression and cleavage activation of caspase-3, up-regulation of IκB and down-regulation of NFκB, STAT3, STAT5b, and JAK2 signal transduction. TMS has potential as a new drug for treatment of non-small cell lung cancer patients with anti-proliferation and apoptosis inducing effect of TMS to A549 cells apparently related to its inhibitory effect on STATs and NF-κB signal transduction. Up-regulation of caspase-3 further supports the potential clinical use of TMS for the treatment of non-small cell lung adenocarcinoma.     

APE1基因沉默增强骨肉瘤U2-OS细胞硼替佐米治疗敏感性的实验研究

  目的  探讨脱嘌呤/脱嘧啶核酸内切酶1(APE1)基因沉默对蛋白酶体抑制剂硼替佐米(bortezomib, PS-341)抑制骨肉瘤U2-OS细胞增殖作用的影响及其生物学机制。  方法  将APE1特异性shRNA的重组质粒, 稳定转染人骨肉瘤U2-OS细胞, 采用聚合酶链反应和免疫印迹法检测转染前后U2-OS细胞中APE1的表达, 采用四甲基偶氮唑盐法观察PS-341和APE1-siRNA对人骨肉瘤U2-OS细胞生长的抑制作用, 采用免疫印迹法检测PS-341和APE1-siRNA对U2-OS细胞中APE1和胞核NF-λB的表达的影响。  结果  细胞稳定转染APE1-siRNA重组质粒后, APE1mRNA和蛋白表达分别下降约46.1%和62.6%, MTT法检测U2-OS细胞增殖受到抑制。转染前后U2-OS细胞PS-341的IC50值分别为371.54 nmoL/L与109.64 nmoL/L, 两者比较差异有统计学意义(P < 0.01)。Western blot结果显示PS-341和APE1-siRNA均抑制U2-OS细胞胞核中NF-λB的表达, 两者联合应用抑制效果更明显。  结论  APE1-shRNA质粒转染骨肉瘤U2-OS细胞后, 肿瘤细胞的增殖率降低, 对PS-341抑制U2-OS细胞的增殖具有协同作用。      

NF-κB在幽门螺杆菌阳性和阴性胃癌中的差异表达及临床意义

  目的  通过探索NF-κB在胃癌标本中的表达情况, 并结合HP感染状态与临床病理资料进行统计学分析, 探讨NF-κB与HP感染状态对胃癌的发生、发展的意义。  方法  采用免疫组织化学的方法检测随机选取的70例胃癌石蜡标本及56例相应癌旁正常组织标本中NF-κB蛋白的表达情况; Q-PCR方法检测8例活体标本(胃癌与相应癌旁正常组织, 其中4例HP+、4例HP-)中NF-κB mRNA的表达水平, 通过Western blot分析8例活体标本中NF-κB蛋白的表达情况。  结果  NF-κB蛋白在HP+胃癌和HP+癌旁组织中的表达率为82.50%和26.32%(χ2=24.867, P<0.05);NF-κB蛋白在HP+和HP-胃癌组织中的表达率为82.50%和53.33%(χ2=6.94, P < 0.05) NF-κB蛋白在胃癌组织中表达阳性率为70.00%(49/70), 与胃癌的转移、浸润相关, 与病理学分级、肿瘤大小无关。活体标本中, HP+胃癌组织中NF-κB的mRNA和蛋白质表达水平明显高于相应的非癌组织, 而在HP-胃癌组织中的表达水平与相应的非癌组织没有差异。  结论  NF-κB蛋白在HP+胃癌中高表达, 而在HP-胃癌、HP+和HP-的癌旁组织中均为低表达NF-κB蛋白的表达与HP+胃癌发展具有相关性。      

全反式维甲酸诱导人肝癌细胞HepG2凋亡的初步研究

[目的]研究全反式维甲酸（ATRA）体外诱导人肝癌细胞株HepG2的凋亡及其作用机制。[方法]ATRA处理HepG2细胞。MTT法分析细胞增殖反应;Hoechst染色法检测细胞凋亡情况;Western blot检测细胞中caspase-9、caspase-3和NF-κB蛋白表达情况。[结果]A-TRA可抑制HepG2细胞增殖并诱导细胞凋亡,可上调细胞中caspase-9、caspase-3表达水平（P〈0.05）。[结论]ATRA可抑制HepG2细胞增殖并诱导细胞凋亡,其作用机制可能与cas-pase-9、caspase-3表达上调有关。     

Effect of TRAF6 on the biological behavior of human lung adenocarcinoma cell

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor family that mediates both tumor necrosis factor receptor and interleukin-1 receptor/Toll-like receptor signaling. A recent study showed that TRAF6 played an important role in tumorigenesis and invasion through activation of nuclear factor kappa B (NF-κB). However, the biological role of TRAF6 remains unknown in lung cancer up to now. To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) and human bronchial epithelial cells were used to detect the expression of TRAF6 protein by western blotting. Results indicated that TRAF6 displayed an upregulation in human lung cancer cell lines. To investigate the effects of TRAF6 on the biological behavior of human lung adenocarcinoma cell, we generated human lung adenocarcinoma A549 cell line in which TRAF6 was depleted. The results showed that downregulation of TRAF6 could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. At the same time, we explored the effects of TRAF6 on the expression of the following proteins: phosphor-NF-κB (p-p65), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Downregulation of TRAF6 could decrease the expression of p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, and invasion of A549 cell line, as well as the inhibition of A549 cell apoptosis by the activation of NF-κB. To make a long story short, the overexpression of TRAF6 might be related to the tumorigenesis and invasion of lung cancer.     

Targeting protein neddylation with an NEDD8-activating enzyme inhibitor MLN4924 induced apoptosis or senescence in human lymphoma cells

Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma.     

MLN4924, a Novel NEDD8-activating enzyme inhibitor,exhibits antitumor activity and enhances cisplatin-induced cytotoxicity in human cervical carcinoma: in vitro and in vivo study

MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to have activity against various malignancies. Here, we investigated the antitumor properties of MLN4924 and MLN4924 in combination with cisplatin on human cervical carcinoma (CC) in vitro and in vivo. Two human CC cell lines, ME-180 and HeLa, were used in this study. The cytotoxic effects of MLN4924 and/or cisplatin were measured by cell viability (MTT), proliferation (BrdU incorporation), apoptosis (flow cytometry with annexin V-FITC labeling), and the expression of cell apoptosis-related proteins (Western blotting). In vivo efficacy was determined in Nu/Nu nude mice with ME-180 and HeLa xenografts. The results showed that MLN4924 elicited viability inhibition, anti-proliferation and apoptosis in human CC cells, accompanied by activations of apoptosis-related molecules and Bid, Bcl-2 phosphorylation interruption, and interference with cell cycle regulators. Moreover, MLN4924 caused an endoplasmic reticulum stress response (caspase-4, ATF-4 and CHOP activations) and expression of other cellular stress molecules (JNK and c-Jun activations). Additionally, MLN4924 suppressed growth of CC xenografts in nude mice. Furthermore, we demonstrated that MLN4924 potentiated cisplatin-induced cytotoxicity in CC cells with activation of caspases. Consistently with this, MLN4924 significantly enhanced cisplatin-induced growth inhibition of CC xenografts. Together, these findings suggest that MLN4924 alone or in combination with cisplatin is of value in treating human CCs.