OBJECTIVES: To investigate the inhibitory effect of pirfenidone (PFD) on growth of bladder cancer xenograft and its regulatory effect on Treg cells in tumor-bearing mice. METHODS: Thirty-two C57BL/6 mice bearing ectopic bladder tumors were randomized into control and PFD groups (n=16). In PFD group, PFD was administered orally at the daily dose of 500 mg/kg, and tumor growth and survival of the mice were monitored. After treatment for 21 days, the tumors and vital organs were harvested for analysis. Immunohistochemistry was used to assess CD3, CD4, CD8, and FOXP3 expressions in the tumors. Flow cytometry and RT-qPCR were used to analyze the percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells and IL-2, IL-10, and IL-35 expressions in the tumors and spleens; organ damage of the mice was examined with HE staining. RESULTS: Compared with the control group, the PFD-treated mice exhibited significantly lower tumor growth rate with smaller tumor volumes at day 21, along with improved survival at day 28. Immunohistochemistry revealed no significant differences in the infiltration of CD3⁺ and CD8⁺ cells between the two groups, but the percentages of CD4⁺ and FOXP3⁺ cells were significantly lower in the tumors of PFD-treated mice. Flow cytometric analysis confirmed a decrease in CD4⁺CD25⁺FOXP3⁺ Treg cells in the tumors from PFD-treated mice, which also had reduced expression levels of IL-2, IL-10 and IL-35 mRNAs in the tumors. No significant differences were found in Treg cell populations or cytokine expressions in the spleen tissues between the two groups. HE staining showed obvious organ damage in neither of the groups. CONCLUSIONS PFD inhibits bladder cancer growth and enhances survival of tumor-bearing mice possibly by suppressing Treg cells in the tumor microenvironment.
Animals ; Urinary Bladder Neoplasms/drug therapy* ; Mice ; T-Lymphocytes, Regulatory/metabolism* ; Mice, Inbred C57BL ; Interleukins/metabolism* ; Interleukin-10/metabolism* ; Cell Line, Tumor ; Interleukin-2/metabolism* ; Xenograft Model Antitumor Assays ; Female

OBJECTIVE: To explore the influences of andrographolide (Andro) on bladder cancer cell lines and a tumor xenograft mouse model bearing 5637 cells. METHODS: For in vitro experiments, T24 cells were stimulated with Andro (0-40 µmol/L) and 5637 cells were stimulated with Andro (0 to 80 µmol/L). Cell growth, migration, and infiltration were assessed using cell counting kit-8, colony formation, wound healing, and transwell assays. Apoptosis rate was examined using flow cytometry. In in vivo study, the antitumor effect of Andro (10 mg/kg) was evaluated by 5637 tumor-bearing mice, and levels of nuclear factor κ B (NF- κ B) and phosphoinositide 3-kinase/AKT related-proteins were determined by immunoblotting. RESULTS: Andro suppressed growth, migration, and infiltraion of bladder cancer cells (P⩽0.05 or P⩽0.01). Additionally, Andro induced intrinsic mitochondria-dependent apoptosis in bladder cancer cell lines. Furthermore, Andro inhibited bladder cancer growth in mice (P⩽0.01). The expression of p65, p-AKT were suppressed by Andro treatment in vitro and in vivo (P⩽0.05 or P⩽0.01). CONCLUSIONS Andrographolide inhibits proliferation and promotes apoptosis in bladder cancer cells by interfering with NF- κ B and PI3K/AKT signaling in vitro and in vivo.
Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Diterpenes/therapeutic use* ; Humans ; Mice ; NF-kappa B/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Urinary Bladder Neoplasms/drug therapy*

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.
Animals ; Apoptosis ; genetics ; Carcinogenesis ; genetics ; metabolism ; pathology ; Carcinoma ; genetics ; metabolism ; pathology ; therapy ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cyclin D1 ; antagonists & inhibitors ; genetics ; metabolism ; Cyclin E ; antagonists & inhibitors ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Injections, Intralesional ; Ki-67 Antigen ; genetics ; metabolism ; Mice ; Mice, Nude ; Polycomb Repressive Complex 1 ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Small Interfering ; administration & dosage ; genetics ; metabolism ; Signal Transduction ; Tumor Burden ; Urinary Bladder ; metabolism ; pathology ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; agonists ; genetics ; metabolism

No abstract available.
Aged ; Anti-Bacterial Agents/therapeutic use ; Bacteria/metabolism ; Catheter-Related Infections/diagnosis/drug therapy/*microbiology ; Color ; Equipment Design ; Escherichia coli Infections/diagnosis/drug therapy/*microbiology ; Female ; Humans ; Intestines/*microbiology ; Pigments, Biological/metabolism ; Treatment Outcome ; Tryptophan/metabolism ; Urinary Bladder Neoplasms/surgery ; Urinary Catheterization/adverse effects/*instrumentation ; *Urinary Catheters ; *Urinary Diversion ; Urinary Tract Infections/diagnosis/drug therapy/*microbiology ; Urine/chemistry/microbiology

OBJECTIVETo evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 (IL-4-SA) bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer.

METHODSIL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 µl of 1 mg/ml NHS-PEO4-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100 µl PBS, GFP-SA+IL-4 or IL-4-SA and incubation for 1 hour. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8(+) tumor-infiltrating lymphocytes were analyzed.

RESULTSThe IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days.On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden.In contrast, 5 out of 10 mice in the IL-4-SA-treated group were tumor-free. The MB49 tumor-specific cytotoxicity from mice in the IL-4-SA group was (11.3 ± 1.2)%, (22.7 ± 1.5)% and (31.0 ± 3.0)% at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. But the corresponding cytotoxicity was (4.3 ± 0.6)%, (9.0 ± 1.0)% and (14.3 ± 1.5)% in the GFP-SA+IL-4 group, and (3.3 ± 0.6)%, (7.3 ± 0.6)%, (12.7 ± 2.1)% in the PBS group. The tumor-specific cytotoxicity in the SA-CD40L group was significantly higher than that in the control groups (P < 0.05). The infiltrating CD8(+) T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups.

CONCLUSIONIntravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.

Administration, Intravesical ; Animals ; Biotinylation ; CD8-Positive T-Lymphocytes ; pathology ; Cell Line, Tumor ; Humans ; Immunotherapy ; methods ; Interleukin-4 ; metabolism ; therapeutic use ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; metabolism ; therapeutic use ; Streptavidin ; metabolism ; therapeutic use ; Urinary Bladder Neoplasms ; metabolism ; therapy

OBJECTIVETo study the clinical and pathologic characteristics of small cell neuroendocrine carcinoma of urinary tract.

METHODSAll cases of urinary tract carcinoma encountered in the General Hospital of People Liberation Army during the period from 1999 to 2010 were retrospectively reviewed. The clinicopathologic data of small cell neuroendocrine carcinomas were further analyzed, with literature review.

RESULTSA total of 16 cases of small cell neuroendocrine carcinoma were identified, including 10 from urinary bladder, 2 from ureter, 3 from renal pelvis, and 1 multifocal tumor involving renal pelvis and ureter. There were altogether 8 males and 8 females. The median age of the patients was 63 years (range = 24 to 79 years). Gross hematuria (11 cases) represented the main presenting symptom. Four patients had flank pain and 4 had urinary irritation symptoms. Seven patients underwent radical cystectomy. Six other patients underwent radical nephroureterectomy, 1 partial cystectomy, 1 TURBT and the remaining case biopsy only. The size of the tumor ranged from 0.8 to 8.0 cm (median = 4.5 cm). Histologically, 15 cases represented mixed small cell neuroendocrine carcinoma (with 13 mixed with transitional cell carcinoma and 2 with adenocarcinoma). Immunohistochemical study showed positive staining for neuroendocrine markers. On presentation, 1 patient was in stage pT1, 7 in stage pT2, 6 in stage pT3, 2 in stage pT4. Six patients died of the disease after operation. The overall survival was 25 months and the 5-year survival rate was 32.4%.

CONCLUSIONSSmall cell neuroendocrine carcinoma of urinary bladder is a highly malignant disease and associated with poor prognosis. The diagnosis relies on detailed histologic examination. Early diagnosis, when coupled with cystectomy or nephroureterectomy and adjuvant chemotherapy, represents the mainstay of management.

Adult ; Aged ; CD56 Antigen ; metabolism ; Carcinoma, Neuroendocrine ; drug therapy ; metabolism ; pathology ; surgery ; Carcinoma, Small Cell ; drug therapy ; metabolism ; pathology ; surgery ; Chemotherapy, Adjuvant ; Cystectomy ; Female ; Follow-Up Studies ; Humans ; Keratins ; metabolism ; Kidney Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Neoplasm Staging ; Nephrectomy ; Phosphopyruvate Hydratase ; metabolism ; Retrospective Studies ; Survival Rate ; Synaptophysin ; metabolism ; Ureteral Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Urinary Bladder Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Urologic Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Young Adult

BACKGROUNDSuperficial urothelial carcinoma (SUC) of the bladder is a common urinary tract tumor in China. There is a high recurrence rate of this tumor even after surgery and intravesical instillation. Previous reports have described a suppression of the immune system in cancer patients. Dendritic cells (DCs) play a pivotal role in the induction of an effective antitumor immune response. The aim of this study was to investigate the effects of surgery and epirubicin intravesical chemotherapy (IC) on peripheral blood DCs in subsets of patients with bladder SUC.

METHODSA total of 66 SUC patients and 38 healthy controls were enrolled in this study. All the patients had undergone transurethral resection (TUR) of their cancer and adjunctive IC after tumor removal. The patients were divided into a non-recurrence group (n = 40) and a recurrence group (n = 26) based on the presence or absence of tumor recurrence. Blood samples were taken preoperatively (PreOP), on postoperative days (POD) 1 and 7, and at postoperative month (POM) 3. Flow cytometric analysis was used for the determination and quantitation of the surface markers CD80 and CD86 in circulating DC subsets.

RESULTSThe preoperative percentages of myeloid dendritic cells (mDCs) and expression of CD80 and CD86 were impaired in SUC patients compared to healthy controls (P < 0.05). The percentages of mDCs and these surface markers decreased significantly on POD 1 and increased on POD 7, remaining higher than the preoperative values in POM 3 (P < 0.05). The percentages of mDCs, and CD80 and CD86 in the non-recurrence group on PreOP, POD 7, and POM 3 were higher than those in recurrence group.

CONCLUSIONSSurgical removal of SUC and adjunctive IC were associated with improved circulating mDC counts and function. Persistent depression of mDC counts and function after treatment in recurrence patients indicated lower antitumor immunity that may lead to tumor recurrence.

Adult ; Aged ; China ; Dendritic Cells ; immunology ; metabolism ; Epirubicin ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Urinary Bladder Neoplasms ; drug therapy ; immunology ; surgery

OBJECTIVETo investigate the treatment efficiency and mechanism of recombinant adenoviral vector carrying LRIG1 gene driven by Survivin promoter for bladder cancer.

METHODSHuman bladder cancer cell line BIU87 and immortalized human bladder epithelial cells SV-HUC-1 were infected with Ad-Surp-LRIG1 and Ad-LRIG, respectively. The selective infection efficiency of Ad-Surp-LRIG1 and Ad-LRIG were evaluated by checking the expression of epidermal growth factor receptor (EGFR). The MTT method was used to test cell growth inhibition ratio of Ad-Surp-LRIG1 and Ad-LRIG. Heterotransplanted models of human bladder cancer cell line BIU87 cells in nude mice were established. The mice were randomly divided into 3 groups during the experiment: Ad-Surp-LRIG1 group received viral supernatant solution of Ad-Surp-LRIG1 by tail vein injection; Ad-LRIG group received viral supernatant solution of Ad-LRIG by tail vein injection; and PBS group received phosphate buffer solution (PBS). The growth of tumors were observed and the growth curve was mapped. The expression of LRIG1 and EGFR were examined by reverse transcription PCR (RT-PCR).

RESULTSWhen Multiplicity of infection was 25, the transfection efficiency of Ad-Surp-LRIG1 was 74.56% in BIU87 cells and 0 in SV-HUC-1 cells (χ² = 58.640, P = 0.000), while the transfection efficiency of Ad-LRIG was 68.27% in BIU87 cells and 72.52% in SV-HUC-1 cells (χ² = 0.075, P = 0.784). The transfection efficiency difference of Ad-Surp-LRIG1 and Ad-LRIG in BIU87 cells was not statistically significant (χ² = 0.016, P = 0.898). Compared with PBS, Ad-Surp-LRIG1 and Ad-LRIG1 could inhibit BIU87 cell growth, the difference was significant in 4 days after transfection (F = 15.960, P = 0.000). There was not significant difference in cell growth rate of Ad-Surp-LRIG1 group and Ad-LRIG1 group. The tumor growth rate in Ad-Surp-LRIG1 group was slower than that in the other 2 groups. The tumor quality in Ad-Surp-LRIG1 was lighter than that in the other two groups, the differences were statistically significant (F = 97.860, P = 0.000), the quality difference in Ad-LRIG1 group and PBS group was not statistically significant difference (t = 1.73, P = 0.06). Compared with Ad-LRIG1 group and PBS group, the mRNA expression of LRIG1 was obviously up-regulated and that of EGFR was down-regulated in Ad-Surp-LRIG1 group (P < 0.01).

CONCLUSIONSThe recombinant adenoviral vector of Ad-Surp-LRIG1 could selectively transfected BIU87 cells, which could inhibit significantly the growth of bladder cancer in vivo and in vitro, the mechanism may be partly LRIG1 can downgrade the expression of EGFR.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Female ; Genetic Therapy ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Membrane Glycoproteins ; genetics ; Mice ; Mice, Nude ; Promoter Regions, Genetic ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transfection ; Urinary Bladder Neoplasms ; metabolism ; pathology ; therapy ; Xenograft Model Antitumor Assays

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Basigin ; analysis ; Biomarkers, Tumor ; analysis ; genetics ; Carcinoma, Transitional Cell ; drug therapy ; genetics ; metabolism ; Gene Expression Profiling ; Humans ; In Situ Hybridization, Fluorescence ; Inhibitor of Apoptosis Proteins ; analysis ; Mutation ; Neoplasm Recurrence, Local ; metabolism ; Nuclear Proteins ; analysis ; Receptor, Fibroblast Growth Factor, Type 3 ; analysis ; genetics ; Tumor Suppressor Protein p53 ; analysis ; genetics ; Urinary Bladder Neoplasms ; drug therapy ; genetics ; metabolism

OBJECTIVETo investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice.

METHODSA total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-alpha fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4(+) and CD8(+) lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival.

RESULTSSA-hTNF-alpha could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SA- hTNF-alpha-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-alpha group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4(+) and CD8(+) lymphocytes were significantly greater in SA-hTNF-alpha group than in the other groups (P<0.05). The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-alpha group than in the other groups (P<0.05).

CONCLUSIONSA-hTNF-alpha immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.

Administration, Intravesical ; Animals ; Biotinylation ; Carcinoma, Transitional Cell ; immunology ; therapy ; Female ; Immobilized Proteins ; therapeutic use ; Immunotherapy ; methods ; Mice ; Mice, Inbred C57BL ; Recombinant Fusion Proteins ; metabolism ; therapeutic use ; Streptavidin ; metabolism ; therapeutic use ; Tumor Necrosis Factor-alpha ; metabolism ; therapeutic use ; Urinary Bladder Neoplasms ; immunology ; therapy