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AIM:To observe the effects of the combination of berberin and epirubicin on the cell cycle of T24 bladder cancer cells and the underlying mechanisms.METHODS:The cancer cells were exposed to epirubicin in the presence or absence of different concentrations of berberin.The viability of the cancer cells was determined by MTT assay.The cell cycle distribution was detected by flow cytometry, and the protein levels of cyclin D1, CDK2, CDK4, P21 and P27 were detected by Western blot.RESULTS:Berberine markedly enhanced the inhibitory effect of epirubicin on the viability of T24 cells and promoted epirubicin-induced cell cycle arrest at G0/G1 phase as compared with the negative control cells.Epirubicin increased the protein expression of P27 and P21, both of which were enhanced by treatment with berberin.In contrast, berberin exposure further decreased the protein expression of cyclin D1, CDK2 and CDK4 in epirubicin-treated T24 cells.CONCLUSION:Berberine significantly promotes epirubicin-induced G0 /G1 phase arrest in human bladder cancer cells by up-regulating P27 and P21 expression and inhibiting the expression of cyclin D1, CDK2 and CDK4.
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AIM:To investigate the effect of small interfering RNA ( siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24.METHODS:The siRNA against ABCE1 was constructed and transfected into the T24 cells with LipofectamineTM 2000.The expression of ABCE1 was detected by RT-PCR and Western blot.Flow cytometry was used to detect the cell cycle.The effects of ABCE1 gene silencing on prolifera-tion, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion as-say, respectively.RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA.The cell cycle was arrested at G0/G1 phase and the cell number in S phase was decreased in the T24 cells.Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly.CONCLUSION:Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.
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Objective To observe the effects of brazilin on proliferation and apoptosis in T24 cells.Methods Trypan blue exclusion test was performed to detect the inhibition of brazilin on the growth of T24 cell lines in vitro cultured within different time.After exposure to different concentrations of brazilin,homogeneous bioluminescence assay was used to detect the inhibitory action of brazilin,Caspase-3 and Caspase-9 activity on T24 cells.Cellular apoptosis was measured by flow cytometry (FCM) and observed by laser scanning confocal microscope.Results Brazilin could significantly inhibit the proliferation of T24 cells after 8 hours,the inhibitory rates of the brazilin at concentration of 25,50,100,200 μg/ml against T24 cells respectively were 43.19 ％,60.73 ％,86.38 ％ and 93.89 ％ (P ＜ 0.05).After exposured to 50 μg/ml of brazilin,the inhibition ration to T24 cells increased with time prolonging (52.72 ％ in 4 h,60.73 ％ in 8 h,91.77 ％ in 24 h,96.41％ in 48 h) (P ＜ 0.05).The activity of Caspase-3 and Caspase-9 increased slightly when brazilin was at 25 μg/ml,but there was no statistical differences compared with that in the control group (P ＞ 0.05).When cells were treated with an increase of the concentration of brazilin from range of 7.5-60 μg/ml for 16 hours,the apoptosis ratio in turn showed a upward trend of 0.15 ％,1.35 ％,2.91％,34.76 ％.It could be seen by laser scanning confocal microscope that the apoptosis occurred in the cells.Conclusion Brazilin can effectively inhibit the proliferation of T24 cells and induce apoptosis in a dose and time dependent manner.
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Objective To investigate the regulating function of p120-catenin (p120ctn) on proliferation and invasion of human bladder cancer cell T24.Methods Special siRNA was used to silence the expression of p120ctn in T24 cells.MTT assay was used to examine the T24 cell growth rate.The invasion of T24 cells and the control cells were measured by transwell chamber assay.Results The silencing of p120ctn could improve the proliferation and the invasiveness of T24 tumor cells.Transwell chamber assay showed that the in vitro invasion function of T24 cells was significant increase after p120ctn-siRNA.Conclusion p120ctn inhibited the bladder cancer proliferation and invasiveness of bladder carcinoma cells.
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Previous researches showed that the expression level of E-Cad in most infiltrating cancer cells was reduced or negative.This study explored whether 4HPR restrained the infiltration of bladder cancer cells through regulating the expression of E-Cad.The infiltrating bladder cancer cells T24 were cultured,and then treated by a proper dosage of drug.Their viability was a determined by MTT method.Western blotting and RT-PCR were adopted to detect the changes of E-Cad gene expression at both protein and mRNA levels.Moreover,immunofluorescent staining and confocal fluorescence microscopy were employed for the observation of the expression of E-Cad.The result showed that,at both mRNA and protein levels,the expression level of E-Cad in T24 cells treated by 4HPR was significantly higher than that of control group,while the β-Cat expression was also relocated from the cell nucleus to cytoplasm.Our findings suggested that the regulatory function of 4HPR on infiltration of bladder cancer cells T24 is at least partly achieved by regulating the expression of E-Cad.
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This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer.Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA,i.e.,pSilencer-LRIG3-siRNA.After confirmation,the vector was transfected into HEK293 cells to make a replication-deficient adenovirus,pAd-LRIG3-siRNA,which was then introduced into bladder cancer T24 cells.RT-PCR,Western-blotting were performed to detect the levels of LRIG3 mRNA and proteins.Cells number was determined by using MTT test.Hoechst33258 staining,transmission microscopy,flow cytometery were conducted to examine the cell apoptosis.Three groups included a blank control group,a negative control group (containing non-interfering plasmids) and a pAd-LRIG3-siRNA group.Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells.The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group (P＜0.01).The siRNA also caused apoptotic changes of some cells,with the apoptosis rate being (17.69±0.75)％,which was significantly different from that of the control group (P＜0.01).It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and,to some extent,inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells.Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.
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The expression of octamer binding factor 4 (Oct4) gene in bladder cancer cell line T24 and its effects on the biological characteristics of the cells were investigated.RT-PCR and Western blot were employed to detect the expression of Oct4 in T24 cells.The changes of biological charac-teristics in T24 cells were analyzed before and after gene-silencing by Boyden chamber and MTT.The results showed that the expression of Oct4 gene was detectable in T24 cells by RT-PCR and Western blot.The expression of Oct4 gene and protein was down-regulated by siRNA,and average number of transwell cells in interference group,negative control group and blank control group was 101.40±4.56,104.20±10.03 and 111.00±11.90,respectively.There was significant difference in the proliferation ability of the cells from 48 h,72 h to 96 h after the interference by siRNA between in-terference group and negative group or blank control group (P<0.05).It was suggested that Oct4 gene was related with proliferation ability ofT24 cells,but not with invasive capability.
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Objective: To investigate the effects of extracellular domain-truncated mutant of hepaCAM gene on biological behaviors of bladder carcinoma cells in vitro. Methods: Human cell line T24 was transfected with pEGFP-N2-hepaCAM, pEGFP-N2-hepa CAM-mt or mock plasmid pEGFP-N2 by lipofectamine mediation. The stable colonies were obtained by G418 screening. Laser confacal microscopy was used to detect the location of hepaCAM-mt in the T24 cells, Western blotting was used to determine the expression of protein in stable cells. Cell adhesion and motility abilities were tested by cell adhesion and detachment assays and wound healing and Matrigel invasion assays. The proliferation ability was investigated by MTT assay. Results: In well-spread cells, mutant proteins were localized on the perinuclear membrane like wild-type hepaCAM protein; after the cells contacted with each other, hepaCAM-mt protein had non-specific expression in the cells, which was significantly different with the localization of the hepaCAM proteins. T24 cell clone which acquired stable expression of hepaCAM-mt protein had decreased adhesion and motility abilities and enhanced proliferation potential compared with the cells with wild-type hepaCAM gene expression. Conclusion: The deletion of extracellular domain in hepaCAM gene was important for the localization of hepaCAM protein but had no significant effect on the proliferation, adhesion, and migration of T24 cells. The extracellular domain of hepaCAM was necessary for exerting normal functions of hepaCAM gene.
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Objective:To investigate the proliferation inhibitory effect of crocin on transitional cell carcinoma of urinary bladde(rTCCB). Methods:MTT assay was used to evaluate the inhibitory effect of Crocin on the proliferation of T24 cells. Microscope was used to observe the morphological changes of the T24 cells. Flow cytometry was used to measure the cell cycle. T24 cells were inoculated in to BALB/c nude mice to establish bladder cancer model. After treatment with Crocin,the inhibitory effects were observed on the growth of tumors. Results:The proliferation of T24 cells were inhibited remarkably. Morphological changes of cells were observed. Flow cytometric profiles revealed that crocin led to the increase of the cells in G0/G1 phase and the decrease of cells in S phase and G2/M(P