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@#Objective: To investigate the role of CpG ODN (CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3 (melanoma antigen gene -3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means (MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTLon target BIU-87 cells (P< 0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced (all P<0.05), and the proliferation ability of xenograft tumor was decreased (P<0.05). Compared with other sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice (P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased（P<0.01）while Bax expression significantly increased（P<0.05 or P<0.01）on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.
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To investigate the inhibitory mechanism of quercetin on growth of human bladder cancer cell line(BIU-87). BIU-87 cells were cultured in vitro, and co-cultured with varying concentrations of quercetin, and the anti-proliferative activity was determined by CCK-8 assay. Apoptosis of quercetin-induced BIU-87 cells and cell cycle were determined by flow cytometry analysis. Expressions of Bal-2 and Bal-xL, and related proteins in TAK1/JNK signal pathway were measured using Western blot analysis. After treatment with quercetin for 24 h and 48 h, the proliferation of BIU-87 cells was significantly suppressed in a dose-dependent manner according to CCK-8 assay(P<0.05). The flow cytometry analysis indicated that each group of quercetin leads to a significant higher percentage of apoptosis of BIU-87 cells than control group after treatment with quercetin for 24 h and 48 h; In G₀/G₁ period, cells reduced, while the amount of cells in G₂/M period increased, and cells in S period remained the same amount. Expressions of Bal-2, Bal-xL, p-TAK1, p-MKK4/7, p-JNK decreased in BIU-87 cells after treatment with quercetin. Quercetin could inhibit the proliferation and promote the apoptosis of BIU-87 cells. The mechanism may be correlated with the inhibition of TAK1/JNK signaling pathway, which led to the further decrease in expressions of Bal-2 and Bal-xL.
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OBJECTIVE:To investigate inhibitory effects of matrine on the proliferation of human bladder cancer BIU-87 cells and its mechanism. METHODS:The cell viability was detected by MTT assay and inhibitory rate of cell proliferation was calculat-ed after human bladder cancer BIU-87 cells were treated with 0(negative control),0.5,1.0,2.0 and 4.0 mg/ml matrine for 24,48 and 72 h,respectively. After treated with 0 (negative control),0.5,1.0,2.0 and 4.0 mg/ml matrine for 48 h,the cell cycle and apoptotic rate were detected by flow cytometry;the expression of Survivin,Caspase-3 and Caspase-7 protein were detected by Western blot assay. RESULTS:Compared with negative control,the proliferation of BIU-87 cells were significantly inhibited after incubated with 1.0-4.0 mg/ml matrine for 24,48 and 72 h(P<0.05 or P<0.01),and inhibitory rate of cell proliferation increased in concentration and time-dependant manner;after treated for 48 h,the percentage of G0/G1 phase cells and apoptotic rate in-creased,while the percentage of cells at S phase and G2/M phase were decreased (P<0.05 or P<0.01);the expression of Cas-pase-3 and Caspase-7 protein increased,while the expression of survivin protein decreased after incubated with 0.5-4.0 mg/ml ma-trine for 48 h. CONCLUSIONS:Matrine can inhibit the proliferation of human bladder cancer BIU-87 cells,block the cell cycle and induce apoptosis;its mechanism may be related to the expression regulation of Survivin,Caspase-3 and Caspase-7.
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Objective To investigate the expression of p 120-catenin in human bladder cell line BIU-87 and T24.And to explore the effect of down-regulation of p120-catenin on proliferation, invasion and adhesion of BIU-87 cell line. Methods The study was from Aug .2010 to May.2011.Real-time RT-PCR and Western blot were used to examine the expression of p 120-catenin in human bladder cell line BIU-87 and T24.There were three study groups:non-transfected group , the group transfected with p 120-catenin siRNA and the group transfected with control siRNA .p120-catenin siRNA was used to decrease the expression of p120-catenin.MTT assay and colony formation assay were used to study the BIU-87 cell growth and cell pro-liferation.The invasion of BIU-87 cells was measured by Transwell chamber assay .Cell adhesion artificial re-constituted basement membrane assay was used to examine the change of BIU-87 cell adhesion capacity . Results In both BIU-87 and T24 cells there were the expressions of p 120-catenin.But the expression in BIU-87 cells (0.11±0.39) was more than that in T24 cells (0.48±0.17).The decreased of p120-catenin ex-pression could enhance the proliferation (182.7±13.4%) and the invasiveness (217.5±15.9) and decrease the adhesion capacity (57.3±6.4%) of BIU-87 cells. Conclusions There is higher expression level of p120-catenin in lower-grade malignant bladder cancer cells .The down-regulation of p120-catenin promotes the bladder cancer proliferation and invasiveness of bladder carcinoma cells , and inhibited the bladder canc-er cell adhesion .
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Objective To explore the influence on the expression of elF4E and heparanase by antisense oligodeoxyribonucleotides (ASODN) transfection in human bladder carcinoma BIU-87 cells.Methods After transfecting the 2.5,5.O,7.5 μg/ml eIF4E ASODN into BIU-87 cells,at 24 h,48 h and 72 h,a cell control group (no transfection),a blank control group (empty liposomes) and a non-sense control group (transfected with non-sense ASODN) were established.The expression of eIF4E,HPA and mRNA were detected by in situ hybridration.The expression of eIF4 and HPA protein were detected by immunocytochemistry.SPSS 13.0 statistical software was used for statistical analysis.Results The expression quantity of eIF4E protein and mRNA were lower in the experimental groups ( protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:3.55 ±0.52,3.52 ±0.51,3.22.±0.44 respectively; 5.0 μg/ml group:3.43 ±0.47,3.41 ± 0.46,3.19 ± 0.41 respectively ; 7.5 μg/ml group:2.36 ± 039,2.33 ± 0.37,2.05 ± 0.30 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:3.19 ±0.41,3.13 ±0.39,2.90 ±0.38 respectively ; 5.0 μg/ml group:3.07 ± 0.39,2.94 ± 038,2.27 ± 0.37 respectively ; 7.5 μg/ml group:2.22 ± 037,2.06 ± 0.30,1.95 ± 0.29 respectively.All data were less than those in the control groups (P ＜0.05).The expression quantity of HPA protein and mRNA were lower in experimental groups (protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:4.44 ±0.55,4.40 ±0.54,3.99 ±0.52 respectively; 5.0 μg/ml group:4.41 ±0.55,4.21 ±0.53,3.77 ±0.50 respectively; 7.5 μg/ml group:4.02 ±0.52,3.98 ±0.52,2.32 ±0.37 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:4.12 ±0.51,3.75 ± 0.50,3.63 ± 0.45 respectively ; 5.0 μg/ml group:4.00 ± 0.51,3.71 ± 0.50,3.54 ± 0.44respectively ; 7.5 μg/ml group:3.87 ± 0.52,3.61 ± 0.49,2.08 ± 0.30 respectively).All data were less than in control groups ( P ＜ 0.05 ).There was a positive correlation on expression of HPA protein and eIF4E protein by inhibitory effect of eIF-4E ASODN (protein r=9.23,mRNA r=9.59,P ＜0.01).Conclusion eIF-4E ASODN might be used to inhibit the expression of eIF-4E gene and HPA gene in bladder cancer BIU-87 cells.
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Summary: The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.
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Objective: To investigate the expression and identification of CH50 polypeptide in bladder cancer cell line BIU-87 by gene transfection. Methods:The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection Lipofectimine TM2000.The expressed product was identified by immunohistochemistry and Western blot method. The expression of the transfected gene was identified by RT-PCR. Results:The BIU-87 cells could produce CH50 polypeptide by transfection. Conclusion:When the vector of pCH510 was transfected into BIU-87 cells in vitro, the cell adhension characteristic would be changed.