Anti-bladder Cancer Research Articles

Up-regulation of BLT2 is critical for the survival of bladder cancer cells

The incidence rates of urinary bladder cancer continue to rise yearly, and thus new therapeutic approaches and early diagnostic markers for bladder cancer are urgently needed. Thus, identifying the key mediators and molecular mechanisms responsible for the survival of bladder cancer has valuable implications for the development of therapy. In this study, the role of BLT2, a receptor for leukotriene B((4)) (LTB((4))) and 12(S)-hydroxyeicosatetraenoic acid (HETE), in the survival of bladder cancer 253J-BV cells was investigated. We found that the expression of BLT2 is highly elevated in bladder cancer cells. Also, we observed that blockade of BLT2 with an antagonist or BLT2 siRNA resulted in cell cycle arrest and apoptotic cell death, suggesting a role of BLT2 in the survival of human bladder cancer 253J-BV cells. Further experiments aimed at elucidating the mechanism by which BLT2 mediates survival revealed that enhanced level of reactive oxygen species (ROS) are generated via a BLT2-dependent up-regulation of NADPH oxidase members NOX1 and NOX4. Additionally, we observed that inhibition of ROS generation by either NOX1/4 siRNAs or treatment with an ROS-scavenging agent results in apoptotic cell death in 253J-BV bladder cancer cells. These results demonstrated that a 'BLT2-NOX1/4-ROS' cascade plays a role in the survival of this aggressive bladder cancer cells, thus pointing to BLT2 as a potential target for anti-bladder cancer therapy.

Role of arsenic trioxide in the anti-bladder cancer and its mechanism

Objective To observe the effects of arsenic trioxide (As2O3) on T24 bladder cancer cells growth, Caspase-3 gene expression, as well as the weight and volume changes of transplantation tumor of hairless mouse. Methods The cell proliferation inhibition was measured by MTT colorimetric assay. Morphological changes in the cell nucleus was measured by fluorescein stain. Morphologic cell apoptosis was analyzed by FCM. The espression of Caspase-3 was observed by immunocytochemistry staining. The weight and volume changes of transplantation tumor of hairless mouse were measured before and after As2 O3 was used. Results As2 O3 could significantly inhibit the growth of T24 cells, which depended on the reaction time and concentration of drug. With the extension of the time, the increase in the rate of apoptosis and expression of casepase-3 increased. The tumor size and tumor analysis of As2O3 group decreased than those of the control group(P＜0. 05). Conclusion As2O3 could significantly inhibit the growth of bladder cancer cells and its anti-cancer mechanisms may be related to increased Caspase-3 protein expression. Key words: Arsenic trioxide; Bladder neoplasms; Carcinoma

Abstract 3799: Rhodiola rosea extracts, salidroside, and metformin decrease the growth of bladder cancer cell lines via inhibition of TSC- mediated translation initiation and induction of autophagy

Abstract Bladder cancer well illustrates the association between aging and cancer, with over 71% of first diagnoses and 85.5% of deaths occurring in patients after 65 years of age. Since cancer and aging share a number of disruptions in molecular pathways and regulatory mechanisms, we examined whether agents with potential anti-aging properties could be useful in bladder cancer chemoprevention. Rhodiola rosea has been in use for centuries in many traditional medicinal practices to increase physical and mental performances, and Rhodiola rosea extracts (RRE) has been the agent in many clinical studies with no reported side effects or drug interactions. Metformin, a commonly prescribed oral and well-tolerated antidiabetic drug, is an insulin-sensitizer. Both RRE and metformin have been reported to have anti-aging effects in model organisms such as Drosophila. In bladder cancer prevention studies, we demonstrated that RRE, one of its active components, salidroside, or metformin significantly inhibit the growth of human urinary bladder cancer cell lines (including RT4, 5637, UMUC3, T24, HT1376, TCCSUP, J82, 253J-P, 253J-BV and 5637), with a minimal effect on normal bladder epithelial cells (TEU-2). In addition, RRE, salidroside and metformin are more effective in the reduction of colony formation of UMUC3 cells in soft agar than in two dimension culture, which suggests their potentials for inhibiting in vivo tumor growth. Using the primary mouse embryo fibroblasts tuberous sclerosis (TSC)-1 or TSC2 knockout and wild-type cells, we showed that TSC2 protein was, at least in part, required for the growth inhibitory effect of RRE, salidroside and metformin. Further study showed that RRE, salidroside and metformin reduce the phosphorylation of the S6 kinase and 4EBP1 which increase the binding of 4EBP1 to m7GTP to stop translation initiation in TSC-positive. UMUC3 but not in TSC-negative RT4 cells. In UMUC3 cells with stably overexperessed EGFP-LC3, we also observed that RRE, salidroside and metformin all significantly increase the percentage of cells with punctuate EGFP-LC3 fluorescence within four to eight hours of treatment, suggesting induction of autophagy. The autophagysome formation by these treatments was further confirmed by vital staining with acridine orange and western blotting analysis of LC3-II cleavage. In summary, our results indicate that RRE, salidroside and metformin share some similar molecular mechanisms in their anti-bladder cancer activity in vitro by involvement of TSC tumor suppressors-mediated inhibition of translational initiation, and by induction of autophagy. Further animal experiments are needed to examine the comparative efficacy of these agents in chemoprevention of bladder carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3799.

Study of recombinant human IFN-α-2b bacilli calmette-guerin activated killer cells and against bladder cancer cell in vitro

Presently, one of the most potent immunotherapies is the application of bacillus Calmette Guerin (BCG) to prevent recurrences of the superficial bladder cancer. Despite its successful use, nonresponders and certain side effects remain a major obstacle. Therefore, current studies aim at developing recombinant BCG (rBCG) strains secreting Th1-like cytokines to improve the effectiveness of the therapy. In this study, a new type of rBCG strain constructed by Tianjin institute of Urology was tested for its immunostimulatory capacity in vitro. Peripheral blood monocytes (PBMC) were stimulated by recombinant BCG and transformed into bacilli Calmette-Guerin activated killer (BAK) cells, and the effect of anticancer BAK cells was studied. Recombinant IFN-α-2b-BCG, wild-type BCG (wBCG), wild-type BCG and IFN-α-2b were coincubated with PBMCs respectively in vitro, and the proliferation of PBMC was detected with MTT in different time. BAK cells have the ability to kill bladder tumor cells, and the antitumor activity of effecter cells was determined by LDH release assay. The result of MTT showed that the proliferation of PBMC in the recombinant BCG group was more powerful than in the other two groups (P < 0.05). The result of LDH release assay showed that the antitumor activity of BAK cells stimulated by Recombinant BCG was the highest in all groups. We conclude that the recombinant BCG can activate more PBMCs to anti-bladder cancer in vitro than wild-type BCG does.

USE OF PROSTATE SPECIFIC ANTIGEN TO MEASURE BLADDER TUMOR GROWTH IN A MOUSE ORTHOTOPIC MODEL

Animal orthotopic tumor models are commonly used in bladder cancer studies. However, to our knowledge there is currently no accurate method to quantify tumor growth inside the bladder in living animals. We used prostate specific antigen (PSA) as a marker to cope with this limitation. Infectious but replication incompetent retroviral particles carrying PSA coding sequence were constructed and infected into MB49 cells, a mouse bladder transitional cell carcinoma line of C57BL/6 origin. Syngeneic mice were intravesically implanted with the novel MB49-PSA transfectants. Tumor burden was evaluated by enzyme-linked immunosorbent assay measurement for PSA in urine and bladder tissues. The MB49-PSA line actively secreted PSA in culture as well as in urine (18 to 2,062 pg/ml) depending on tumor mass. Immunofluorescence staining confirmed PSA expression in MB49-PSA derived orthotopic tumors. Urinary PSA production paralleled tumor growth and was detectable prior to the development of a palpable tumor. Although urinary PSA did not tightly correlate with tumor mass, all bladders (total of 16 tested) weighing 34 mg or greater (18 to 21 mg for age and sex matched normal bladders) showed 18 pg/ml or greater urinary PSA. In contrast, bladder tissue PSA correlated more with tumor mass in general and it was measurable even before the detection of urinary PSA. This MB49-PSA orthotopic tumor model also demonstrated its usefulness for evaluating the antibladder cancer agents gemcitabine and mitomycin. This novel MB49-PSA line may serve as a useful tool for bladder cancer study because its growth inside the bladder can be noninvasively measured in living animals even during early stages of tumor growth.

The Essential Role of Interferon-γ During Interleukin-12 Therapy for Murine Transitional Cell Carcinoma of the Bladder

Recombinant interleukin (IL)-12 and adenoviral IL-12 gene therapy have been shown to be potent therapeutic interventions for murine transitional cell carcinoma (TCC) of the bladder in vivo. We investigated the mechanisms through which IL-12 induces antibladder cancer immunity. The ability of IL-12 to enhance interferon-gamma (IFN-gamma) expression, a major T-helper type 1 cytokine, was analyzed in murine serum, urine and splenocyte cultures. MB49, a murine TCC line, was treated with IFN-gamma and evaluated for its proliferation, surface molecule expression and sensitivity to splenocyte mediated cytotoxicity. Neutralizing antiIFN-gamma antibody was applied to test the role of IFN-gamma in the IL-12 therapy of MB49 tumor. IL-12 was observed to significantly increase IFN-gamma concentrations in serum and urine as well as in splenocyte cultures. While IL-12 had no direct activity against TCC in vitro, IFN-gamma showed potent dose dependent antiproliferative and pro-apoptotic activity, which was further enhanced by supplementation of tumor necrosis factor-alpha. In addition, IFN-gamma substantially up-regulated the expression of surface immune molecules on TCC cells, including MHC-I, MHC-II, ICAM-I, B7.1, B7.2 and Fas. Maximum splenocyte mediated cytotoxicity against TCC was enhanced by pretreatment of target bladder cancer cells with IFN-gamma plus tumor necrosis factor-alpha. Furthermore, IL-2 in combination with IL-12 further enhanced splenocyte mediated cytotoxicity. The in vivo antibladder cancer activity of IL-12 was abolished by concurrent treatment with antibodies to IFN-gamma. This study strongly suggests that IFN-gamma has an essential role in IL-12 induced antibladder tumor immunity. Activation of host effector immune cells by IL-12 is also required for induction of optimal tumor destruction in IL-12 therapy.

Interleukin-12 Immunotherapy of Murine Transitional Cell Carcinoma of the Bladder: Dose Dependent Tumor Eradication and Generation of Protective Immunity

The antitumor activity of interleukin (IL)-12 has been demonstrated in a number of tumor models but barely tested in bladder cancer models. We evaluated the antibladder cancer activity of this cytokine in syngeneic mice bearing subcutaneous, metastatic and orthotopic tumors. Mice were implanted subcutaneously, intravenously or orthotopically with syngeneic transitional cell carcinoma (TCC) of the bladder. The tumor bearing mice were then treated with IL-12 locally or systemically and monitored for tumor regression and survival. In the subcutaneous model dose dependent suppression of tumorigenesis was observed when IL-12 was administered subcutaneously at a distal site with the MB49 line being more sensitive than MBT-2. IL-12 (10 days) above 50 ng daily was tumor inhibitory, while doses of 500 or 1000 ng daily prolonged survival and cured 70% and 75% of subjects, respectively. Upon re-challenge with parental tumor cells mice previously cured with IL-12 (1000 vs 500 ng daily) exhibited specific protection (70% vs 35% rejection) that was dependent on the earlier dose of cytokine. IL-12 administered intraperitoneally at a dose of 250 ng daily was more potent than subcutaneous administration and complete regression was observed. Metastatic TCC in the lungs and orthotopic tumors in the bladder also favorably responded to systemic or intravesical IL-12 therapy, respectively. Addition of IL-2 to IL-12 therapy increased tumor regression, long-term survival and rejection of re-challenged parental tumor. IL-12 is exceptionally effective for treating murine bladder TCC in subcutaneous, metastatic and orthotopic models. The antibladder cancer activity of this cytokine should be tested in human bladder cancer therapy.

Radiochemical synthesis and preliminary evaluation of anti‐bladder cancer monoclonal antibody BDI‐1 labeled with rhenium‐188

AbstractThe anti‐human bladder cancer monoclonal antibody BDI‐1 was radio‐labeled with rhenium‐188 by direct labeling methods using SnCl2 as reductant and MDP as stannous stabilizer or by indirect method using NHS‐ECM ester as bifunctional chelator. Radiochemical yields of 30±7.23% and 87.4±5.67% and radiochemical purity of more than 95% were achieved. The inmmunoreactive fraction was 58.7%. The results showed that radionuclide 188Re might be employed using the same labeling methodology with 99mTc labeling of BDI‐1 for radiommunoimaging (RII) and radioimmunotherapy (RIT) and that may be useful in radioimmunodetection and RIT of human bladder carcinoma in vivo. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Two-Dimensional Immunoelectrophoretic Analysis of Urinary Ultraconcentrates: Antigenic Differences Between Bladder Cancer Positive Patients, Normal Individuals, and Patients With Urinary Tract Infections

Urine ultraconcentrates (100-fold) from bladder cancer patients, patients suffering from urinary tract infection, and normal individuals were analyzed using polyacrylamide gel electrophoresis (PAGE) and two-dimensional immunoelectrophoresis. A combined sample of normal urine was resolved into 1 to 3 protein bands by PAGE, whereas a single concentrated bladder cancer urine was resolved into 10-12 protein bands. Yet, this same concentrated urine sample was resolved into 17-20 antigen peaks by two-dimensional immunoelectrophoresis (2DIEP) against antihuman serum. A significant (P less than 0.05) increase was observed in the relative antigen concentration when comparing 2DIEP profiles of bladder cancer urines to normal controls. A significant increase in the relative antigen concentration and the number of antigen peaks was also found when comparing immunoelectrophoretic patterns obtained from ultraconcentrated urine specimens of bladder cancer positive urine and normal controls using a rabbit antibladder cancer urine antisera (P less than 0.002 and P less than 0.02, respectively). In addition, significant (P less than 0.02) antigenic differences were found when comparing concentrated urine samples from bladder cancer positive individuals to those with urinary tract infection. The bladder cancer group demonstrated 8/9 positive results for relative antigen concentrations greater than 3.0. Fifteen of 16 normal or urinary tract infected individuals combined had relative antigen concentrations less than 3.0. These differences were highly significant (P less than 0.001). No differences were found between concentrated bladder cancer and normal urine specimens tested against rabbit antinormal urine antisera.