倍半萜烯内酯对鼻咽癌细胞内核因子-кB活化的影响

背景与目的:探讨倍半萜烯内酯化合物对人鼻咽癌(nasopharyngeal carcinoma,NPC)细胞内核因子-кB(NF-кB)信号转导活化的影响.材料与方法:采用小白菊内酯(parthenolide,PN)作为受试物,以对PN诱导转归敏感的CNE1细胞给予TNF-α预诱导建立模型,PN处理后提取胞质和胞核蛋白,分别检测IкBα降解、NF-кB p65亚单位活化后核内迁移,电泳迁移率改变试验(EMSA)检测核内活化NF-кB的DNA结合活性.进行PN剂量和作用时间依赖关系分析.结果:阴性对照组IкBα蛋白存在于胞质中,PN处理组使TNF-α诱导的胞质IкBα蛋白降解被抑制、胞核内蛋白含量减少;相应地,阴性对照组p65亚单位在胞质中含量高于胞核内,PN处理组抑制TNF-α诱导的胞质p65核转位;同步进行的EMSA可见,PN处理组NF-кB核结合活性比,TNF-α诱导组明显降低.随PN处理时间(0.5～4h)和剂量(5～25 μmol/L)增加,胞质中IкBα蛋白降解的抑制作用增强(其蛋白含量增加),胞核内p65亚单位蛋白减少,EMSA结合活性降低,呈明显的剂量和时间依赖性(P均＜0.05).结论:PN可影响NPC细胞内NF-кB因子的活化,提示PN对TNF-α诱导NF-кB信号的抑制作用可能是PN诱导NPC细胞凋亡敏感性的分子机制之一.     

NF-κB在TNF-α诱导的Hela细胞凋亡中的作用

目的：探讨核因子-κB(nuclear factorkappa B，NF-κB)对肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)诱导的宫颈癌Hela细胞凋亡的影响。方法：培养宫颈癌Hela细胞。不同处理因素按指定时间作用后，RT一PCR检测NF-κB p65 mRNA的表达。免疫印迹和免疫组化检测NF-κB p65活性的变化，TUNEL法检测细胞凋亡。结果：静息状态下。在Hela细胞中NF-κB p65具有微弱活性水平。TNF-α诱导Hela细胞NF—κB活化与其诱导细胞凋亡明显相关，NF-κB p65单抗能显著抑制TNF-α(10ng／mL)诱导的Hela细胞NF-κB活化，抑制率为49．69％，并增强其诱导Hela细胞凋亡的作用，凋亡增加率为57．52％。结论：TNF-α在诱导宫颈癌Hela细胞凋亡的同时活化NF-κB。NF-κB p65单抗可通过抑制NF-κB活化以增强TNF-α诱导Hela细胞凋亡的作用。     

核转录因子κBp65蛋白及其抑制蛋白IκBα表达与食管鳞癌浸润转移的关系

目的:探讨核转录因子κB(NF-κB)p65蛋白及其抑制蛋白IκBα表达与食管鳞癌浸润转移的关系.方法:用免疫组化SP法检测了61例食管鳞癌中NF-κBp65、IκBα蛋白的表达.结果:p65、IκBα蛋白的胞浆表达与食管鳞癌的组织学分级、浸润深度和淋巴结转移无关,但p65蛋白的胞核表达与食管鳞癌的组织学分级、浸润深度和淋巴结转移均呈正相关(P值均＜0.01),IκBα蛋白的胞核表达与食管鳞癌的淋巴结转移有关(P＜0.05).在食管鳞癌中IκBα蛋白的胞浆和胞核阳性率均高于p65蛋白,但差异均无统计学意义,也无相关关系.结论:NF-κBp65蛋白的胞核表达与食管鳞癌的分级、浸润和淋巴结转移有关,IκBα蛋白的胞核表达与食管鳞癌的淋巴结转移有关.     

食管鳞癌及癌旁粘膜中核转录因子κB p65及IκBα基因和蛋白表达及意义

目的:探讨核转录因子κB(NF-κB)p65基因及其抑制基因IκBα的表达与食管鳞癌发生的关系。方法:用RT-PCR及免疫组化SP法检测了61例食管鳞癌及其正常粘膜上皮、癌旁粘膜上皮中NF-κBp65基因、IκBα基因mRNA及蛋白的表达。结果:食管鳞癌中p65mRNA、IκBαmRNA的表达量明显高于正常上皮(P<0.01),p65蛋白、IκBα蛋白胞浆表达或胞核表达率均明显高于正常上皮(P<0.01)。癌旁粘膜中,NF-κBp65蛋白、IκBα蛋白在癌旁正常上皮中的胞浆或胞核阳性率均明显低于不典型增生上皮、原位癌和浸润癌(P均<0.05)。结论:NF-κBp65、IκBαmRNA表达和蛋白表达均与食管鳞癌的发生有关。     

HCV C 蛋白调控肝门部胆管癌细胞中NF-κB活性的研究

目的探讨HCV C蛋白在肝门部胆管癌细胞中对核转录因子κB(NF-κB)调控作用.方法利用稳定表达HCV C蛋白的QBC939细胞株(QBC939 HCV C ),通过NF-κB报道基因分析法、凝胶迁移率分析(EMSA)检测HCV C蛋白增强肝门部胆管癌细胞NF-κB的DNA结合活性和反式激活活性.RT-PCR、Western blot检测HCV C蛋白对核转录因子κB抑制蛋白α(IκBα)mRNA及p50、p65、IκBα蛋白表达及IκBα蛋白磷酸化的影响.结果①EMSA结果显示QBC939HCV C 细胞系中NF-κB相对信号密度明显比QBC939细胞高.②将pNF-κB-lun表达质粒转染稳定表达HCV C蛋白胆管癌细胞系中,通过单光子检测仪检测荧光素酶活性,结果显示QBC939HCV C 细胞中活化NF-κB为12.8倍,而QBC939细胞中NF-κB为2.6倍.③RT-PCR显示IκBα mRNA在QBC939 HCV C 细胞和QBC939细胞中的表达无明显差异;Western blot结果显示稳定表达HCV C蛋白的QBC939 HCV C 细胞株的胞核中的p50、p65蛋白高水平表达,而未转染HCV C蛋白的QBC939细胞仅检测出极低水平的p50、p65蛋白.在QBC939HCV C 细胞胞浆中IκBα蛋白低水平表达,QBC939细胞高水平表达,但IκBα蛋白磷酸化水平则相反.结论HCV C蛋白通过增加IκBα降解激活的NF-κB在肝门部胆管癌的发生中起重要作用.     

食管鳞癌细胞中NF-κB与IκBα的mRNA、蛋白表达

目的：核转录因子NF—kappaB（NF—κB）是一种重要的转录因子，参与调控多种与炎症、抗凋亡、肿瘤形成和转化有关的基因表达。本研究通过检测食管鳞癌细胞中NF—κB与IκBαmRNA、蛋白的表达及NF—κB的DNA结合活性．分析NF—κB在食管鳞癌细胞中的激活状态及其在食管鳞癌细胞的生存及转化中的作用。方法：采用Western blotting法检测两株食管鳞癌细胞胞质中NF—κB亚单位p50和p65、阻遏物IKBα及其磷酸化IKBα的蛋白表达．检测细胞核中p50和p65的蛋白表达并采用EMSA法检测其与DNA的结合活性。使用RT-PCR法检测p50、p65、IκBαmRNA的表达：结果：NF—κB的亚单位p50、p65、IκBα及磷酸化IκBα在食管鳞癌细胞质中均表达．p50、p65蛋白在细胞核中也表达并具有较高的DNA结合活性.RT-PCR结果表明．p50、p65、IκBα的mRNA在细胞中也存在过表达。结论：本研究发现NF—κ在两株食管鳞癌细胞系中被激活，基因的过表达及IκBα的磷酸化在维持NF—κB的活化中起着重要的作用．     

NF-κB在TNF-α诱导的Hela细胞凋亡中的作用

目的探讨核因子-κB(nuclear factor-kappa B,NF-κB)对肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)诱导的宫颈癌Hela细胞凋亡的影响。方法培养宫颈癌Hela细胞,不同处理因素按指定时间作用后,RT-PCR检测NF-κBp65mRNA的表达,免疫印迹和免疫组化检测NF-κB p65活性的变化,TUNEL法检测细胞凋亡。结果静息状态下,在Hela细胞中NF-κB p65具有微弱活性水平,TNF-α诱导Hela细胞NF-κB活化与其诱导细胞凋亡明显相关,NF-κB p65单抗能显著抑制TNF-α(10ng/mL)诱导的Hela细胞NF-κB活化,抑制率为49.69%,并增强其诱导Hela细胞凋亡的作用,凋亡增加率为57.52%。结论TNF-α在诱导宫颈癌Hela细胞凋亡的同时活化NF-κB。NF-κB p65单抗可通过抑制NF-κB活化以增强TNF-α诱导Hela细胞凋亡的作用。     

EGCG影响TNF-α诱导的NF-κB/p65入核及促凋亡效应

背景与目的:研究表明,表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate, EGCG)抗肿瘤作用机制尚不十分清楚,本研究旨在观察EGCG调节TNF-α诱导的胃癌SGC-7901细胞核转录因子κB/p65(NF-κB/p65)的入核并发挥诱导凋亡的作用.方法:Hoechst染色法检测EGCG处理SGC-7901细胞前后凋亡的形态学改变;Western blot方法观察EGCG作用前后NF-κB/p65蛋白在胞质内及核内的变化;流式细胞术检测EGCG调节NF-κB/p65入核前后细胞凋亡的变化;DNA ladder方法观察EGCG诱导细胞DNA断裂情况.结果:Hoechst染色法显示EGCG处理后细胞形态改变:核致密浓染或呈碎块状、苍白色.Western blot方法检测发现,10 ng/ml的TNF-α作为NF-κB/p65入核的诱导剂分别作用细胞30、60、90和120 min后,核内NF-κB/p65明显增多并在60 min达高峰;60 μg/ml的EGCG预处理细胞不同时间(0、12、24、36和48 h)后,再以 TNF-α处理60 min,检测发现核内NF-κB/p65明显下降并在24 h达低谷; 不同浓度EGCG(40、60、80和100 μg/ml) 预处理细胞24 h后,再加TNF-α处理60 min,核内NF-κB/p65呈时间依赖性下降.流式细胞术显示,10 ng/ml的TNF-α处理细胞60 min后,细胞凋亡率为3.7%,60 μg/ml EGCG处理细胞24 h后再以TNF-α处理细胞,凋亡率为16.6%;NF-κB通路阻断剂吡咯啉烷二甲基硫脲 (PDTC) 100 μmol/L预处理30 min后再以TNF-α处理细胞60 min,细胞凋亡率为21.4%.EGCG 60 μg/ml处理细胞24 h、PDTC处理细胞30 min后再以TNF-α处理细胞60 min,DNA凝胶电泳出现明显梯形条带.结论:EGCG能够抑制TNF-α诱导的NF-κB/p65入核,并可能通过此机制发挥诱导细胞凋亡的作用.     

NF-κB信号通路在食管鳞癌细胞系中的激活

目的核转录因子NF-kappaB(NF-κB)是一种重要的转录因子,参与调控多种与炎症、抗凋亡、肿瘤形成和转化有关的基因表达。本研究拟通过检测NF-κB信号通路在食管鳞癌细胞系中是否存在,分析NF-κB在食管鳞癌细胞中的激活状态及其对食管鳞癌细胞的生存及转化作用。方法采用免疫细胞化学法和Western blotting法检测两株食管鳞癌细胞系中NF-κB亚单位p50和p65,阻遏物IκBα及其上游激酶IKKβ的蛋白表达。并采用EMSA法检测两株食管鳞癌细胞核中NF-κB与DNA的结合活性。结果食管鳞癌细胞中存在激活的NF-κB信号通路,p50、p65、IκBα及其上游激酶IKKβ的蛋白在细胞质中均表达,并且p50/p65在细胞核中具有较高的DNA结合活性。结论本研究发现NF-κB信号通路在两种食管鳞癌细胞系被激活,提示激活的NF-κB信号通路可能在食管鳞癌发生中起重要作用。     

三氧化二砷诱导K562细胞凋亡过程中IκB-α,NF-κB蛋白表达的研究

目的探讨三氧化二砷(As2O3)诱导慢性粒细胞性白血病K562细胞凋亡过程中IκB-α以及NF-κB的表达变化.方法应用不同剂量As2O3诱导K562细胞凋亡,以Western-Blot免疫印迹电泳检测NF-κB,IκB-α的表达,流式细胞术检测K562细胞凋亡及分析IκB-α的阳性表达率变化.结果 As2O3可诱导K562细胞凋亡,随着As2O3浓度从1 μmol/L到4 μmol/L的增高细胞凋亡率由16.0%增加到60.6%,胞浆中IκB-α阳性表达率则由88.1%减少到49.2%,胞核中p65量逐渐增加.结论 As2O3可诱导K562细胞凋亡,在此过程中伴有NF-κB的激活,在一定剂量范围内激活的程度随着砷剂浓度的增加而增强.砷剂激活NF-κB通过其抑制蛋白IκB-α的降解.     

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-KB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI doublelabeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blotting.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

Interference of Fisetin with Targets of the Nuclear Factor-κB Signal Transduction Pathway Activated by Epstein-Barr Virus Encoded Latent Membrane Protein 1

Fisetin is an effective compound extracted from lacquer which has been used in the treatment of variousdiseases. Preliminary data indicate that it also exerts specific anti-cancer effects. However, the manner in whichfisetin regulates cancer growth remains unknown. In this study, we elucidated interference of fisetin with targetsof the nuclear factor κB signal transduction pathway activated by Epstein-Barr virus encoding latent membraneprotein 1 (LMP1)in nasopharyngeal carcinoma (NPC) cells, Results showed that fisetin inhibited the survivalrate of CNE-LMP1 cells and NF-κB activation caused by LMP1. Fisetin also suppressed nuclear translocationof NF-κB (p65) and IκBα phosphorylation, while inhibiting CyclinD1, all key targets of the NF-κB signaltransduction pathway. It was suggested that interference effects of fisetin with signal transduction activated byLMP1 encoded by the Epstein-Barr virus may play an important role in its anticancer potential.     

Bioimaging analysis of nuclear factor-κB activity in Philadelphia chromosome-positive acute lymphoblastic leukemia cells reveals its synergistic upregulation by tumor necrosis factor-α-stimulated changes to the microenvironment

To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.     

An RNAi screen identifies USP2 as a factor required for TNF-α-induced NF-κB signaling

Tumor necrosis factor α (TNF-α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin-dependent processes are central to the regulation of TNF-α and nuclear factor κB (NF-κB) signaling. We performed a targeted siRNA screen for ubiquitin-specific proteases (USPs) and identified USP2 as a modulator of TNF-α-induced NF-κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes and IL-8 secretion. Our study also provides evidence for isoform-specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together, our results implicate USP2 as a novel positive regulator of TNF-α-induced NF-κB signaling and show that its expression is altered in tumor cells.     

Combination treatment using adenovirus vector-mediated tumor necrosis factor-alpha gene transfer and a NF-κB inhibitor for pancreatic cancer in mice

Gene therapy using an adenoviral vector expressing tumor necrosis factor-alpha (TNF-α) is a new therapeutic approach for pancreatic cancer. However, the efficacy of TNF-α is limited, because it activates nuclear factor-κB (NF-κB). We investigated the combined use of AxCAhTNF-α, adenoviral vector-expressing human TNF-α, and nafamostat mesilate, a serine-protease inhibitor, a NF-κB inhibitor, using pancreatic cancer in mice. In vitro, nafamostat mesilate inhibited TNF-α-induced NF-κB activation and enhanced TNF-α-induced apoptosis in human cancer cell line (MIAPaCa-2). In vivo, we assessed combination treatment of AxCAhTNF-α and nafamostat mesilate using xenograft models in nude mice by subcutaneous injection of MIAPaCa-2. When the implanted tumor size reached 8.0mm, TNF-α group (n=12), was injected AxCAhTNF-α intra-tumorally once a week, while combination group (n=12), was injected AxCAhTNF-α intra-tumorally once a week and nafamostat mesilate intraperitoneally thrice a week. In combination group, tumor growth was significantly slower, and the number of apoptosis cells was significantly greater than those in AxCAhTNF-α group (p<0.05). In conclusion, adenovirus vector-mediated TNF-α gene therapy combined with nafamostat mesilate was effective for pancreatic cancer in mice.