Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P<0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

The Role of IκBα in TNF-α-induced Apoptosis in Hepatic Stellate Cell Line HSC-T6

To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTF methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones （P〈0.01） and it was also true of the inhibition rate （P〈0.01）. It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.     

Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-κB signaling pathway in nasopharyngeal carcinoma

Objectives To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1(LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-KB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).Methods A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.Results LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMPI. Moreover, LMPl-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the KB site KB1 or KB5 was disrupted,whereas mutation of κB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.Conclusion LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-κB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.     

Adenovirus-mediated overexpression of novel mutated IκBα inhibits nuclear factor κB activation in endothelial cells

Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.     

Expression of Nuclear Factor-κB in Mouse Uterus during Peri-implantation

To investigate the expression of the subunit p65 of NF-κB and inhibitor kappa B alpha (IκBα) in mouse uterus during peri-implantation, thereby investigating whether transient activation of nuclear factor-κB (NF-κB) takes place during embryo implantation in mice. Immunohistochemical technique was used to examine the expression and localization of p65 in endometrium or deciduas, and Western blot analysis was employed to detect the levels of IκBα protein in mouse endometrium or deciduas. P65 protein was detected in stromal cells, epithelial cells of endometrium as well as in myometrium. Staining was predominately seen in the cytoplasm of the cells. Staining intensity for p65 was stronger in the epithelial compartment than the stromal compartment and myometrium. Staining intensity increased slightly during pregnancy, and it reached a high level on pregnancy day 5 and day 8. In contrast to p65, the level of IκBα protein was lowest on pregnancy day 5 in all groups. Our results suggested that NF-κB may regulate embryo implantation by its transient activation in mice.     

EB病毒LMP1在鼻咽癌细胞中通过IκBα的降解活化NF-κB

目的　阐明鼻咽癌细胞中EB病毒编码的潜伏膜蛋白 1(LMP1)活化核转录因子NF κB的机制。方法　利用强力霉素Dox诱导表达LMP1的鼻咽癌细胞株Tet on LMP1 HNE2为实验模型 ,首先应用免疫印迹方法测定Dox诱导后不同时相LMP1的表达动力学以及IκBs蛋白量及功能的改变。进而用间接免疫荧光法检测NF κB的亚细胞定位。最后采用瞬间共转染及报道基因活性分析分析NF κB的活性。结果　在鼻咽癌细胞Tet on LMP1 HNE2中 ,Dox处理 15分钟后LMP1的表达迅速升高并维持与较高水平直至 12 0分钟。LMP1的诱导性表达导致IκBα的磷酸化并降解 ,但IκBα蛋白总量无改变。继IκBα的磷酸化并降解 ,NF κB(P6 5 )自胞浆易位至胞核且活性升高。IκBα的显性负性突变子抑制NF κB(P6 5 )的核易位及报道活性。LMP1的诱导性表达并未引起IκBβ蛋白水平变化。结论　在鼻咽癌细胞Tet on LMP1 HNE2中 ,EB病毒LMP1通过IκBα的磷酸化并降解激活核转录因子NF κB的活性 ,并且 ,LMP1诱导的NF κB活性能被IκBα的显性负性突变子完全抑制。IκBβ在此信号传导途径中无改变。LMP1表达前后IκBα蛋白总量维持恒定可能是由于NF κB的活化迅速启动了IκBα的重头合成这一自身调节环路所致。     

EB病毒LMP1促进Tet-on-LMP1 HNE2鼻咽癌细胞增殖

目的探讨EB病毒编码的潜伏膜蛋白1（LMP1）对Tet-on-LMP1 HNE2鼻咽癌细胞生物学效应的影响.方法采用Western blot方法、流式细胞术DNA含量分析法、细胞生长曲线、软琼脂集落形成试验和四唑盐（MTT）比色法检测不同剂量的DOX诱导下Tet-on-LMP1 HNE2细胞在不同时间点的增殖效应.结果在LMP1表达逐渐增强的情况下,Tet-on-LMP1 HNE2细胞由G0/G1期加速向S期的行进;细胞生长速度逐渐加快（P〈0.05）;软琼脂集落形成率逐渐上升（P〈0.05）;细胞吸光度值逐渐升高（P〈0.05）.结论EB病毒LMP1促进Tet-on-LMP1 HNE2鼻咽癌细胞增殖.     

核转录因子κB及其抑制基因在自体移植静脉中的表达

目的　研究核转录因子 κB (NF κB)及其抑制因子IκB基因在自体移植静脉中的表达 ,及其在内膜增生中的作用。方法　Wistar大鼠 80只 ,建立自体移植静脉模型 ,术后随机分为 6、2 4h ,3、7d ,2、4、6、8周等 8组 ,于相应时点取材 ,进行病理形态学研究 ,半定量逆转录PCR检测NF κBp6 5mRNA和IκBβmRNA表达的变化 ,原位杂交方法检测NF κBp6 5mRNA表达 ,联合应用Western蛋白印迹和免疫组织化学方法检测p6 5、IκBα和IκBβ的蛋白质表达。 结果　移植静脉术后 6h即出现p6 5mRNA和IκBβmRNA表达增强 ,基因表达值分别为 16 %± 4 %和 31%± 9% ,与正常静脉比较差异有非常显著意义 (P <0 0 1) ;p6 5mRNA表达 3～ 7d达高峰 ,术后 3、7d的表达值分别为 37%± 12 %和34%± 10 % ,IκBβmRNA表达则于术后 1～ 2周达到高峰 ,表达值分别为 5 3%± 17%和 4 9%± 10 % ,与其余各时点比较差异有非常显著意义 (P <0 0 1)。p6 5蛋白质阳性表达 1周达高峰 32 %± 13% ,IκBα、IκBβ蛋白质术后 2周达高峰 ,阳性率分别为 35 %± 11%和 4 4 %± 13% (P <0 0 1)。IκBα、β蛋白质术后 6～ 2 4h表达量有所降低 ,为正常静脉的 1/3～ 1/2。结论　移植静脉中存在NF κB系统及其抑制因子IκB基因的活化。核转录因子κB可能     

核因子—kB调控白细胞介素1β刺激人肾小球系膜细胞表达细胞间粘 …

目的 研究因子－ｋＢ（ＮＦ－ｋＢ）对白细胞介素１β（ＩＬ－１β）引起的体外培养的人肾小球系膜细胞表达细胞间粘附分子（ＩＣＡＭ－１）的基因调控机理。方法 采用凝胶迁移率法观测ＮＦ－ｋＢ的活化。以Ｎｏｒｔｈｅｒｎ杂交方法检测细胞间粘附分子的基因表达,用细胞ＥＬＩＳＡ法检测ＩＣＡＭ－１蛋白水平的表达。结果 ｒｈＩＬ－１β１０ｎｇ／ｍｌ刺激肾小球系膜细胞１,２,４小时,均引起ＮＦ－ｋＢ活化,且１小时为最强     

抑制核转录因子κBp65对鼻咽癌CNE2细胞的生物学影响

目的  探讨核转录因子κBp65（NF-κBp65）沉默对鼻咽癌的生物学作用。方法  构建NF-κBp65特异性小干扰核糖核酸（siRNA）质粒表达载体（pGPU6/RFP/Neo-NF-κBp65）和阴性对照质粒表达载体（pGPU6/RFP/Neo-NC）。采用非脂质体脂类转染剂将重组质粒表达载体转入人鼻咽癌CNE2细胞,建立NF-κ Bp65沉默的稳定转染CNE2细胞株（NF-κBp65沉默组）和阴性对照细胞株（阴性对照组）;蛋白质印迹法（Western blot）分析癌细胞中NF-κBp65基因的表达水平,MTT实验及流式细胞术分析癌细胞的增殖能力和细胞周期变化;复制裸鼠移植瘤模型,分析沉默NF-κBp65对癌细胞成瘤性的影响。结果  成功获得稳定转染CNE2细胞株,NF-κBp65沉默组细胞中NF-κBp65蛋白表达水平降低;NF-κBp65沉默后,CNE2细胞周期阻滞于S期,增殖速度减慢;NF-κBp65沉默组裸鼠移植瘤生长慢,重量轻,与阴性对照组和空白对照组比较,差异有统计学意义（F =14.386,P <0.05）。结论  NF-κBp65沉默能降低鼻咽癌CNE2细胞的恶性生物学行为。

     

LPS-induced NF-κB activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB in LPS-induced acinar cell injury. Methods Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-κB’s subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P&lt;0.05). NF-κB p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation.     

Gadd45α、NF-κB在胃癌中表达情况及其相关性研究

目的  通过分析Gadd45α、NF-κB在不同幽门螺杆菌（HP）感染状态胃癌中的差异表达来探讨两者表达的相关性及其对胃癌发生的意义。方法  随机选取本院70例胃癌石蜡标本及56例相应癌旁正常组织标本来检测Gadd45α、NF-κB蛋白的表达情况;分析Gadd45α蛋白与NF-κB在胃癌中表达的相关性。结果  NF-κB在HP+胃癌、HP-胃癌、HP+癌旁组织中的阳性表达率分别为82.5%、53.33%和26.32%,Gadd45α蛋白在HP+胃癌、HP-胃癌、HP+癌旁组织中的阳性表达率分别为77.5%、46.67%和34.21%,经过χ2检验比较阳性表达的差异有统计学意义（P <0.05）。Gadd45α与NF-κB在胃癌中表达呈正相关（P <0.05）。结论  NF-κB、Gadd45α蛋白在胃癌组织中高表达,且Gadd45α与NF-κB蛋白两者在胃癌中的表达呈正相关。

     

克雷伯氏肺炎杆菌内毒素诱导小鼠β—防御素—4基因表达及其信号传递

目的 探讨用克雷伯氏肺炎杆菌内毒素（LPS）对小鼠β－防御素表达的影响及其相关的信号传导通路。方法 分别给予C3H／HeJ和C3H／HeN小鼠腹腔注射LPS（4mg/kg),于不同时间点采取气管、肺、肾管组织,提取总RNA。用RT－PCR检测各组织β-防御素－3和／或β-防御素－4mRNA的表达,并对扩增出的cDNA片段进行测序;同步用Western blot法检测该两系小鼠肺脏1－kBa磷酸化情况（p-I-kBa)和I－kBa的量。结果 （1）经LPS处理24小时后的C3H／HeN小鼠,其肺脏表达β-防御素－4mRNA而C3H／HeJ小鼠未见表达。（2）与未给予LPS处理小鼠比较,经LPS处理4小时后,C3H／HeN小鼠肺组织p-kBa含量明显增高;LPS处理后8小时,p-I-kBa以及I-kBa含量均呈减少趋势;至第24小时,p-I-kBa和I－kBa含量均明显减少。而C3H／HeJ小鼠肺组织p-I-kBa及I－kBa含量均未见相应变化。结论 克雷伯氏肺炎杆菌内毒素能诱导C3H／HeN小鼠肺β-防御素－4的表达,其诱导表达作用与Toll受体蛋白－4介导的NF－kB活化级联信号传递通路有关。