NF-κB靶向性圈套策略对膀胱肿瘤生物学行为的影响
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摘要
第一部分核因子κB和uPA在人膀胱移行细胞癌中的表达及临床意义
目的核转录因子-κB(NF-κB)的激活是恶性肿瘤演变过程中的一个重要起始环节,其家族蛋白被认为与肿瘤的发生、发展,侵袭转移和预后有关。本试验研究不同因素人膀胱移行细胞癌组织中NF-κB和尿溶酶纤溶酶激活剂(uPA)的表达情况,评价两者之间表达的相关性,探讨NF-κB在膀胱癌发生、发展中的作用。
方法应用免疫组织化学二步法检测38例膀胱移行细胞癌和10例非癌膀胱粘膜标本中NF-κB p65和uPA蛋白的表达情况,同时结合患者的性别、年龄、临床分期、病理分级、初发还是复发、有无淋巴结转移等因素进行分析,判断上述因素对膀胱癌组织中NF-κB p65和uPA表达的影响,并分析NF-κB p65表达和uPA之间的相关性。
结果38例膀胱癌组织中NF-κB p65阳性率为71.1%(27/38),10例非癌膀胱粘膜标本中NF-κB p65阳性率为10%(1/10),NF-κB p65的过度活化与膀胱癌淋巴结的转移(P<0.01)和病理分级(P<0.05)有关,而患者性别﹑年龄、肿瘤复发和临床分期等因素对NF-κB p65的表达无明显影响;肿瘤组织uPA的表达明显高于良性病变组织(P<0.01),uPA与肿瘤病理分级(p<0.05)、临床分期(p<0.05)和淋巴结的转移(P<0.01)密切相关;经Spearman相关分析显示,在不同恶性程度的膀胱癌中,NF-κB p65表达与uPA之间呈正相关(r=0.89、P<0.01)。
结论NF-κB可能通过上调与肿瘤侵袭转移有关的因子(如PA)的表达,有助于肿瘤的恶性演变。NF-κB的过度活化可能在膀胱移行细胞癌的恶性进展,侵袭转移中发挥重要作用,可能成为未来膀胱癌的治疗中的一个重要靶子。
第二部分核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87生物活性的影响
一、核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87增殖力和侵袭性的影响
目的研究NF-κB环状哑铃型圈套寡核苷酸(CD-ODN)转染对NF-κB活性和膀胱癌细胞BIU87增殖活性和侵袭力的影响,探索膀胱癌基因治疗的新靶点。
方法激光共聚焦法观察BIU87细胞内NF-κB核转位的情况,脂质体介导法将CD-ODN体外转染BIU87细胞(CD-ODN-BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV-β-gal)转化大肠杆菌DH5α并扩增、抽提、纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;并用四甲基偶氮唑蓝比色法(MTT法)和细胞侵袭小室分别检测不同处理组BIU87细胞体外增殖和侵袭活性的变化,细胞克隆形成试验检测CD-ODN对BIU87细胞系干细胞的影响。
结果激光共聚焦技术显示,膀胱癌细胞系BIU87中存在明显的NF-κB过度核转位;荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;MTT发现,CD-ODN-BIU87细胞在第24、48、72和96小时的吸光度值A明显低于两对照组(P<0.01或p<0.05),以正常BIU87细胞为对照,CD-ODN-BIU87在第24、48、72和96小时的细胞生长抑制率分别为27%、46%、28%和11%;细胞侵袭试验显示,CD-ODN-BIU8细胞组、BIU87细胞组和CD-ODN-mut-BIU87细胞组细胞浸润穿透ECM层的数目分别为8.5±1.3、25.4±2.6和24.7±2.1,CD-ODN-BIU87细胞组明显少于两对照组(P<0.05);细胞克隆形成试验显示细胞经ODN干预并继续培养一周后,CD-ODN组、对照组和CD-ODN-mut组的克隆形成数目分别为26.3士3.2、52.3士4.5和48.7士3.1,克隆形成率分别为(8.8±1.1)%、(17.4±1.5)%和(16.2±1.0)%,CD-ODN组与两对照组比较差异有显著性(P<0.01)。
结论膀胱癌细胞系BIU87中存在NF-κB的过度活化,CD-ODN能明显的抑制NF-κB和DNA的联结活性,降低膀胱肿瘤细胞体外增殖活性和侵袭力,为基因治疗膀胱肿瘤提供了新的思路。
二、核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87凋亡的影响
目的研究NF-κB环状哑铃型圈套寡核苷酸(CD-ODN)转染对NF-κB活性和人膀胱癌细胞系BIU87凋亡的影响,探索膀胱癌基因治疗的新途径。
方法脂质体介导法将NF-κB新型诱骗剂CD-ODN体外转染BIU87细胞(CD-ODN- BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV -β-gal)转化大肠杆菌DH5α并扩增、抽提和纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;流式细胞仪检测各组细胞早期凋亡和细胞周期的变化。
结果荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;流式细胞仪Annexin V/PI双染色法检测发现ODN转染细胞48小时后,CD-ODN-BIU87,CD-ODN-mut-BIU87和BIU87的细胞凋亡率分别为(15.81±0.36)%,(6.76±0.96)%和(5.21±0.71)%。经统计学分析,CD-ODN-BIU87细胞的细胞凋亡率明显高于其它两对照组(P<0.01);细胞周期的检测显示, ODN转染细胞48小时后,CD-ODN-BIU87、CD-ODN-mut-BIU87和BIU87处于细胞周期G0-G1期的细胞数目分别为(42.34±2.91)%、(20.34±1.99)%和(16.28±2.26)%,CD-ODN-BIU87停滞于G0-G1期的细胞数目明显多于其它两组(P<0.05),上述三组细胞处于S期的细胞数目分别为(42.68±2.51)%、(60.06±2.61)%和(65.12±4.45)%,而CD-ODN-BIU87处于S期的细胞数目明显少于其它两组(P<0.05)。
结论CD-ODN能特异性抑制BIU87肿瘤细胞内NF-κB活性,促进肿瘤细胞早期凋亡,对肿瘤细胞产生G1-S阻滞作用。这提示,CD-ODN可能通过阻断NF-κB信号转导通路来调控细胞周期,促进肿瘤细胞的早期凋亡,而以NF-κB为靶点的基因治疗有其可行性。
第三部分NF-κB圈套策略CD-ODN对膀胱癌细胞系BIU87NF-κB信号通路的作用
目的探讨NF-κB的活化与尿溶酶纤溶酶激活剂(uPA)和Bcl-2——两个被确认与肿瘤细胞的侵袭转移和凋亡有关的因子——之间的关系,进一步揭示膀胱癌的形成机制,探讨膀胱癌基因治疗的新途径。方法脂质体介导法将NF-κB新型诱骗剂CD-ODN体外转染BIU87细胞(CD-ODN-BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV-β-gal)转化大肠杆菌DH5α并扩增、抽提和纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;PCR检测ODN转染24小时后各处理组uPA mRNA表达情况,Western-blot检测各处理组uPA和Bcl-2蛋白的表达情况。
结果荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;PCR检测发现BIU87细胞中uPA mRNA呈强表达,转染CD-ODN后表达明显被抑制,而转染其突变体无此作用;Western-blot检测显示在BIU87和CD-ODN-mut-BIU87细胞中均有uPA和Bcl-2蛋白的明显表达,而在CD-ODN-BIU87细胞中这两种蛋白的表达被明显抑制。
结论uPA和Bcl-2基因上均含有NF-κB的结合位点,NF-κB诱骗剂CD-ODN能抑制uPA和Bcl-2等与肿瘤的增殖、凋亡、侵袭和转移有关的因子的表达,可能是通过特异性的抑制NF-κB的活性来实现的,为开发新的基因治疗膀胱癌药物提供了理论依据。
Part 1 Expression and Clinical Significance of nuclear factor kappa B and urokinase-type plasminogen activator in Human Transitional Cell Carcinoma of Bladder
Object Activation of transcription factor nuclear factor kappa B(NF-κB) has been showen to be an important original stage in malignant tumor evolution. NF-κB family proteins has been deemed to be involved to proliferation, angiogenesis, invasiveness, apoptosis, metastasis and prognosis of tumor cells.Our task is to study the expression of NF-κBp65 in transitional cell carcinoma(TCCs) of bladder and the relationship between NF-κBp65 expression and urokinase-type plasminogen tor(uPA) in tumor tissues.
Methods The expression of NF-κB and uPA protein in 38 TCCs of bladder and 10 non-carcinoma bladder mucosa were detected by immunohistochemical technique. Gender and age of patients,tumor grades and staging,initial tumor or recurring, lymph node metastasis or not were taken into account. The influences of the factors above on NF-κBp65 and uPA expression and the relation between them were analyzed.
Results In the 38 bladder cancers, 71.1%(27/38) was positive for NF-κBp65 expression. 10% (1/10) was positive for NF-κBp65 expression in all bladder benign lesions. The expression of NF-κBp65 was well correlated with tumor grades(P<0.05) and lymph node metastasis(P<0.01).The uPA in bladder cancer was higher than that in benign lesions(P< 0.01 ) . uPA expression significantly correlated with tumor grades(P<0.05),staging (P<0.05)and lymph node metastasis(P<0.01). There was a positive correlation between NF-κBp65 expression and uPA in bladder cancer(r=r=0.89、P<0.01).
Conclusion The results obtained indicated that constitutively activation of NF-κB and the over- expression of uPA were correlated with biological behaviors of TCCs of bladder and suggested that NF-κB activity was related to tumor progression due to its transcriptional regulation of uPA and NF-κB may be an important target for the development of future anti-invasion therapy in cases of TCCs of bladder.
Part 2 Effects of nuclear factor kappa B decoy strategy-circular dumbbell oligodeoxynucleotides on Biological Activities of Human Bladder Cancer Cell Lines BIU87
1、Effects of Nuclear Factor kappa B“decoy strategy”-Circular Dumbbell Oligodeoxynucleotides on Proliferation and Invasion of Human Bladder Cancer Cell Lines BIU87
Object To study the effects of nuclear factor kappa B(NF-κB)“decoy strategy”-circular dumbbell oligodeoxynucleotides(CD-ODN) transfection on activation of NF-κB and proliferation and invasion of human bladder cancer cell lines BIU87,and to bring a new method of gene therapy for bladder cancer.
Methods Laser confocal scanning technique was applied to study NF-κB nuclear translocation. CD-ODN were transfected into human bladder cancer cell line BIU87(CD-ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD-ODN-mut-BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid- pCMV-β-gal was introduced into E.coli DH5α, amplified and prepared , and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. Cell proliferation and invasion ability were measured by MTT and cell invasion assay.Drug sensitivity of bladder cancer stem cell to CD-ODN wal detected by cell clone formation counting assay. Results It was confirmed by laser confocal scanning technique that the phenomenon of NF-κB nuclear translocation is obviously in BIU87. Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups. MTT founded, absorbance of the 24th,48th,72th and 96th hours of CD-ODN-BIU87 were significantly lower than those of control groups(P<0.05 and P<0.01),and using normal BIU87 as control groups,inhibition rates of cell growth of the first、second、third and fourth day were 27%、46%、28% and 11%,respectively;Cell invasion assay showed, number of CD-ODN-BIU87 cell penetrating ECM was 8.5±1.3,obviously lower than those of control groups, 25.4±2.6 and 24.7±2.1(P<0.05). Cell clone formation counting assay detection found that the counting of clone formation of CD-ODN-BIU87, CD-ODN-mut-BIU87 and BIU87 were 26.3士3.2, 52.3士4.5 and 48.7士3.1,respectively, and the rate ratio of clone formation of them were (8.8±1.1)%, (17.4±1.5)% and (16.2±1.0)%, respectively. Both counting and rate ratio of clone formation counting of CD-ODN-BIU87 significantly decreased compared with control groups(P<0.05).
Conclusion Collectively, the date showed NF-κB is constitutively activated in human bladder cancer cell and NF-κB“decoy strategy”-CD-ODN can efficiently suppress DNA binding activity of NF-κB and inhibit proliferation and invasion ability of bladder cancer cells so as to inhibit the occurrence and development of tumors.
2、Effects of Nuclear Factor kappa B“decoy strategy”-Circular Dumbbell Oligodeoxynucleotides on Apoptosis of Human Bladder Cancer Cell Lines BIU87
Object To study the effects of NF-κB“decoy strategy”-circular dumbbell oligodeoxynucleotides (CD-ODN) transfection on activation of NF-κB and apoptosis of human bladder cancer cell lines BIU87,and to bring a new method of gene therapy for bladder cancer.
Methods CD-ODN were transfected into human bladder cancer cell line BIU87 (CD-ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD-ODN -mu t- BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid- pCMV-β-gal was introduced into E.coli DH5α, amplified and prepared, and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. Flow cytometry was applied to analyze the cell cycle and apoptosis of BIU87.
Results Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups. Flow cytometry Annexin V/PI double-staining assay showed apoptosis rates of CD-ODN-BIU87,CD-ODN-mut-BIU87 and BIU87 were (15.81±0.36)%,(6.76±0.96)% and(5.21±0.71)%, respectively, and apoptosis rate of CD-ODN-BIU87 significantly increased(P<0.01).The cell cycle assay found the cell counting of CD-ODN-BIU87, CD-ODN-mut-BIU87 and BIU87 in G0-G1 of cell cycl were (42.34±2.91)%,(20.34±1.99)% and(16.28±2.26)%,respectively,and in S were (42.68±2.51)%, (60.06±2.61)% and(65.12±4.45)%,respectively. CD-ODN-BIU87 cell in G0-G1 of cell cycle was more than those of control groups(P<0.05), and cell in S ,less(P<0.01). It was clear that CD-ODN block cell cycle at G1-S.
Conclusion Transfection of CD-ODN might induce apoptosis of bladder cancer cells so as to inhibit the occurrence and development of tumor possibly by blocking the NF-κB signaling pathway. It may be a new kind of treatment strategy for bladder cancer.
Part 3 Effects of Circular Dumbbell Oligodeoxynucleotides Transfection on NF-κB Signal Pathway of Bladder Cancer Cell BIU87
Object Activation of transcription factor NF-κB has been showen to be an important original stage in malignant tumor evolution. Activated NF-κB might translocate into the nucleus, resulting in expression of target genes, so as to effect the biological activities of tumor. Our task is to study relationship between NF-κBp65 activity and uPA expression and Bcl-2——two factors haven been considered to be related to invasion and apoptosis of tumor cell,investigate the mechanism of bladder cancer, and to bring a new method of gene therapy for bladder cancer.
Methods CD-ODN were transfected into human bladder cancer cell line BIU87(CD - ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD–ODN -mut-BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid(pCMV-β-gal) was introduced into E.coli DH5αand amplified and prepared ,and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. RT-PCR was applied to measure the expression level of uPAmRNA; Western-blot was applied to analysis the expression of uPA protein and Bcl-2.
Results Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups.The expression level of mRNA and protein of uPA were inhibited evidently after CD-ODN transfected, and Bcl-2 protein expression of CD-ODN-BIU87 cell decreased obviously as well.
Conclusion There are NF-κB binding sites on both uPA and Bcl-2 gene promoter, NF-κB“decoy stategy”-CD-ODN might decrease invasion-related factors such as uPA and apoptosis-related factors such as Bcl-2, possibly due to its blocking to the signal transduction path of NF-κB and suggest NF-κB may be an importment target for the development of future gene therapy in case of bladder cancinoma.
目的核转录因子-κB(NF-κB)的激活是恶性肿瘤演变过程中的一个重要起始环节,其家族蛋白被认为与肿瘤的发生、发展,侵袭转移和预后有关。本试验研究不同因素人膀胱移行细胞癌组织中NF-κB和尿溶酶纤溶酶激活剂(uPA)的表达情况,评价两者之间表达的相关性,探讨NF-κB在膀胱癌发生、发展中的作用。
方法应用免疫组织化学二步法检测38例膀胱移行细胞癌和10例非癌膀胱粘膜标本中NF-κB p65和uPA蛋白的表达情况,同时结合患者的性别、年龄、临床分期、病理分级、初发还是复发、有无淋巴结转移等因素进行分析,判断上述因素对膀胱癌组织中NF-κB p65和uPA表达的影响,并分析NF-κB p65表达和uPA之间的相关性。
结果38例膀胱癌组织中NF-κB p65阳性率为71.1%(27/38),10例非癌膀胱粘膜标本中NF-κB p65阳性率为10%(1/10),NF-κB p65的过度活化与膀胱癌淋巴结的转移(P<0.01)和病理分级(P<0.05)有关,而患者性别﹑年龄、肿瘤复发和临床分期等因素对NF-κB p65的表达无明显影响;肿瘤组织uPA的表达明显高于良性病变组织(P<0.01),uPA与肿瘤病理分级(p<0.05)、临床分期(p<0.05)和淋巴结的转移(P<0.01)密切相关;经Spearman相关分析显示,在不同恶性程度的膀胱癌中,NF-κB p65表达与uPA之间呈正相关(r=0.89、P<0.01)。
结论NF-κB可能通过上调与肿瘤侵袭转移有关的因子(如PA)的表达,有助于肿瘤的恶性演变。NF-κB的过度活化可能在膀胱移行细胞癌的恶性进展,侵袭转移中发挥重要作用,可能成为未来膀胱癌的治疗中的一个重要靶子。
第二部分核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87生物活性的影响
一、核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87增殖力和侵袭性的影响
目的研究NF-κB环状哑铃型圈套寡核苷酸(CD-ODN)转染对NF-κB活性和膀胱癌细胞BIU87增殖活性和侵袭力的影响,探索膀胱癌基因治疗的新靶点。
方法激光共聚焦法观察BIU87细胞内NF-κB核转位的情况,脂质体介导法将CD-ODN体外转染BIU87细胞(CD-ODN-BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV-β-gal)转化大肠杆菌DH5α并扩增、抽提、纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;并用四甲基偶氮唑蓝比色法(MTT法)和细胞侵袭小室分别检测不同处理组BIU87细胞体外增殖和侵袭活性的变化,细胞克隆形成试验检测CD-ODN对BIU87细胞系干细胞的影响。
结果激光共聚焦技术显示,膀胱癌细胞系BIU87中存在明显的NF-κB过度核转位;荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;MTT发现,CD-ODN-BIU87细胞在第24、48、72和96小时的吸光度值A明显低于两对照组(P<0.01或p<0.05),以正常BIU87细胞为对照,CD-ODN-BIU87在第24、48、72和96小时的细胞生长抑制率分别为27%、46%、28%和11%;细胞侵袭试验显示,CD-ODN-BIU8细胞组、BIU87细胞组和CD-ODN-mut-BIU87细胞组细胞浸润穿透ECM层的数目分别为8.5±1.3、25.4±2.6和24.7±2.1,CD-ODN-BIU87细胞组明显少于两对照组(P<0.05);细胞克隆形成试验显示细胞经ODN干预并继续培养一周后,CD-ODN组、对照组和CD-ODN-mut组的克隆形成数目分别为26.3士3.2、52.3士4.5和48.7士3.1,克隆形成率分别为(8.8±1.1)%、(17.4±1.5)%和(16.2±1.0)%,CD-ODN组与两对照组比较差异有显著性(P<0.01)。
结论膀胱癌细胞系BIU87中存在NF-κB的过度活化,CD-ODN能明显的抑制NF-κB和DNA的联结活性,降低膀胱肿瘤细胞体外增殖活性和侵袭力,为基因治疗膀胱肿瘤提供了新的思路。
二、核因子-κB圈套策略—CD-ODN对膀胱癌细胞系BIU87凋亡的影响
目的研究NF-κB环状哑铃型圈套寡核苷酸(CD-ODN)转染对NF-κB活性和人膀胱癌细胞系BIU87凋亡的影响,探索膀胱癌基因治疗的新途径。
方法脂质体介导法将NF-κB新型诱骗剂CD-ODN体外转染BIU87细胞(CD-ODN- BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV -β-gal)转化大肠杆菌DH5α并扩增、抽提和纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;流式细胞仪检测各组细胞早期凋亡和细胞周期的变化。
结果荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;流式细胞仪Annexin V/PI双染色法检测发现ODN转染细胞48小时后,CD-ODN-BIU87,CD-ODN-mut-BIU87和BIU87的细胞凋亡率分别为(15.81±0.36)%,(6.76±0.96)%和(5.21±0.71)%。经统计学分析,CD-ODN-BIU87细胞的细胞凋亡率明显高于其它两对照组(P<0.01);细胞周期的检测显示, ODN转染细胞48小时后,CD-ODN-BIU87、CD-ODN-mut-BIU87和BIU87处于细胞周期G0-G1期的细胞数目分别为(42.34±2.91)%、(20.34±1.99)%和(16.28±2.26)%,CD-ODN-BIU87停滞于G0-G1期的细胞数目明显多于其它两组(P<0.05),上述三组细胞处于S期的细胞数目分别为(42.68±2.51)%、(60.06±2.61)%和(65.12±4.45)%,而CD-ODN-BIU87处于S期的细胞数目明显少于其它两组(P<0.05)。
结论CD-ODN能特异性抑制BIU87肿瘤细胞内NF-κB活性,促进肿瘤细胞早期凋亡,对肿瘤细胞产生G1-S阻滞作用。这提示,CD-ODN可能通过阻断NF-κB信号转导通路来调控细胞周期,促进肿瘤细胞的早期凋亡,而以NF-κB为靶点的基因治疗有其可行性。
第三部分NF-κB圈套策略CD-ODN对膀胱癌细胞系BIU87NF-κB信号通路的作用
目的探讨NF-κB的活化与尿溶酶纤溶酶激活剂(uPA)和Bcl-2——两个被确认与肿瘤细胞的侵袭转移和凋亡有关的因子——之间的关系,进一步揭示膀胱癌的形成机制,探讨膀胱癌基因治疗的新途径。方法脂质体介导法将NF-κB新型诱骗剂CD-ODN体外转染BIU87细胞(CD-ODN-BIU87),以转染了CD-ODN突变体(CD-ODN-mut)的BIU87细胞(CD-ODN-mut-BIU87)和仅转染空脂质体的BIU87细胞为对照;将携有4个NF-κB的串联重复识别元件的荧光素酶报告基因重组质粒(pNF-κB -LUC+)和β-半乳糖苷酶表达质粒(PCMV-β-gal)转化大肠杆菌DH5α并扩增、抽提和纯化,然后将其与CD-ODN瞬时共转染入BIU87细胞,检测不同处理组NF-κB活性的改变;PCR检测ODN转染24小时后各处理组uPA mRNA表达情况,Western-blot检测各处理组uPA和Bcl-2蛋白的表达情况。
结果荧光素酶报告质粒的方法证实CD-ODN-BIU87细胞组NF-κB的活性明显低于转染其突变体组和对照组;PCR检测发现BIU87细胞中uPA mRNA呈强表达,转染CD-ODN后表达明显被抑制,而转染其突变体无此作用;Western-blot检测显示在BIU87和CD-ODN-mut-BIU87细胞中均有uPA和Bcl-2蛋白的明显表达,而在CD-ODN-BIU87细胞中这两种蛋白的表达被明显抑制。
结论uPA和Bcl-2基因上均含有NF-κB的结合位点,NF-κB诱骗剂CD-ODN能抑制uPA和Bcl-2等与肿瘤的增殖、凋亡、侵袭和转移有关的因子的表达,可能是通过特异性的抑制NF-κB的活性来实现的,为开发新的基因治疗膀胱癌药物提供了理论依据。
Part 1 Expression and Clinical Significance of nuclear factor kappa B and urokinase-type plasminogen activator in Human Transitional Cell Carcinoma of Bladder
Object Activation of transcription factor nuclear factor kappa B(NF-κB) has been showen to be an important original stage in malignant tumor evolution. NF-κB family proteins has been deemed to be involved to proliferation, angiogenesis, invasiveness, apoptosis, metastasis and prognosis of tumor cells.Our task is to study the expression of NF-κBp65 in transitional cell carcinoma(TCCs) of bladder and the relationship between NF-κBp65 expression and urokinase-type plasminogen tor(uPA) in tumor tissues.
Methods The expression of NF-κB and uPA protein in 38 TCCs of bladder and 10 non-carcinoma bladder mucosa were detected by immunohistochemical technique. Gender and age of patients,tumor grades and staging,initial tumor or recurring, lymph node metastasis or not were taken into account. The influences of the factors above on NF-κBp65 and uPA expression and the relation between them were analyzed.
Results In the 38 bladder cancers, 71.1%(27/38) was positive for NF-κBp65 expression. 10% (1/10) was positive for NF-κBp65 expression in all bladder benign lesions. The expression of NF-κBp65 was well correlated with tumor grades(P<0.05) and lymph node metastasis(P<0.01).The uPA in bladder cancer was higher than that in benign lesions(P< 0.01 ) . uPA expression significantly correlated with tumor grades(P<0.05),staging (P<0.05)and lymph node metastasis(P<0.01). There was a positive correlation between NF-κBp65 expression and uPA in bladder cancer(r=r=0.89、P<0.01).
Conclusion The results obtained indicated that constitutively activation of NF-κB and the over- expression of uPA were correlated with biological behaviors of TCCs of bladder and suggested that NF-κB activity was related to tumor progression due to its transcriptional regulation of uPA and NF-κB may be an important target for the development of future anti-invasion therapy in cases of TCCs of bladder.
Part 2 Effects of nuclear factor kappa B decoy strategy-circular dumbbell oligodeoxynucleotides on Biological Activities of Human Bladder Cancer Cell Lines BIU87
1、Effects of Nuclear Factor kappa B“decoy strategy”-Circular Dumbbell Oligodeoxynucleotides on Proliferation and Invasion of Human Bladder Cancer Cell Lines BIU87
Object To study the effects of nuclear factor kappa B(NF-κB)“decoy strategy”-circular dumbbell oligodeoxynucleotides(CD-ODN) transfection on activation of NF-κB and proliferation and invasion of human bladder cancer cell lines BIU87,and to bring a new method of gene therapy for bladder cancer.
Methods Laser confocal scanning technique was applied to study NF-κB nuclear translocation. CD-ODN were transfected into human bladder cancer cell line BIU87(CD-ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD-ODN-mut-BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid- pCMV-β-gal was introduced into E.coli DH5α, amplified and prepared , and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. Cell proliferation and invasion ability were measured by MTT and cell invasion assay.Drug sensitivity of bladder cancer stem cell to CD-ODN wal detected by cell clone formation counting assay. Results It was confirmed by laser confocal scanning technique that the phenomenon of NF-κB nuclear translocation is obviously in BIU87. Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups. MTT founded, absorbance of the 24th,48th,72th and 96th hours of CD-ODN-BIU87 were significantly lower than those of control groups(P<0.05 and P<0.01),and using normal BIU87 as control groups,inhibition rates of cell growth of the first、second、third and fourth day were 27%、46%、28% and 11%,respectively;Cell invasion assay showed, number of CD-ODN-BIU87 cell penetrating ECM was 8.5±1.3,obviously lower than those of control groups, 25.4±2.6 and 24.7±2.1(P<0.05). Cell clone formation counting assay detection found that the counting of clone formation of CD-ODN-BIU87, CD-ODN-mut-BIU87 and BIU87 were 26.3士3.2, 52.3士4.5 and 48.7士3.1,respectively, and the rate ratio of clone formation of them were (8.8±1.1)%, (17.4±1.5)% and (16.2±1.0)%, respectively. Both counting and rate ratio of clone formation counting of CD-ODN-BIU87 significantly decreased compared with control groups(P<0.05).
Conclusion Collectively, the date showed NF-κB is constitutively activated in human bladder cancer cell and NF-κB“decoy strategy”-CD-ODN can efficiently suppress DNA binding activity of NF-κB and inhibit proliferation and invasion ability of bladder cancer cells so as to inhibit the occurrence and development of tumors.
2、Effects of Nuclear Factor kappa B“decoy strategy”-Circular Dumbbell Oligodeoxynucleotides on Apoptosis of Human Bladder Cancer Cell Lines BIU87
Object To study the effects of NF-κB“decoy strategy”-circular dumbbell oligodeoxynucleotides (CD-ODN) transfection on activation of NF-κB and apoptosis of human bladder cancer cell lines BIU87,and to bring a new method of gene therapy for bladder cancer.
Methods CD-ODN were transfected into human bladder cancer cell line BIU87 (CD-ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD-ODN -mu t- BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid- pCMV-β-gal was introduced into E.coli DH5α, amplified and prepared, and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. Flow cytometry was applied to analyze the cell cycle and apoptosis of BIU87.
Results Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups. Flow cytometry Annexin V/PI double-staining assay showed apoptosis rates of CD-ODN-BIU87,CD-ODN-mut-BIU87 and BIU87 were (15.81±0.36)%,(6.76±0.96)% and(5.21±0.71)%, respectively, and apoptosis rate of CD-ODN-BIU87 significantly increased(P<0.01).The cell cycle assay found the cell counting of CD-ODN-BIU87, CD-ODN-mut-BIU87 and BIU87 in G0-G1 of cell cycl were (42.34±2.91)%,(20.34±1.99)% and(16.28±2.26)%,respectively,and in S were (42.68±2.51)%, (60.06±2.61)% and(65.12±4.45)%,respectively. CD-ODN-BIU87 cell in G0-G1 of cell cycle was more than those of control groups(P<0.05), and cell in S ,less(P<0.01). It was clear that CD-ODN block cell cycle at G1-S.
Conclusion Transfection of CD-ODN might induce apoptosis of bladder cancer cells so as to inhibit the occurrence and development of tumor possibly by blocking the NF-κB signaling pathway. It may be a new kind of treatment strategy for bladder cancer.
Part 3 Effects of Circular Dumbbell Oligodeoxynucleotides Transfection on NF-κB Signal Pathway of Bladder Cancer Cell BIU87
Object Activation of transcription factor NF-κB has been showen to be an important original stage in malignant tumor evolution. Activated NF-κB might translocate into the nucleus, resulting in expression of target genes, so as to effect the biological activities of tumor. Our task is to study relationship between NF-κBp65 activity and uPA expression and Bcl-2——two factors haven been considered to be related to invasion and apoptosis of tumor cell,investigate the mechanism of bladder cancer, and to bring a new method of gene therapy for bladder cancer.
Methods CD-ODN were transfected into human bladder cancer cell line BIU87(CD - ODN-BIU87) by Lipofectamine, With CD-ODN-mut transfected BIU87(CD–ODN -mut-BIU87)and normal BIU87 as control groups. The recombinant plasmid of firefly luciferase report gene containing 4 tandem duplicational identification elements of NF-κB (pNF-κB-LUC+) and an eukaryotic expression plasmid(pCMV-β-gal) was introduced into E.coli DH5αand amplified and prepared ,and then purified, eventually cotransfected transiently into BIU87 with CD-ODN to detecting NF-κB DNA binding activity. RT-PCR was applied to measure the expression level of uPAmRNA; Western-blot was applied to analysis the expression of uPA protein and Bcl-2.
Results Luciferase reporter showed the activation of NF-κB of CD-ODN-BIU87 significantly decreased compared with control groups.The expression level of mRNA and protein of uPA were inhibited evidently after CD-ODN transfected, and Bcl-2 protein expression of CD-ODN-BIU87 cell decreased obviously as well.
Conclusion There are NF-κB binding sites on both uPA and Bcl-2 gene promoter, NF-κB“decoy stategy”-CD-ODN might decrease invasion-related factors such as uPA and apoptosis-related factors such as Bcl-2, possibly due to its blocking to the signal transduction path of NF-κB and suggest NF-κB may be an importment target for the development of future gene therapy in case of bladder cancinoma.
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14. Hinz M, Krappmann D, Eichten A, et al. NF-kappaB function in growth control: regulation of cyclin Dl expression and Go/G1-to-S-phase transition. Mol Cell Biol, 1999,19:2690-2698.
1. Yu L, Yuan JL, Wu GJ, et al. Expression and significance of Bcl-2 and Bag-1 in renal cell carcinoma. J Fourth Mil Med Univ, 2002, 23:1303-1305.
2. Yang LJ, Wang WL.Expression of Bcl-2 and Bax proteins in hepatocellar carcinoma tissue.J Fourth Mil Med Univ, 2002,23:337-340.
3. Hosoya T, Takeuchi H, Kanesaka Y, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters, 1999, 461:136-140.
4. Mahabeleshwar GH, Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem, 2003,278: 6209-6221.
5. Sen R, Baltimore D. Multiple nuclear factors interect the immuno-globulin enhancer sequences. Cell, 1986,46: 705.
6. Sasaki N, Morisaki T, Hashizume K, et al. Nuclear Factor-κB p65 (RelA) Transcription Factor Is Constitutively Activated in Human Gastric Carcinoma Tissue. Clin Cancer Res, 2001, 7:4136-4142.
7. Bours V, Dejardin E, Goujon-Letawe F, et al. The NF-kappaB transcription factor and cancer: high expression of NF-kappaB and IkappaB related proteins in tumor cell lines. Biochem Pharmacol,1994,47:145-149.
8. Nakshatri H, Bhat-Nakshatri P, Martin D A, et al. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol, 1997, 17: 3629-3639.
9. Wang W, James L. Abbruzzese DB, et al. The Nuclear Factor-kappaB RelA Transcription Factor Is Constitutively Activated in Human Pancreatic Adenocarcinoma Cells. Clin Cancer Res, 1999, 5:119-127.
10. Li Y, Rizvi SM, Ranson M,et al. 213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model[J]. Br J Cancer ,2002 ,86:1197-1203.
11. Shariat SF, Monoski MA, Andrews B, et al. Association of plasma urokinase- type plasminogen activator and its receptor with clinical outcome in patients undergoing radical cystectomy for transitional cell carcinoma of the bladder. Urology, 2003, 61: 1053-1058.
12. Turco MC, Romano MF, Petrella A,et al. NF-kB /Rel-mediated regulation of apaptosis in hematologic malignancies and normal hematopoietic progenitors. Leukemia, 2004,18: 11-17.
13. Viatour P, Bentires AM,Chatiot A,etal. NF-kappaB2/p100 induces Bcl- 2 expression. Leukemia, 2003, 17: 1349-1359.
1. Grumont RJ, Gerondakis S. Rel induces interferon regulatory factor4 (IRF-4) expression in lymphocytes: modulation of interferon- regulated gene expression by Rel/nuclear factor kappa B.J ExpMed, 2000, 1919:1281-1292.
2. Chen F,Castranova V,Shi X,et al.New insights into the role of nuclear factor-κB,a ubiquitous transcription factor in the initiation of diseases.Clin Chem,1999,45:7-17.
3. Raffi G,Raelene G,Mathis G,et al. Rel/NF-κB transcription factors: key mediators of B-cell activation. Immunol Rev 2000, 176:134-140.
4. Aradhya S, Nelson DL. NF-kappaB signaling and human disease.Curr Opin Genet Dev, 2001,11:300-306.
5. Tak PP, Firestein GS. NF-kappaB:a key role in inflammatory diseases. J Clin Invest,2001,107:7-11.
6. Baldwin AS Jr. Series introduction: the transcription factor NF kappa B and human disease.J Clin Invest,2001,107:3-6.
7. Sen R, Baltimore D. Multiple nuclear factors interect the immuno-globulin enhancer sequences. Cell, 1986,46: 705.
8. Thompson JE, Phillips RJ, Erdjument-Bromage H,et al.IkappaB-beta regulates the persistent response in a biphasi activation of NF-kappaB. Cell,1995,80:573-582.
9. Silverman N, Maniatis T. NF-kappaB signaling pathways in mammalian and insect innate immunity.Genes Dev, 2001, 15:2321-2342.
10. Zandi E, Karin M. Bridging the Gap: Composition, regulation and physiological function of IκB kinase complex. Mol Cell Biol,1999,19: 4547-4551.
11. Magnani M, Crinelli R, Bianchi M, et al. The ubiquitindependent proteolytic system and other potential targets for the modulation of nuclear factor-kB.Curr Drug Targets,2000, 1:387-399.
12. Zhang G, Ghosh S. Molecular mechanisms of NF-kappaB activation induced by bacterial lipopolysaccharide through Tol-likereceptors. J Endotoxin Res, 2000,6 453-457.
13. Gorlich D, Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol,1999,15:607-660.
14. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. [J]. Am J Respir Cell Biol, 1997,17:3-9.
15. Tai DI, Tsai SL, Chen YM. Hepatitis C Virus Infection: Implications for pathogenesis and hepatocarcinogenesis. Hepatology, 2000,31: 785-787.
16. Gilmore TD. Multiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel.Oncogene,1999,18:6925-6937.
17. Chen F, Castranora V,Shi XL.New insights into the role of NF-κB,an obigutious transcription factor in the initiation of disease.Clin Chem,1999, 45:7-17.
18. He Z, Xin B,Yang X. Nuclear factor-kappaB activation is involved in LMP1-mediated transformation and tumorigenesis of rat-1 fibroblasts. Cancer Research, 2000,60: 1845-1851.
19. Wu MX, Ao Z,Prasad KV,et al.IEX-1L,an apoptosis inhibitor involved in NF-kappaB- mediated cell survival.Science,1998,281:998-1001.
20. Zong WX,Edelstein LC, Chen C,et al.The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Dev, 1999,13:382-387.
21. Feuillard J, Schuhmacher M, Kohanna S, et al.Inducible loss of NF-kappaB activity is associated with apoptosis and bcl-2 down-regulation in Epstein-Barrvirus-transformed B lymphocytes. Blood,2000,95:2068-2075
22. Wester GA, Perkins ND. Tanscriptional cross talk between NF-kappaB and p53. Mol Cell Biol,1999, 19: 3485-3495.
23. Huang Y,Johnson KR, Norris JS, et al. Nuclear factor-kappaB/IkappaB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells. Cancer Res, 2000, 60:4426-4432.
24. Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene, 1999, 18: 6910-6924.
25. Wang CY, Cusack JC Jr, Liu R, et al. Control of inducible chemoresistance: Enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med, 1999, 5:412-417.
26. Bentires-Alj M, Hellin AC, Ameyar M, et al. Stable inhibition of nuclear factor kappaB in cancer cells does not increase sensitivity to cytotoxic drugs. Cancer Res, 1999, 59: 811-815.
27. Dumont A, Hehner SP, Hofmann TG, et al.Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB, Oncogene, 1999, 18: 747-757.
28. Kasibhatla S, Genestier L, Green DR. Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB. J Biol Chem,1999,274:987-992.
29. Guttridge DC, Albanese C, Reuther JY, et al. NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol, 1999, 19: 5785-5799.
30. Hinz M, Krappmann D, Eichten A, et al. NF-kappaB function in growth control: Regulation of cyclin D1 expression and G0/G1-to S-phase transition.Mol Cell Biol, 1999, 19: 2690-698.
31. Ghosh S, MayM J ,Kopp EB. NF-kappaB and Rel proteins: evolutio arily conserved mediators of immune responses. Annu Rev Immuno 1998,16:225-260.
32. Sumitomo M, Tachibana M, Ozu C, et al.Induction of apoptosis of cytokine-producing bladder cancer cells by adenovirus-mediated IkappaBalph overexpression. Hum Gene Ther,1999,10:37-47.
33. Chen S, Fribley A, Wang CY. Potentiation of tumor necrosis factor mediated apoptosis of oral squamous cell carcinoma cells by adenovirus mediated gene transfer of NF-kappaB inhibitor.J Dent Res,2002,81:98- 102.
34. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappa B. J Clin Invest,2001,107:241-246.
35. Duffey DC, Crowl Bancroft CV, Chen Z, et al. Inhibition of transcription factor nuclear factor-kappaB by a mutant inhibitor kappaBalpha attenuates resistance of human head and neck squamous cell carcinoma to TNF-alpha caspase-mediated cell death. Br J Cancer, 2000, 83:1367-1374.
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38. May MJ,D’Acquisto F, Madge LA, et al.Selective inhibition of NF-kappa B activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science, 2000, 289:1550-1554.
39. Morishita R, Higaki J,Tomita N,et al. Application of transcription factor“decoy”strategy as means of gene therapy and study of gene expression in cardiovascular disease.Circ Res,1998,82:1023-1028.
40. Tomita N,Morishita R,Tomita S,et al.Transcription factor decoy for nuclear factor- kappaB inhibits tumor necrosis factor-alpha-induced expresion of interleukin-6 and intracellular adhesion molecule-1 in endotheli cells.JHypertens,1998,16 (7):993 -1000.
41. Morishita R, Aoki M, Kaneda Y. Decoy oligodeoxynucleotides as novel cardiovascular drugs for cardiovascular disease. Ann N Y Acad Sci, 2001, 947:294-301.
42. Manninen HI, Makinen K. Gene therapy techniques for peripheral arterial disease. Cardiovasc Intervent Radiol,2002, 25:98-108.
43. Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-kappaB pathway in the treatment of inflammation and cancer.J Cli Invest,2001,107:135-142.
44. Holmes-McNary M, Baldwin AS Jr. Chemopreventive properties of transresveratrol are associated with inhibition of activation of the IkappaB kinase. Cancer Res, 2000,61; 3477 -3483.
45. Gupta,-Sanjay; Afaq,-Farrukh; Mukhtar,-Hasan. Involvement of nuclear factor kappaB, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells. Oncogene, 2002, 21:3727-3738.
46. Mouria M, Gukovskaya AS, Jung Y, et al. Food-derived polyphenols inhibit pancreaticcancer growth through mitochondrial cytochrome C release and apoptosis. Int-J- Cancer, 2002,98:761-769.
47. Lee IK, Ahn JD, Kim HS, et al. Advantages of the circular dumbbe decoy in gene therapy and studies of gene regulation. Curr Drug Targets, 2003, 4:619-623.
48. Chu BC, Orgal LE. The stability of different forms of double-stranded decoy DNA in serum and nuclear extracts Nucleic Acids Res, 1992, 20: 5857-5858.
49. Hosoya T, Takeuchi H, Kanesaka Y, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters, 1999, 461: 136-140.
50. Ahn JD, Morishita R, Kaneda Y, et al. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo.Circ Res, 2002 90, 1325-1332.
51. Ahn JD, Morishita R, Kaneda Y, et al. Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia . Gene Therapy, 2002,9:1682-1692.
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4. Waddick KG, Uckun FM. Innovative treatment programs against cancer:ⅡNuclear facter-kappa B (NF-kappaB) as a samolecular target [J]. Biochem Pharmacol, 1999,57: 9
5. Dong Z, Nemeth JA, Cher ML,et al. Differential regulation of matrix metalloproteinase -9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression in co-culture of prostate cancer and stromal cells[J]. Int J Cancer, 2001,93:507
6. Giri DK, Aggarwal BB. Constitutive activation of NF-kappa B cause resistance to apoptosis in human cutaneous T cell lymphoma HUT-78 cells. Atocrine role of tumor necrosis factor and reactive oxygen intermediate[J]. J Biol Chem, 1998,273:14008.
7. Gara A, Aggarwal BB. Buclear transcription factor-B as a target for cancer drugdevelopment [J]. Leukemia, 2002, 16:1053.
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11. Tomita N, Morishita R, Lan HY, et al. In vivo administration of a nclear transcription factor-kappa B decoy suppresses experimental crescetic glomerulonephritis. J Am Soc Nephrol, 2000,11:1244-1252.
12. Lee IK, Ahn JD, Kim HS, et al. Advantages of the circular dumbbe decoy in gene therapy and studies of gene regulation. Curr Drug Targets, 2003, 4:619-623.
13. Takeshi H, Hiroaki T, Yoshiyuki K, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters,1999 461:136-140.
1.Nobuhiko S, Takashi M, Kentaro H et al. Nuclear factor-kappaB p65 (RelA) transcription factor is constitutively activated in human gastric carcinoma tissue. Clin Cancer Res, 2001 , 7:4136.
2.Oya M, Takayanagi A, Horiguchi A et al. Increased nuclear factor-kappa B activation is related to the tumor development of renal cell carcinoma. Carcinogenesis,2003 ,24:377.
3.Reuning U, Wilhelm O, Nishiguchi T et al. Inhibition of NF-κB -Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1. Nucleic Acids Res,1995,23: 3887.
4.Wang W, Abbruzzese J L, Evans D B et al. Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA. Oncogene,1999, 18: 4554.
5.Handel M L, Mcmorrow L B, Gravallese E M. NF-κB in rheumatoid synovium. Arthritis Rheum, 1995,38:1762.
6.Miyamoto S, Chiao P J, Verma I M et al. Enhanced IκB degradation is responsible for constitutive activity in mature B-cell linds. Mol Cell Biol, 1994,14:3276.
7.Shariat S F, Monoski M A, Andrews B et al. Association of plasma urokinase-type plasminogen activator and its receptor with clinical outcome in patients undergoing radical cystectomy for transitional cell carcinoma of the bladder. Urology, 2003 ;61: 1053.
8. Seddighzadeh M, Steineck G, Larsson P et al. Expression of uPA and uPAR is associated with the clinical course of urinary bladder neoplasms. Int J Cancer, 2002 ,99:721.
1. Hosoya T, Takeuchi H, Kanesaka Y, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters, 1999, 461:136-140.
2. Mahabeleshwar GH, Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem, 2003,278: 6209-6221.
3. Nobuhiko S, Takashi M, Kentaro H et al. Nuclear factor-kappaB p65 (RelA) transcription factor is constitutively activated in human gastric carcinoma tissue. Clin Cancer Res, 2001 ,7:4136.
4. Oya M, Takayanagi A, Horiguchi A et al. Increased nuclear factor-kappa B activation is related to the tumor development of renal cell carcinoma. Carcinogenesis,2003 ,24:377.
5.文博,杨为民,周四维,等.核因子κB和uPA在人膀胱移行细胞癌中的表达及临床意义.华中科技大学学报(医学版),2005, 2:225-228.
6. Das R, Philip S, Mahabeleshwar GH, et al. Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression. IUBMB Life, 2005 ,57:441-447.
7. Brown DA, Kang SH, Gryaznov SM, et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem 1994 26801-26805.
8. Ahn JD, Morishita R, Kaneda Y,et al. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ. Res, 2002,90 ,1325-1332.
9. Ahn JD, Morishita R, Kaneda Y, et al. Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia. Gene Therapy, 2002. 9:1682-1692.
10. Kawamura I, Morishita R, Tsujimoto S, et al. Intravenous injection of oligodeoxynucleotides to the NF-kappaB binding site inhibits hepaticmetastasis of M5076 reticulosarcoma in mice. Gene Ther, 2001,8:905-912.
11. Euning U, Wilhelm O, Nishiguchi T, et al. Rel transcription factors contribute to elevated urokinase expression in human ovarian carcinoma cells. Eur J Biolchem, 1999, 259: 143-148.
12. Das R, Philip S, Mahabeleshwar GH, et al. Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression. IUBMB Life, 2005 ,57:441-447.
1. Ichikawa H, Takada Y, Shishodia S, et al. Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through suppression of nuclear factor- {kappa} B (NF-{kappa}B) activation and NF-{kappa}B-regulated gene expression. Mol Cancer Ther, 2006, 5:1434-1445.
2. Lee IK, Ahn JD, Kim HS, et al. Advantages of the circular dumbbe decoy in gene therapy and studies of gene regulation. Curr Drug Targets, 2003, 4:619-623.
3. Hosoya T, Takeuchi H, Kanesaka Y, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters, 1999,461:136-140.
4. Mahabeleshwar GH, Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem, 2003,278: 6209-6221.
5. Sen R, Baltimore D. Inducibility of the immunoglobulin enhancer blinding protein NF-B by a posttranslational mechanisn [J]. Cell,1986, 46(5):705-716.
6. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene reguation. Am J Respir Cell Biol, 1997,17:3-9.
7. Turco MC, Romano MF, Petrella A,et al. NF-kB /Rel-mediated regulation of apaptosis in hematologic malignancies and normal hematopoietic progenitors. Leukemia, 2004, 18: 11-17.
8. tomita N, Morishita R, Lan HY, et al. In vivo administration of a nuclear transcription factor-kappa B decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol, 2000, 11:1244-1252.
9. Takeshi H, Hiroaki T, Yoshiyuki K, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters,1999 461:136-140
10. Cailin C, Leonard C, Edelstein, et al. The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor bcl-x[J]. Mole Cell Biol, 2002, 20(8):2687～2695.
11. Viatour P, Bentires AM, Chariot A, et al. NF-kappaB2/P100 induces Bcl-2 expression [J]. Leukemia, 2003,17(7):1349～1359.
12. Wester GA, Perkins ND.Transcriptional crosstalk between NF-kappa B and p53[J].Mol Cell Biol, 1999, 19: 3485-3495.
13. Ravi R, Mookerjee B, van Hensbergen Y, et al. p53 mediated repression of nuclear factor kappa B Rela via the transcriptional integrator p300[J].Cancer Res, 1998,58: 4531 4536.
14. Hinz M, Krappmann D, Eichten A, et al. NF-kappaB function in growth control: regulation of cyclin Dl expression and Go/G1-to-S-phase transition. Mol Cell Biol, 1999,19:2690-2698.
1. Yu L, Yuan JL, Wu GJ, et al. Expression and significance of Bcl-2 and Bag-1 in renal cell carcinoma. J Fourth Mil Med Univ, 2002, 23:1303-1305.
2. Yang LJ, Wang WL.Expression of Bcl-2 and Bax proteins in hepatocellar carcinoma tissue.J Fourth Mil Med Univ, 2002,23:337-340.
3. Hosoya T, Takeuchi H, Kanesaka Y, et al. Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides. FEBS Letters, 1999, 461:136-140.
4. Mahabeleshwar GH, Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem, 2003,278: 6209-6221.
5. Sen R, Baltimore D. Multiple nuclear factors interect the immuno-globulin enhancer sequences. Cell, 1986,46: 705.
6. Sasaki N, Morisaki T, Hashizume K, et al. Nuclear Factor-κB p65 (RelA) Transcription Factor Is Constitutively Activated in Human Gastric Carcinoma Tissue. Clin Cancer Res, 2001, 7:4136-4142.
7. Bours V, Dejardin E, Goujon-Letawe F, et al. The NF-kappaB transcription factor and cancer: high expression of NF-kappaB and IkappaB related proteins in tumor cell lines. Biochem Pharmacol,1994,47:145-149.
8. Nakshatri H, Bhat-Nakshatri P, Martin D A, et al. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol, 1997, 17: 3629-3639.
9. Wang W, James L. Abbruzzese DB, et al. The Nuclear Factor-kappaB RelA Transcription Factor Is Constitutively Activated in Human Pancreatic Adenocarcinoma Cells. Clin Cancer Res, 1999, 5:119-127.
10. Li Y, Rizvi SM, Ranson M,et al. 213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model[J]. Br J Cancer ,2002 ,86:1197-1203.
11. Shariat SF, Monoski MA, Andrews B, et al. Association of plasma urokinase- type plasminogen activator and its receptor with clinical outcome in patients undergoing radical cystectomy for transitional cell carcinoma of the bladder. Urology, 2003, 61: 1053-1058.
12. Turco MC, Romano MF, Petrella A,et al. NF-kB /Rel-mediated regulation of apaptosis in hematologic malignancies and normal hematopoietic progenitors. Leukemia, 2004,18: 11-17.
13. Viatour P, Bentires AM,Chatiot A,etal. NF-kappaB2/p100 induces Bcl- 2 expression. Leukemia, 2003, 17: 1349-1359.
1. Grumont RJ, Gerondakis S. Rel induces interferon regulatory factor4 (IRF-4) expression in lymphocytes: modulation of interferon- regulated gene expression by Rel/nuclear factor kappa B.J ExpMed, 2000, 1919:1281-1292.
2. Chen F,Castranova V,Shi X,et al.New insights into the role of nuclear factor-κB,a ubiquitous transcription factor in the initiation of diseases.Clin Chem,1999,45:7-17.
3. Raffi G,Raelene G,Mathis G,et al. Rel/NF-κB transcription factors: key mediators of B-cell activation. Immunol Rev 2000, 176:134-140.
4. Aradhya S, Nelson DL. NF-kappaB signaling and human disease.Curr Opin Genet Dev, 2001,11:300-306.
5. Tak PP, Firestein GS. NF-kappaB:a key role in inflammatory diseases. J Clin Invest,2001,107:7-11.
6. Baldwin AS Jr. Series introduction: the transcription factor NF kappa B and human disease.J Clin Invest,2001,107:3-6.
7. Sen R, Baltimore D. Multiple nuclear factors interect the immuno-globulin enhancer sequences. Cell, 1986,46: 705.
8. Thompson JE, Phillips RJ, Erdjument-Bromage H,et al.IkappaB-beta regulates the persistent response in a biphasi activation of NF-kappaB. Cell,1995,80:573-582.
9. Silverman N, Maniatis T. NF-kappaB signaling pathways in mammalian and insect innate immunity.Genes Dev, 2001, 15:2321-2342.
10. Zandi E, Karin M. Bridging the Gap: Composition, regulation and physiological function of IκB kinase complex. Mol Cell Biol,1999,19: 4547-4551.
11. Magnani M, Crinelli R, Bianchi M, et al. The ubiquitindependent proteolytic system and other potential targets for the modulation of nuclear factor-kB.Curr Drug Targets,2000, 1:387-399.
12. Zhang G, Ghosh S. Molecular mechanisms of NF-kappaB activation induced by bacterial lipopolysaccharide through Tol-likereceptors. J Endotoxin Res, 2000,6 453-457.
13. Gorlich D, Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol,1999,15:607-660.
14. Blackwell TS, Christman JW. The role of nuclear factor-kappa B in cytokine gene regulation. [J]. Am J Respir Cell Biol, 1997,17:3-9.
15. Tai DI, Tsai SL, Chen YM. Hepatitis C Virus Infection: Implications for pathogenesis and hepatocarcinogenesis. Hepatology, 2000,31: 785-787.
16. Gilmore TD. Multiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel.Oncogene,1999,18:6925-6937.
17. Chen F, Castranora V,Shi XL.New insights into the role of NF-κB,an obigutious transcription factor in the initiation of disease.Clin Chem,1999, 45:7-17.
18. He Z, Xin B,Yang X. Nuclear factor-kappaB activation is involved in LMP1-mediated transformation and tumorigenesis of rat-1 fibroblasts. Cancer Research, 2000,60: 1845-1851.
19. Wu MX, Ao Z,Prasad KV,et al.IEX-1L,an apoptosis inhibitor involved in NF-kappaB- mediated cell survival.Science,1998,281:998-1001.
20. Zong WX,Edelstein LC, Chen C,et al.The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Dev, 1999,13:382-387.
21. Feuillard J, Schuhmacher M, Kohanna S, et al.Inducible loss of NF-kappaB activity is associated with apoptosis and bcl-2 down-regulation in Epstein-Barrvirus-transformed B lymphocytes. Blood,2000,95:2068-2075
22. Wester GA, Perkins ND. Tanscriptional cross talk between NF-kappaB and p53. Mol Cell Biol,1999, 19: 3485-3495.
23. Huang Y,Johnson KR, Norris JS, et al. Nuclear factor-kappaB/IkappaB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells. Cancer Res, 2000, 60:4426-4432.
24. Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene, 1999, 18: 6910-6924.
25. Wang CY, Cusack JC Jr, Liu R, et al. Control of inducible chemoresistance: Enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med, 1999, 5:412-417.
26. Bentires-Alj M, Hellin AC, Ameyar M, et al. Stable inhibition of nuclear factor kappaB in cancer cells does not increase sensitivity to cytotoxic drugs. Cancer Res, 1999, 59: 811-815.
27. Dumont A, Hehner SP, Hofmann TG, et al.Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB, Oncogene, 1999, 18: 747-757.
28. Kasibhatla S, Genestier L, Green DR. Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor kappaB. J Biol Chem,1999,274:987-992.
29. Guttridge DC, Albanese C, Reuther JY, et al. NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol, 1999, 19: 5785-5799.
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