EGCG干预LMPl激活的AP-1信号转导通路

目的 探讨EGCG对鼻咽癌细胞激活蛋白1(AP-1)信号转导通路的干预作用，阐明在鼻咽癌细胞中茶多酚干预EB病毒潜伏膜蛋白1(LMPl)活化的AP-1信号转导通路中靶分子的机制。方法 采用EB病毒阴性及阳性的鼻咽癌细胞系CNE1和CNE1-LMP1细胞。利用四甲基偶氮唑蓝(MTT)法观察EGCG对CNE1和CNE1-LMP1细胞的生存率的影响。采用瞬间转染及报道基因法观察EGCG对AP-1活性的作用。利用间接免疫荧光法观察EGCG对JNK核移位的影响，再分别提取CNE1和CNE1-LMP1的胞浆及胞核蛋白，Western blot分析EGCG抑制JNK的核移位后，胞浆及胞核蛋白中JNK的变化。采用Western blot分析EGCG对c-Jun的磷酸化水平的影响。采用瞬间转染及报道基因法观察EGCG对cyclinD1启动子活性的影响，并用Western blot分析EGCG对cyclinD1蛋白表达的作用。结果 EGCG对鼻咽癌细胞的抑制作用有剂量依赖性，并可抑制AP-1的活性。EGCG能抑制JNK的核移位，并抑制c-Jun的磷酸化。EGCG对AP-1信号通路下游的靶基因cyclinD1的启动子活性及其蛋白表达都有抑制作用。结论 EGCG对信号转导通路上的AP-1、JNK、c-Jtm、cyclinD1多个靶点分子具有干预作用。LMP1是EB病毒这种编码的蛋白，因此，EGCG抑制与病毒相联系的信号转导通路，可能是EGCG抑制与病毒相关的肿瘤的分子机制之一。     

EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响

背景与目的:已证实EB病毒编码的潜伏膜蛋白1(latentmembraneprotein,LMP1)具有转化致瘤作用,在鼻咽癌的发生发展中起重要作用。LMP1的转化致瘤作用可能与细胞凋亡有关,本实验目的是观察EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响,探索LMP1在鼻咽癌发生发展中的可能机制。方法:用电转染的方法将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,用荧光显微镜检测报道基因GFP的表达情况;用免疫组化法及Westernblot法检测目的基因LMP1的表达情况;用流式细胞仪、荧光染色及DNA片段分析等方法检测在有地塞米松磷酸钠或无血清饥饿诱导的情况下LMP1对CNE1细胞凋亡的影响。结果:CNE1G细胞(转染空载质粒PAT-GFP的CNE1细胞)及CNE1GL(转染目的基因质粒PAT-GFP-LMP1的CNE1细胞)有绿色荧光蛋白表达,CNE1细胞(未经转染的CNE1细胞)无绿色荧光蛋白表达;CNE1GL细胞LMP1呈阳性表达,而CNE1及CNE1G细胞均为阴性。CNE1、CNE1G及CNE1GL3组细胞分别用地塞米松磷酸钠或无血清1640培养液处理后均有凋亡出现,且两种诱导方法均能使CNE1GL组细胞的凋亡指数(apoptosisindex,AI)较CNE1及CNE1G组明显增高(P<0.05),而CNE1与CNE1G组的凋亡指数无明显差别。3组细胞常规培养下则无凋亡出现。结论:LMP1基因成功导入CNE1细胞     

EB病毒潜伏膜蛋白1通过蛋白激酶C调节Annexin Ⅰ的丝氨酸磷酸化

背景与目的:在利用磷酸化蛋白质富集结合蛋白质组学技术分析EB病毒潜伏膜蛋白1(latent membrane protein 1,LMP1)调节的磷酸化蛋白质分子时,我们发现了包括Annexin Ⅰ在内的25个新的受EB病毒LMP1调节的磷酸化蛋白质分子.本实验探讨LMPl调节Annexin Ⅰ磷酸化的信号通路.方法:采用鼻咽癌细胞系CNE1和LMP1稳定表达的细胞系CNE1-LMP1,Western blot检测Annexi Ⅰ的蛋白表达水平;免疫沉淀结合Western blot检测Annexin Ⅰ的丝氨酸和酪氨酸磷酸化;激酶分析实验检测蛋白激酶C(protein kinase C,PKC)的活性.结果:LMP1对Annexin Ⅰ的蛋白表达水平没有影响,LMP1上调Annexin Ⅰ的丝氨酸磷酸化水平,而对酪氨酸磷酸化水平没有影响.在CNE1细胞中PKC的相对活性为0.97±0.05,CNE1-LMP1细胞中的PKC相对活性为1.22±0.10,CNE1与CNE1-LMP1细胞之间PKC的活性差异有统计学意义(P＜0.01,n=6),表明LMP1可以上调PKC活性.Annexin Ⅰ的丝氨酸磷酸化水平随PKC活性的变化而变化.结论:EB病毒LMP1通过PKC信号通路调节Annexin Ⅰ丝氨酸磷酸化.     

茶多酚诱导鼻咽癌细胞cyclin D1表达下调

目的：探索茶多酚及其单体抑制鼻咽癌增殖的分子机制。方法：应用光学显微镜、MTT测定、流式细胞仪、报告基因和Western blot分析等方法,研究茶多酚及其单体对细胞增殖和细胞周期的影响及分子机理。结果：茶多酚处理鼻咽癌细胞CNE1－LMP124h后,从裂相细胞显著减少,细胞增殖受到抑制,细胞的生存率从100％下降到38．1％（200μg/ml);S期细胞明显减少,从22．20％下降至13．16％（200μg/ml) ,G0/G1细胞所占百分率增加,从68．50％上升至74．08％,细胞出现G1／S阻滞,同时,cyclinD1基因启动子转录活性显著下调,与对照组比下降4－5倍,但相同浓度的茶多酚与（-)-epigallocatechin-3-gallate(EGCG)处理没有显著性差别,cyclinD1蛋白表达水平明显降低,采用荧光酶双报告基因系统分析,发现茶多酚处理12－14h后,AP－1、NFkB反式激活活性均匀显著下降,与对照组（0μg/ml)比分别下降5－6倍、7－8倍,且呈明显量效关系。结论：茶多酚能抑制鼻咽癌细胞CNE－LMP1增殖,茶多酚诱导致cyclinD1基因的转当和表达下调可能在其中起着重要作用。     

鼻咽癌细胞系CNE1和CNE2Z中EBV-LMP 1的表达

目的：探讨人高分化鼻咽癌细胞系CNE1和人低分化鼻咽癌细胞系CNE2Z中EB病毒编码的LMP 1基因表达情况。方法：用免疫组化法及Western bolt法分别检测鼻咽癌细胞系CNE1和CNE2Z中LMP 1基因的表达情况。结果：免疫组化及Western bolt法均显示,CNE2Z细胞中LMP 1呈强阳性表达,而CNE1细胞中LMP 1呈阴性表达。结论：人高分化鼻咽癌细胞系CNE1中没有LMP 1的表达而人低分化鼻咽癌细胞系CNE2Z中LMP 1呈阳性表达,鼻咽癌的恶性程度可能与LMP 1的表达有关。     

EB病毒LMP1对人鼻咽癌细胞转移能力的影响

背景与目的:EB病毒编码的潜伏膜蛋白1(latent membrane protein 1,LMP1)在鼻咽癌的浸润和转移过程中起重要的作用。本实验目的在于研究EB病毒LMP1对人鼻咽癌细胞转移能力的影响和探讨其中的相关机制。方法:应用免疫细胞化学RT-PCR法和Western blot检测LMP1、E-cadherin和intercellular adhesion molecule-1 (ICAM-1)在人高分化鼻咽癌细胞系CNE1和转染了LMP1基因的CNE1-GL细胞中的表达。应用细胞-细胞粘附实验、细胞-基质粘附实验、划痕实验和细胞运动实验观察LMP1对鼻咽癌细胞粘附和运动能力的影响。结果:免疫细胞化学结果显示:LMP1在CNE1和CNE1-GL细胞中的阳性率分别为0和(96.60±3.03)%(P<0.01);E-cadherin蛋白的阳性率分别为(37.47±1.50)%和(19.53±1.92)%(P<0.01);ICAM-1蛋白的阳性率分别为(5.27±1.45)%和(93.33±4.23)%(P<0.01)。RT-PCR和Westernblot结果表明,与CNE1细胞相比较,CNE1-GL细胞中E-cadherin的mRNA和蛋白表达明显降低(P<0.01),而ICAM-1的mRNA和蛋白表达则明显升高(P<0.01)。肿瘤细胞同质粘附实验显示,CNE1-GL细胞的同质粘附能力较CNE1细胞弱(P<0.05)。肿瘤细胞-基质粘附实验得出CNE1-GL细胞的异质粘附能力(A值=0.60±0.03)较CNE1细胞(A值=0.46±0.01)强(P<0.01)。肿瘤细胞运动实验显示,穿过PVPF膜的CNE1-GL细胞数多于CNE1细胞(119.3±6.0 vs 46.3±7.0,P<0.05)。结论:LMP1通过抑制E-cadherin表达、促进ICAM-1表达从而抑制肿瘤细胞的同质粘附能力、增强其异质粘附能力和运动能力来参与鼻咽癌的侵袭和转移。     

EB病毒潜伏膜蛋白1对鼻咽癌CNE1细胞增殖的影响

目的探讨EB病毒潜伏膜蛋白1（LMP1）对人高分化鼻咽癌CNE1细胞增殖的影响。方法采用电穿孔基因转染技术。将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,以载体质粒转染及CNE1细胞为对照组,用免疫组化检测LMP1蛋白的表达;用细胞体外增殖实验检测细胞OD比值;用裸小鼠成瘤实验观察移植瘤体积倍增时间和生长率。结果内动物实验细胞移植后CNE1GL组潜伏期缩短,第7、8、9周时,其平均肿瘤体积生长显著高于对照组（P〈0.05）;体外细胞增殖实验3组细胞OD比值差异无显著性（P〉0.05）。结论EBV-LMP1对CNE1细胞的体内增殖能力可能有促进作用。     

鼻咽癌细胞系CNE1和CNE2Z中EBV-LMP1的表达

目的:探讨人高分化鼻咽癌细胞系CNE1和人低分化鼻咽癌细胞系CNE2Z中EB病毒编码的LMP 1基因表达情况.方法:用免疫组化法及Western bolt法分别检测鼻咽癌细胞系CNE1和CNE2Z中LMP 1基因的表达情况.结果:免疫组化及Western bolt 法均显示,CNE2Z细胞中LMP 1呈强阳性表达,而CNE1细胞中LMP 1呈阴性表达.结论:人高分化鼻咽癌细胞系CNE1中没有LMP 1的表达而人低分化鼻咽癌细胞系CNE2Z中LMP 1呈阳性表达,鼻咽癌的恶性程度可能与LMP 1的表达有关.     

EBV-LMP1对鼻咽癌细胞系CNE1细胞转移相关因素的影响

背景与目的:已证实EB病毒(Epstein-Barrvirus,EBV)编码的潜伏膜蛋白1(latentmembraneprotein1,LMP1)能够诱导鼻咽癌细胞中基质金属蛋白酶9(matrixmetalloproteinase-9,MMP-9)的表达。本实验的目的是观察EB病毒潜伏膜蛋白1(EBV-LMP1)对鼻咽癌细胞系CNE1细胞转移相关因素的影响,探讨LMP1在鼻咽癌侵袭、转移过程中的作用。方法:用免疫组化法及Westernblot法检测CNE1-GL(转染LMP1基因的CNE1细胞)和CNE1细胞中MMP-9的表达情况;用细胞-基质粘附实验、细胞运动实验和肿瘤细胞重组基底膜侵袭实验检测LMP1对CNE1细胞粘附、运动及侵袭能力的影响。结果:免疫组化法及Westernblot法结果均显示CNE1-GL细胞中MMP-9的表达明显高于CNE1细胞(P<0.05);肿瘤细胞-基质粘附实验结果显示,CNE1-GL的粘附能力(平均吸光度值为1.2508±0.0711),高于CNE1细胞(平均吸光度值为0.9519±0.068),两者相比差异有显著性(P<0.01)。运动实验及重组基底膜侵袭实验结果均显示,穿过游离的聚乙烯吡咯烷酮膜(polyvinylpyrroli-done-free,PVP-F)的CNE1-GL细胞数明显高于CNE1细胞(P<0.01)。结论:LMP1能够诱导CNE1细胞中MMP-9的表达,且增强CNE1细胞与基底膜的粘附能力、运动能力及侵袭能力。     

EB病毒编码潜伏膜蛋白1通过转录因子c-Jun和Ets1间信息交流调控鼻咽癌中基质金属蛋白酶9的表达

目的　探讨在EBV -LMP1作用下,基质金属蛋白酶9(MMP 9)启动子区相邻的AP 1( 5 33)和Ets( 5 4 0 )结合位点对其转录活化的影响,并确定LMP1可通过c -Jun和Ets1间信息交流(cross -talk)调控鼻咽癌细胞中MMP 9的表达。方法　应用定点突变技术,在野生型MMP- 9 -CAT质粒的基础上,建立MMP- 9启动子区相邻Ets( 5 4 0 )结合位点和AP- 1 (- 5 33)结合位点单独突变体和共同突变体;在四环素调控表达LMP1的鼻咽癌细胞系L7中,比较LMP1对这些突变体报道基因活性的影响。通过针对c- Jun、Ets1的硫代反义寡核苷酸进行阻断,采用GelatinZymography观察c -Jun、Ets1及其cross- talk对LMP1介导的MMP 9表达的影响。结果　与野生型MMP -9 -CAT质粒比较,突变体的报道基因活性均降低(P <0 .0 1 ) ,以MMP- 9 -CATAP -1 ( 5 33) /Ets( 5 4 0 )mt降低最为显著;在c Jun和Ets1反义寡核苷酸单独或共同阻断后,LMP1介导的MMP -9活性均降低,以共同阻断的作用最为明显。结论　EBV -LMP1可以通过转录因子c -Jun和Ets1间cross talk调控鼻咽癌细胞中MMP 9的表达。     

JNK interacting protein 1 (JIP-1) protects LNCaP prostate cancer cells from growth arrest and apoptosis mediated by 12-0-tetradecanoylphorbol-13-acetate (TPA)

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10(-9)-10(-7) mol l(-1)) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10(-9) mol l(-1)) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10(-12)-10(-8) mol l(-1)) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10(-9)-10(-8) mol l(-1)) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway.     

A dominant role for the c-Jun NH2-terminal kinase in oncogenic ras-induced morphologic transformation of human lung carcinoma cells

Oncogenic (activated) Ras is a signal transducer that activates multiple effector-mediated signaling pathways leading to altered cell morphology, growth and differentiation, and neoplastic transformation. Activating mutations of Ras family genes have been detected in many types of human cancers, including lung cancer. However, the signaling mechanisms by which oncogenic Ras controls cancer cell growth is poorly characterized. This study evaluates the role of two specific signaling pathways, the c-Jun NH2-terminal kinase (JNK) pathway, and the extracellular signal-regulated kinase (ERK) pathway, in oncogenic Ras-induced morphological transformation of NCI-H82 human small cell lung cancer cells. In the NCI-H82 cell line, oncogenic Ras causes a marked and sustained activation of JNK but only has a modest effect on activation of the ERK pathway. The persistent JNK activation is associated with Ras-induced changes in cell morphology and enhanced transforming activity. Furthermore, JNK activation correlates with the induction of c-Jun expression, c-Jun phosphorylation on serines 63 and 73, and increased AP-1 activity. Deregulation of the JNK pathway using a dominant-negative mutant of JNK1, JNK1(APF), completely reverses the oncogenic Ras-induced transformed phenotype, including morphological reversion and inhibition of anchorage-independent growth and low-serum growth. Moreover, expression of JNK1(APF) leads to a decrease in c-Jun/AP-1 activity. In contrast, inhibition of ERK activation via a pharmacological approach using a mitogen-activated protein kinase/ERK kinase-specific inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one is unable to reverse the Ras-induced transformed morphology and c-Jun/AP-1 induction. These results demonstrate that the JNK/c-Jun/AP-1 pathway plays an essential role in mediating oncogenic Ras function in lung carcinoma cells.     

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.