The effect of endogenous transforming growth factor β_1 on the growth of bladder cancer cells in vitro

Objective To investigate the influence of endogenous transforming growth factor β1(TGFβ1) on the cell cycle regulation and proliferation of bladder cancer. Methods A constructed replication defective retroviral vector pRevTβ-AS, which carried antisense RNA of TGFβ1.was transfected to a bladder cancer cell line EJ. The proliferation and clone-formation of transferred cells were observed in vitro,and the alteration of cell cycle was also detected by flow cytometric analysis. Results TGFβ1 antisense RNA was transferred into EJ cell and expressed efficiently. After the inhibition of target gene expression in EJ cells, the reduced growth and clone-formation rates were demonstrated, and the proliferative indexes were decreased by 12 % . The ratios of GO and G1 stage cells to June 2003 Vol12 No2 the antisense RNA-transfected EJ cells were increased, simultaneously,the ratio of S stage cells to the antisense RNA-transfected EJ cells ratios were decreased, compared with the control group. Conclusion The

Effect of temsirolimus on bladder cancer cells in vitro and in vivo

Objective To examine the effects of temsirolimus, an inhibitor of mammalian target of rapamycin,on bladder cancer cell lines T24 and BIU-87 in vitro and in vivo for purpose of evaluating the probability of mTOR targeted therapy for bladder cancer. Methods After

Over-expression of LRIG3 Suppresses Growth and Invasion of Bladder Cancer Cells

The purpose of this study was to investigate the impact of leucine-rich repeats and immu- noglobulin-like domains 3 （LRIG3） on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRIG3 and the blank plasmid serving as control were transfected into the bladder cancer cell lines, T24, EJ and BIU-87, and the expression levels of LRIG3 mRNA and protein were de- tected by using real-time PCR and Western blotting. The changes in the cell cycle and apoptosis were examined by using flow cytometry. The invasive ability was measured by Transwell assay, and CCK-8 assays were used to measure the proliferation of cells. As compared with the control group, the LRIG3 mRNA and protein expression levels in LRIG3 cDNA-transfected group were raised significantly （P〈0.05）. The average number of cells with up-regulated LRIG3 passing through the inserted filter was decreased significantly as compared with the control group （P〈0.05）. Up-regulation of LRIG3 also could inhibit proliferation and induce apoptosis of T24, EJ and BIU-87 cells. Except BIU-87, the T24 and EJ cells transfected with LIRG3 eDNA were arrested in G0/G1 phase compared to the control group （P〈0.05）. In conclusion, the over-expression of LRIG3 could influence the cell cycle and invasion, in- hibit proliferation and induce apoptosis in the three bladder cancer cell lines.

Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells

The effects of combined RNA interference(RNAi) of human telomerase RNA(hTR) and human telomerase reverse transcriptase(hTERT) genes on telomerase activity in a bladder cancer cell line(BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,phTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,BIRC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA,NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

Procaine Inhibiting Human Bladder Cancer Cell Proliferation by Inducing Apoptosis and Demethylating APAF1 CpG Island Hypermethylated

Studies have shown that aberrant DNA methylation of apoptotic protease activating factor-1（APAF1） is an important epigenetic mechanism of gene regulation in the progression of bladder cancer.In this article,we have proved that procaine,an inhibitor of DNA methyltransferases,could inhibit the proliferation of T24 and 5637 human bladder cancer cells by inducing their apoptosis.The mechanism studies reveal that procaine could induce demethylation of APAF1 gene in T24 or 5637 cells,subsequently activating caspase-3/9.It was also shown that the serum soluble fas ligand（sFasL） was activated,and the expression of matrix metallopeptidase 9（MMP-9） was down-regulated.Procaine seems to induce cell death by different pathways,and it might be used as a potential agent for bladder cancer treatment.

Preliminary Study of the in vitro Growth Inhibition of Human Bladder Cancer Cell Line BIU-87 by Arsenic Trioxide

To study the effects of arsenic trioxide (As2O3) on the in vitro growth of human bladder cancer cells and the mechanisms. The growth inhibition rates of human bladder cancer cell line BIU87 by various concentrations of As2O3 were detected by using MTT method. Cell apoptosis was detected by in situ terminally labeled transferase technique and bcl-2 gene expression of BIU-87 cells was observed by SABC immunohistochemical method. The results showed that As2O3 could inhibit the growth of BIU-87 effectively in a dose-dependent manner. After drug's action, the apoptotic bladder cancer cells were obviously increased, which depended on the prolongation of the action time and Bcl-2 expression of BIU-87 cells was decreased significantly. It was suggested that As2O3 could significantly inhibit the growth of bladder human cancer cells. Inducing cell apoptosis by down- regulating the expression of hcl-2 gene might be one of its action mechanisms.

Effects of Gene Tranfection with CH50 Polypeptide on the Invasion Ability of Bladder Cancer Cell Line BIU-87

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.

CLINICAL SIGNIFICANCES OF THE EXPRESSION OF METALLO- THIONEIN IN FIVE TYPES EPITHELIUM CELL CANCER

Objective： To study the expressions of （CSC）, bladder transitional cell cancer metallothionein and the significances in cervical squamous cell cancer （BTC）, esophageal squamous cell cancer （ESC）, gastral tubular adenocarcinoma （GC） and large intestinal tubular adenocarcinoma （LIC）. Methods： lmmunohistochemical method was used to examine the expression rates of MT in five types of cancer tissue. Results： The expression rates of MT were 75.00% （24/32） in ESC, 52.27% （46/88） in GTC, 59.46% （44/74） in LIC, 64.86% （48/74） in BTC and 58.57% （41/70） in CSC respectively. The positive rates of MT expression were higher in low differentiation and deep muscular group than those in medium or high differentiation and superficial muscular invasion group （P〈0.05）. Conclusion： The expression of MT is related to differentiation degree and invasion degree.