| Part 1 Expression and Clinical Significance of nuclear factor kappa B and urokinase-type plasminogen activator in Human Transitional Cell Carcinoma of BladderObject 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 BIU871,Effects of Nuclear Factor kappa B"decoy strategy"-Circular Dumbbell Oligodeoxynucleotides on Proliferation and Invasion of Human Bladder Cancer Cell Lines BIU87Object 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 BIU87Object 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 BIU87Object 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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