Bladder Cancer Cell Line BIU-87 Research Articles

Ferroptosis-Related Long Noncoding RNAs Have Excellent Predictive Ability for Multiomic Characteristics of Bladder Cancer.

Background The role of ferroptosis-related long non-coding RNAs (lncRNAs) in bladder cancer remains elusive. This study is aimed at examining the prognostic role of ferroptosis-related lncRNAs in bladder cancer. Materials and Methods The transcriptomic matrix and clinical information of patients with bladder cancer were obtained from The Cancer Genome Atlas (TCGA) database. A ferroptosis-related lncRNA signature was developed via the least absolute shrinkage and selection operator (LASSO) analysis using data from the training cohort, and the signature was further validated using data from the test cohort. The role of AC006160.1, the most significant lncRNA in the risk signature, was examined in various cell lines including SV-HUC-1, BIU-87, HT-1376, T24, RT4, RT-112, 5637, and UMUC3. The pcDNA3.1-AC006160.1 plasmid was constructed and transfected into the bladder cancer cell lines T24 and BIU-87. In addition, cell proliferation, colony formation, transwell, and wound healing assays were performed to examine the biological function of AC006160.1 in T24 and BIU-87 cell lines. Results Two clusters were identified through consensus clustering based on prognostic ferroptosis-related lncRNAs. A 5-lncRNA risk signature was successfully constructed using data from the training cohort and validated using data from the test cohort. The risk signature had excellent ability to predict survival outcomes, clinical stages, pathological grades, expression of immune checkpoints, and immunotherapeutic responses in bladder cancer samples. Furthermore, AC006160.1 expression was found to be lower in the cancer cell lines BIU-87, T24, RT4, RT-112, and 5637 than in the normal control cell line SV-HUC-1. Cell proliferation, colony formation, transwell migration, and wound healing assays validated that overexpression of AC006160.1 significantly inhibited the proliferation and invasion abilities of both T24 and BIU-87 cells. Drug sensitivity analysis revealed that patients with high expression of AC006160.1 were sensitive to metformin and methotrexate, and the results were further validated via in vitro drug experiments. Conclusions Ferroptosis-related lncRNAs play a vital role in predicting the multiomic characteristics of bladder cancer. The lncRNA AC006160.1 serves as a protective factor for the development of bladder cancer.

Association between TOP2A, RRM1, HER2, ERCC1 expression and response to chemotherapy in patients with non-muscle invasive bladder cancer

PurposeThis study aimed to detect the expression levels of topoisomerase IIα (TOP2A), ribonucleotide reductase catalytic subunit M1 (RRM1),c-erbB-2 (HER2) and excision repair cross complementing group 1 (ERCC1) in non-muscular invasive bladder cancer (NMIBC) and explore the correlation between the expression of these genes and NMBIC sensitivity to pirarubicin or gemcitabine treatment. Materials and methodsNMIBC patient tissues and the bladder cancer cell lines BIU-87 and KK47 were selected for the exploration of drug sensitivity in vitro. Immunohistochemistry was used to examine protein expression in tissues. Reverse transcription-polymerase chain reaction (RT-qPCR) and a Western blot assay were used to detect the mRNA and protein levels in cells. The cell IC50 value was evaluated by an MTT assay. Flow cytometry was used to sort the cell subpopulations. ResultsIn the pirarubicin-treated group, the patients with high TOP2A expression experienced lower recurrence rates than those with low TOP2A expression, whereas TOP2A and HER2 co-expression resulted in higher recurrence rates. The patients with low RRM1 expression, especially those with low ERCC1 expression, experienced lower recurrence rates than the patients with high RRM1 expression in the gemcitabine-treated group. Tumour cells with high TOP2A expression were highly sensitive to pirarubicin, and TOP2A+ HER2- cells were more sensitive to pirarubicin than TOP2A+ HER2+ cells. Cells with low RRM1 expression levels were sensitive to gemcitabine, and RRM1−ERCC1- cells were more sensitive to gemcitabine than RRM1−ERCC1+ cells. ConclusionHigh TOP2A expression or low RRM1 expression could predict the sensitivity of NMIBC to pirarubicin or gemcitabine treatment. HER2 and ERCC1 expression may affect the effect of TOP2A and RRM1, thus affecting the efficacy of chemotherapeutic drugs.

Effect the Mechanism of Emodin Combined with Cp-1 on Proliferation and Apoptosis of Bladder Cancer Cell Line BIU-87

To investigate the effect of emodin combined with Cp-1 on the proliferation and apoptosis of bladder cancer cell line BIU-87. BIU-87 cells were randomly divided into control group, Cp-1 group, emodin group, Cp-1 combined with emodin group. Flow cytometry was used to detect the apoptosis of BIU-87 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to detect the proliferation of BIU-87 cells. Western blot and reverse transcription–polymerase chain reaction were used to detect the recepteur d’origine nantais, Caspase-3 and B-cell lymphoma 2 expressions. Compared with control group, the cell survival rate of Cp-1 group, emodin group and Cp-1 combined with emodin groupwas significantly decreased, especially in Cp-1 combined with emodin group, which was significantly lower than that of Cp-1 group and emodin group (p<0.05), while the cell apoptosis rate of Cp-1 group, emodin group and Cp-1 combined with emodin group was significantly increased, especially in Cp-1 combined with emodin group, which was significantly higher than that of Cp-1 group and emodin group (p<0.05) and the expression of recepteur d’origine nantais decreased significantly (p<0.05). Inhibition of recepteur d’origine nantais expression suppressed B-cell lymphoma 2 and increased Caspase-3 significantly (p<0.05). Our results indicated that emodin combined with Cp-1 could enhance the apoptosis and inhibit proliferation of bladder cancer cells. The mechanism may be that emodin can inhibit the expression of recepteur d’origine nantais, down regulated the expression of B-cell lymphoma 2, which increased the expression of caspase-3 in bladder cancer cells and promoted the apoptosis of bladder cancer cells.

Isoflurane promotes epithelial-to-mesenchymal transition and metastasis of bladder cancer cells through HIF-1α-β-catenin/Notch1 pathways

AimsEpithelial-to-mesenchymal transition (EMT) facilitates cell migration and invasion, and contributes to metastasis in bladder cancer. Within the perioperative period, anesthetic such as isoflurane have been found to affect cancer prognosis. In the study, we reported the tumor-promoting effect of isoflurane in bladder cancer. Materials and MethodsHuman bladder cancer cell lines T24 and BIU-87 were exposed to isoflurane at different concentrations. The immunofluorescent staining of Ki67, Annexin V-FITC/PI staining, Transwell invasion assays and wound-healing assays were performed to assess cell proliferation, apoptosis, invasion and migration. Expressions of EMT markers (E-cadherin, N-cadherin and Vimentin) and metastatic markers (Snail-1, Slug-1 and MMP-2/9) were determined by immunoblotting. Orthotopic tumor models and mice given tail vein injection of T24 cells were developed with or without 4-h exposure to 2% isoflurane. Key findingsWe found isoflurane promoted bladder cancer cell proliferation, invasion and migration but reduce apoptosis in a concentration-dependent manner. In addition, isoflurane was shown to increase HIF-1α and its nuclear accumulation in bladder cancer cells. HIF-1α knockdown inhibited bladder cancer cell proliferation and delayed EMT, which was reversed in the presence of 4-h exposure to 2% isoflurane. Likewise, we found isoflurane modulated β-catenin/Notch1 pathways via HIF-1α. In vivo studies showed that isoflurane exposure accelerated formation of orthotopic bladder tumor and promoted hepatic metastases from carcinoma of the bladder. SignificanceTaken together, our study demonstrates that a frequently used anesthetic can exert a protumorigenic effect on bladder cancer. Isoflurane may serve as an important contributory factor to high recurrence following surgery.

MiR-155 affects proliferation and apoptosis of bladder cancer cells by regulating GSK-3β/β-catenin pathway.

GSK-3β negatively regulates Wnt/β-catenin signaling pathway. The abnormal miR-155 expression is associated with bladder cancer. Bioinformatics analysis revealed a complementary binding site between miR-155 and GSK-3β mRNA. This study investigated the role of miR-155 in the proliferation and apoptosis of bladder cancer cells. The dual luciferase reporter gene assay validated the targeted regulation between miR-155 and GSK-3β. Tumor tissues and adjacent tissues were collected from bladder cancer patients and the expression of miR-155 and GSK-3β mRNA was detected by RT-PCR. Bladder cancer cell line BIU-87 cells were cultured in vitro and divided into miR-NC group and miR-155 inhibitor group. The expressions of miR-155, GSK-3β and β-catenin were compared, cell apoptosis was detected by flow cytometry, and cell proliferation was detected by EdU staining. Compared with adjacent tissues, miR-155 expression was significantly increased in bladder cancer tissues, and GSK-3β mRNA expression was significantly decreased. There was a targeted regulatory relationship between miR-155 and GSK-3β. Compared with SV-HUC-1 cells, miR-155 expression in bladder cancer BIU-87 and 5637 cells was significantly increased, and GSK-3β expression was significantly decreased. Transfection of miR-155 inhibitor significantly increased GSK-3β expression in BIU-87 and 5637 cells, decreased β-catenin expression, increased cell apoptosis, and decreased cell proliferation. The increased expression of miR-155 plays a role in reducing the expression of GSK-3β and in promoting the pathogenesis of bladder cancer. Inhibition of miR-155 can up-regulate the expression of GSK-3β, inhibit the activity of Wnt/β-catenin pathway, attenuate proliferation and promote apoptosis of bladder cancer cells.

Effects of miR-1280 expression on the cell cycle and proliferation of bladder cancer by activating p21 gene expression

Objective To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line. Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24, 5637, J82, BIU-87 and normal bladder epithelial cells SV-HUC-1. miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280. The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR. Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter. Western blotting was used to detect the expressions of p21, cell cycle-dependent kinase 1 (CDK1), Cyclin A2 mRNA and protein in the two groups. Cell cycle was detected by flow cytometry, and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay. Results The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24, 5637, J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503±0.094, 0.611±0.054, 0.567±0.077, 0.257±0.032 and 1.014±0.090 respectively, with a significant difference (F=1.880, P<0.001). Compared with bladder cancer cell lines T24, 5637 and J82 cells, the expression of miR-1280 in BIU-87 cell was the lowest (P=0.026, P=0.003, P=0.008). Compared with the control group, the expre-ssion of miR-1280 in BIU-87 cell was significantly increased (1 041.000±157.500 vs. 1.023±0.118, t=6.606, P<0.001), and the expression of p21 mRNA was also significantly increased (5.280±0.660 vs. 1.007±0.070, t=6.440, P<0.001). Western blotting showed that p21 protein expression was up-regulated, CDK1 and Cyclin A2 protein expressions were down-regulated. ChIP experiments showed that compared with the miR-NC transfection group, the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246±0.171 vs. 0.519±0.087, t=3.787, P=0.009). The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360%±3.064% vs. 46.970%±3.971%, t=4.263, P=0.005). The results of MTT showed that compared with the control group, the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826±0.099 vs. 1.224±0.057, t=3.505, P=0.013). Conclusion miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene, blocking the progression of cell cycle and inhibiting cell proliferation, which provides a new direction for bladder cancer targeted therapy theory. Key words: MicroRNAs; Urinary bladder neoplasms; Proto-oncogene proteins p21 (ras); RNA activation

The influence and mechanisms of purple sweet potato anthocyanins on the growth of bladder cancer BIU87 cell

Objective: To observe the effect of purple sweet potato anthocyanins on the proliferation of bladder cancer cell line BIU87 and to investigate the molecular mechanisms. Methods: Bladder cancer BIU87 cells were cultured and exposed to anthocyanins at the different concentrations of 100, 200, 400, and 800 μg/ml respectively. The growth inhibition of anthocyanins on BIU87 cells were evaluated by morphometry and cell counting kit-8 (CCK-8) assay, and the cell apoptosis rate was detected by Flow cytometry (FCM). Results: Morphometry showed that the number of BIU87 cells decreased, the volume shrank, the intercellular space enlarged, the ability of cell adherence weakened, and the cell shape changed when the concentration of anthocyanins increased. CCK-8 assay showed that when 100, 200, 400, 800 μg/ml anthocyanins treated BIU87 cells for 48 h, the absorbance was 24 ± 0.07, 1.15 ± 0.11, 0.90 ± 0.08, 0.56 ± 0.09, respectively. Compared with the control group, anthocyanins-treated groups significantly inhibited the proliferation of BIU87 cells (P<0.05). FCM test showed that after treatment with different doses of anthocyanins, the apoptosis rate was 7.31%, 11.11%, 25.96%, 36.28%, respectively, and with the concentration of anthocyanins being higher, the apoptosis rate of BIU87 cells was being higher. Conclusion: Purple sweet potato anthocyanins can inhibit the growth of bladder cancer BIU87 cells through inducing cell apoptosis in a dose-dependent manner.

MicroRNA-34a Attenuates Metastasis and Chemoresistance of Bladder Cancer Cells by Targeting the TCF1/LEF1 Axis

Background/Aims: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear. Methods: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules. Results: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis. Conclusions: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.

Activation of a c-Jun N-terminal kinase-mediated autophagy pathway attenuates the anticancer activity of gemcitabine in human bladder cancer cells.

The role of autophagy in the anticancer activity of gemcitabine (GEM) in bladder cancer is unclear. The aim of this study is to determine whether GEM activates autophagy, the role of autophagy in the anticancer activity of GEM, and the underlying mechanism by which GEM induces autophagy. Human bladder cancer cell lines T24 and BIU87 were treated with GEM in vitro. Cell viability was measured using the Cell Counting Kit-8 assay. Apoptosis was detected by annexin V assay and western blot. Autophagy was measured by western blot and transmission electron microscopy. c-Jun N-terminal kinase (JNK) activation was detected by western blot. Chemical inhibitors were used for intervention of JNK and autophagy. GEM killed bladder cancer cells, which was associated with apoptosis induction. Autophagy was effectively activated by GEM. Suppressing autophagy in GEM-treated cells significantly decreased cell viability, which was associated with increased apoptosis. GEM-induced JNK activation and suppressed B-cell lymphoma 2 expression. The JNK inhibitor SP600125 inhibited GEM-induced autophagy activation and increased GEM's cytotoxicity. GEM kills bladder cancer cells through apoptosis. Meanwhile, JNK-mediated autophagy was activated, which protects the cells against apoptosis. Therefore, inhibition of autophagy could be exploited to enhance the anticancer efficacy of GEM for treating bladder cancer.

Tea Polyphenol inhibits autophagy to sensitize Epirubicin-induced apoptosis in human bladder cancer cells.

Resistance to anticancer agents such as Epirubicin (EPI) becomes a great challenge for treating bladder cancer. However, the mechanism by which chemoresistance arised is still elusive. In the present study, we showed evidence that EPI induced cytoprotective autophagy in bladder cancer cell lines T24 and BIU87. In addition, EPI robustly activated JNK-mediated phosphorylation of Bcl-2 and disruption of Bcl-2/Beclin-1 complex. Furthermore, the green tea derivative tea polyphenol (TP) inhibited EPI-induced autophagy and promoted apoptosis induced by EPI in bladder cancer cells. These results revealed a pathway for EPI-induced autophagy that involved in JNK/Bcl-2/Beclin-1 in bladder cancer cells, and that TP synergistically promoted EPI-induced apoptosis at least partly through autophagy inhibition. Thus, TP could be utilized in combination with EPI to improve EPI-based bladder cancer therapy.

Lycium barbarum polysaccharides inhibit proliferation and migration of bladder cancer cell lines BIU87 by suppressing Pi3K/AKT pathway.

The aim of this study was to verify whether Lycium barbarum polysaccharides inhibits proliferation and migration of BIU87 cells through Pi3K/AKT pathway. Different concentrations of Lycium barbarum polysaccharides were used to incubate with BIU87cells. LY-294002 and IGF-1 were used to inhibit and activate Pi3K/AKT pathway respectively. MTT were used to investigate the proliferation of BIU87cells. Transwell chambers and wound healing were used to test the migratory ability of BIU87cells. Western blotting were used to investigate the expressions of P21,P27,MMP-2, MMP-9, AKT and p-AKT in BIU87cells. Compared with the control group, the proliferation and migration of BIU87cells and the expression of p-AKT were significantly decreased in the study group; the inhibitory effect of the downregulation of p-AKT by LY-294002on the induction of BIU87cells proliferation and migration was identical to that of Lycium barbarum polysaccharides; upregulation of p-AKT by IGF-1 reversed the Lycium barbarum polysaccharides-induced inhibition of BIU87cells dedifferentiation. In conclusion, LBP inhibits the proliferation and migration of BIU87 cells by suppressing Pi3K/AKT signaling pathway.

A new non-muscle-invasive bladder tumor-homing peptide identified by phage display in vivo.

Bladder cancer is common and widespread, and its incidence is increasing. Many new diagnostic methods combined with state-of-the-art technology have been introduced in cystoscopy to collect real-time images of the bladder mucosa for diagnosis, but often miss inconspicuous early-stage tumors. Fluorophore-labeled peptides with high sensitivity and specificity for cancer would be a desirable tool for the detection and treatment of tiny or residual bladder tumors. Phage display and the human non-muscle-invasive bladder cancer cell line BIU-87 were used to identify a peptide. The isolated phage display peptide (CSSPIGRHC, named NYZL1) was tested in vitro for its binding specificity and affinity. Accumulation into xenograft tumors in a nude mouse model was analyzed with FITC-labeled NYZL1. NYZL1, with strong tumor-homing ability, was identified by in vivo phage library selection in the bladder cancer model. The NYZL1 phage and synthetic FITC-labeled NYZL1 peptides bound to tumor tissues and cells, but were hardly detected in normal control organs. Notably, accumulation of FITC-NYZL1 in bladder tumor cells was time-dependent. Biodistribution studies of xenografts of BIU-87 cells showed accumulation of injected FITC-NYZL1 in the tumors, and the bound peptide could not be removed by perfusion after 24 h. The mouse model of bladder tumor showed increased fluorescence intensity in the tumor-bearing bladder in comparison with normal bladder tissues after 4–6 h. In conclusion, NYZL1 may represent a lead peptide structure applicable in the development of optical molecular imaging.

Rab23 is overexpressed in human bladder cancer and promotes cancer cell proliferation and invasion.

Rab23 overexpression has been implicated in several human cancers. However, its expression pattern and biological roles in human bladder cancer have not been elucidated. In this study, we examined Rab23 expression in 93 bladder cancer specimens and analyzed its correlation with clinicopathological parameters. We found that Rab23 was overexpressed in 45 of 93 (48.3%) cancer specimens. Significant association was found between Rab23 overexpression and tumor invasion depth (p = 0.0027). Rab23 overexpression also negatively correlated with FGFR3 protein expression (p = 0.021). We found that Rab23 expression was lower in normal bladder transitional cell line SV-HUC-1 than in bladder cancer cell lines BIU-87, 5637, and T24. We knocked down Rab23 expression in T24 cancer cells and transfected a Rab23 plasmid in the BIU-87 cell line. Rab23 depletion inhibited cell growth rate and invasion, while its overexpression resulted in increased cell growth and invasion. In addition, we demonstrated that Rab23 depletion decreased and its transfection upregulated expression of cyclin E, c-myc, and MMP-9. Furthermore, we showed that Rab23 knockdown inhibited NF-κB signaling and its overexpression upregulated NF-κB signaling. BAY 11-7082 (NF-κB inhibitor) partly inhibited the effect of Rab23 on cyclin E and MMP-9 expression. In conclusion, the present study demonstrated that Rab23 overexpression facilitates malignant cell growth and invasion in bladder cancer through the NF-κB pathway.

Evaluation of Su Fu'ning Lotion's Inhibitory Effects on Bladder Cancer Cells In Vitro and In Vivo by Intravesical Instillation.

Bladder cancer is a common malignant tumor with a very high recurrence rate after surgery. Intravesical instillation can help clear up the residual tumor cells after surgery and thereby reduce the recurrence rate. To establish a bladder tumor transplantation animal model and to evaluate the inhibitory effects of a novel perfusate, Su Fu'ning Lotion (SFN), on bladder tumor. SFN was compared with several commonly used chemotherapy drugs, including mitomycin (MMC) and pirarubicin (THP) for anticancer effects on the bladder cancer cell lines T24, BTT, and BIU-87 and SFN half inhibitory concentrations (IC50) were determined after 48 hours of treatment. In addition, bladder cancer orthotopic transplantation tumor models were established in BALB/C nude mice and T739 mice, and SFN anticancer effects were assessed in vivo, with normal saline and MMC as negative and positive controls, respectively. SFN, MMC, and THP were all lethal to bladder cancer cells, in vitro, with SFN and THP significantly superior to MMC. IC50 values for SFN were 13.22, 11.22, and 12.5 µg/mL on T24, BTT, and BIU-87 cells, respectively. In vivo, SFN significantly reduced the mouse bladder wet weight and prolonged the animal survival compared with controls (P < .05), suggesting that SFN significantly inhibited T24/BTT cell growth in mice. SFN inhibited the bladder cancer cell proliferation in vitro and in vivo and significantly prolonged the survival of mice with bladder cancer xenografts, indicating that SFN could be used as a perfusate after surgery for removal of residual bladder cancers cells.

Numb inhibits proliferation and migration of human bladder cancer cell line BIU-87 by negative modulating CXCR4

Objective To study the effect of Numb gene on CXCR4 expression and proliferation and migration of bladder cancer. Methods Human bladder cancer cells were divided into 3 groups: experimental group (transfected with Numb-ORF plasmid), negative-control group (transfected with blank vector), and blank-control group (no DNA transfected in). The expressions of Numb and CXCR4 were detected by real-time PCR and Western blotting respectively. Cell proliferation and migration were measured by MTS and Transwell assay respectively. Results Numb expressions in experimental group, negative-control group and blank-control group were 31.044±3.350, 4.401±0.567 and 4.287±0.341 respectively, with a statistical significance (F=183.418, P=0.000). CXCR4 levels in experimental group, negative-control group and blank-control group were 0.344±0.167, 0.996±0.148 and 1.010±0.106 respectively, with a statistical significance (F=21.355, P=0.002). In MTS assay, the absorb values in experimental group, negative-control group and blank-control group were 0.615±0.057, 0.987±0.063 and 0.957±0.066, with a statistical difference (F=33.210, P=0.001). In Transwell assay, the numbers of migratory cells in experimental group, negative-control group and blank-control group were 164.667±19.858, 670.133±38.760 and 667.533±27.610, with a statistical significance (F=286.788, P=0.000). Conclusion Overexpression of Numb can suppress the ability of proliferation and migration of the BIU-87 cells through down-regulation of CXCR4 expression in human bladder cancer. Key words: Urinary bladder neoplasms; Receptors, CXCR4; Cell proliferation; Cell migration assays

Effect of recombinant human programmed cell death 5 protein on cell apoptosis of human bladder cancer cell line BIU87 induced by cisplatin and its mechanism

Objective To observe the effect of recombinant human programmed cell death 5 (rhPDCD5) protein on the apoptosis of human bladder cancer cell line BIU87 induced by cisplatin(DDP) and to investigate its mechanism. Methods Inhibition rates of bladder cancer cell line BIU87 intervened by rhPDCD5 protein and/or DDP with different concentrations were tested by cell counting kit-8(CCK-8). The morphological changes of cells were detected by Hoechst 33342 staining.The cells apoptosis rates were examined by flow cytometry.The changes of expression of the cysteinyl aspartate specific proteinase-3 (Caspase-3)and B-cell lymphoma/Leukemia-2(bcl-2) were measured by Western blotting analysis. Results CCK-8 showed that rhPDCD5 protein or/and DDP could inhibit the proliferation of BIU87 cells effectively.Hoechst 33342 staining showed that the positive cells rate of cells treated with rhPDCD5 protein and DDP(31. 62±2. 33)% was obviously higher than that only treated with rhPDCD5 protein(9. 61± 0. 42)% or DDP(14. 12±0. 24)%, the difference was significant(F= 221. 42, P<0. 01).The flow cytometry(FCM)showed that the apoptosis rate of cells treated with rhPDCD5 protein and DDP(58. 74± 3. 48)% was obviously increased than that only treated with rhPDCD5 protein(18. 84±1. 23)% or DDP (21. 25±2. 27)%, the difference was significant(F= 228. 93, P<0. 01).Compared with the group only treated with rhPDCD5 protein or DDP, the expression of Caspase-3 of the group treated with rhPDCD5 protein and DDP was significantly up-regulated(F= 12. 60, P<0. 01)and the expression of bcl-2 was significantly down-regulated(F= 12. 20,P<0. 01). Conclusion rhPDCD5 protein can induce the apoptosis of BIU87 cells,and accelerate the apoptosis of BIU87 cells induced by DDP.rhPDCD5 protein may reduce the expression of bcl-2 protein and increase activation of Caspase-3 to play its role in promoting apoptosis. Key words: Programmed cell death 5; Cisplatin; Bladder neoplasm; Apoptosis

Notch-1 regulates proliferation and differentiation of human bladder cancer cell lines by inhibiting expression of Krüppel-like factor 4.

Inhibition of Notch signaling pathways, consisting of 4 highly conserved receptors (Notch 1-4), induces expression of Krüppel-like transcription factors (KLFs) linked to bladder cancer tumorigenesis and metastasis. Effects of Notch-1 knockdown on cell proliferation, differentiation and KLF4 levels in bladder cancer cell lines were investigated. PsiRNA1‑mediated Notch-1 and KLF4 knockdown models and control model without the psiRNA1 vector were constructed using bladder cancer cell lines T24 and BIU87. Cell proliferation, apoptosis and expression of Notch-1 and KLF4 were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay with AnnexinV-FITC/PI staining, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, respectively. Proliferation was assessed in Notch-1 and/or KLF4 knockdown. The results showed that Notch-1 mRNA and protein expression levels were significantly lower following psiRNA1 vector transfection in both cell lines (P<0.05). Growth and proliferation of both cell lines were significantly inhibited by Notch-1 knockdown (P<0.05), and more G0/G1 arrest and apoptosis were observed compared to those in the control groups (P<0.05). The effects were time-dependent, peaking between 24-48h and declining by 72h. KLF4 expression was significantly higher in the Notch-1 knockdown group than in control cells (P<0.05). Notch-1 knockdown cell proliferation was significantly lower than that of Notch-1 and KLF4 knockdown (P<0.05). In conclusion, Notch-1 may act as an oncogene, regulating the proliferation and differentiation of bladder cancer cells by inhibiting KLF4. Pending further exploration of pathway variations and crosstalk, these pathways may be useful targets for bladder cancer therapy.

Anticancer activity of Luteolin and its synergism effect with BCG on human bladder cancer cell line BIU-87

To investigate the anticancer activity of Luteolin (Lu) and its synergism effect with bacillus calmette-guerin (BCG) on human bladder cancer cell line BIU-87. Cultured BIU-87 cells were treated with different concentrations of Lu alone or the combination of Lu with BCG. MTT assay was used to measure the cell proliferation inhibition, and IC50 was calculated. Cell cycle and apoptosis were analyzed by flow cytometry with propidium iodide (PI) staining and Annexin-V FITC/PI dual parameter markers to clarify the mechanism of inhibiting cell proliferation and inducing apoptosis. Caspase-3 and phosphorylated c-Jun N-terminal kinases (P-JNK) expression were measured to detect the apoptosis signal pathways of Lu in cancer cells. Both Lu and BCG apparently inhibited the cell proliferation and induced the apoptosis dose-dependently, and microscope observation showed morphological changes in the apoptosis. Flow cytometry indicated that Lu arrested the cell cycle at G2 phase (P<0.05). It sensitized BCG-induced cytotoxicity and cell apoptosis, and upregulated expression of caspase-3 and activation of JNK (P<0.05). As an effective anticancer agent, Lu can sensitize the effect of BCG by inducing the cell cycle arrest and apoptosis. This synergism effect is achieved by activation of caspase-3 and JNK. Combination of Lu with BCG may be one of the potential treatment for bladder cancer.

Intercellular Transfer of P-Glycoprotein from the Drug Resistant Human Bladder Cancer Cell Line BIU-87 Does Not Require Cell-to-Cell Contact

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Bcl-2 Overexpression Inhibits Generation of Intracellular Reactive Oxygen Species and Blocks Adriamycin-induced Apoptosis in Bladder Cancer Cells

Resistance to induction of apoptosis is a major obstacle for bladder cancer treatment. Bcl-2 is thought to be involved in anti-apoptotic signaling. In this study, we investigated the effect of Bcl-2 overexpression on apoptotic resistance and intracellular reactive oxygen species (ROS) generation in bladder cancer cells. A stable Bcl-2 overexpression cell line, BIU87-Bcl-2, was constructed from human bladder cancer cell line BIU87 by transfecting recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. The sensitivity of transfected cells to adriamycin (ADR) was assessed by MTT assay. Apoptosis was examined by flow cytometry and acridine orange fluorescence staining. Intracellular ROS was determined using flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were also investigated by the xanthinoxidase and visible radiation methods using SOD and CAT detection kits. The susceptibility of BIU87-Bcl-2 cells to ADR treatment was significantly decreased as compared with control BIU87 cells. Enhanced expression of Bcl-2 inhibited intracellular ROS generation following ADR treatment. Moreover, the suppression of SOD and CAT activity induced by ADR treatment was blocked in the BIU87-Bcl-2 case but not in their parental cells. The overexpression of Bcl-2 renders human bladder cancer cells resistant to ADR-induced apoptosis and ROS might act as an important secondary messenger in this process.