Urinary bladder cancer is the tenth most common cancer worldwide with high morbidity and mortality. The majority of bladder cancers are urothelial carcinomas. More than half are papillomas or the ...papillary urothelial carcinomas (stages Ta and T1), which have a relatively good prognosis. Squamous cell carcinomas have a variable survival rate, while carcinomas in situ (Tis) can progress to muscle-invasive urothelial carcinomas (T2) with a poor prognosis. The most challenging feature of bladder cancer is its high recurrence rate, ranging from 50% to 90% of cases. The N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model is an invaluable experimental tool for bladder cancer research, as BBN-induced bladder cancer in rodents resembles human bladder cancer in its morphological, biological, and molecular features. We present here a detailed protocol for the treatment of mice and the main expected results.
Bladder cancer (BC) is the most common malignant disease of the urinary tract. Recurrent high grade non muscle invasive BC carries a serious risk for progression and subsequent metastases. The most ...common preclinical mouse model for bladder cancer relies on administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to mice. BBN-induced tumors in mice recapitulate the histology of human BC and were characterized with an overexpression of markers typical for basal-like cancer subtype in addition to a high mutational burden with frequent mutations in Trp53, similar to human muscle invasive BC.
Bladder cancer was induced in C57BL/6J male mice by administering the BBN in the drinking water. A thorough histopathological analysis of bladder specimen during and post BBN treatment was performed at 2, 4, 16, 20 and 25 weeks. RNA sequencing and qPCR was performed to assess the levels of expression of immunologically relevant genes at 2 weeks and 20 weeks during and post BBN treatment.
We characterized the dynamics of the inflammatory response in the BBN-induced BC in mice. The treatment with BBN had gradually induced a robust inflammation in the first 2 weeks of administration, however, the inflammatory response was progressively silenced in the following weeks of the treatment, until the progression of the primary carcinoma. Tumors at 20 weeks were characterized with a marked upregulation of IL18 when compared to premalignant inflammatory response at 2 weeks. In accordance with this, we observed an increase in expression of IFNγ-responsive genes coupled to a pronounced lymphocytic infiltrate during the early stages of malignant transformation in bladder. Similar to human basal-like BC, BBN-induced murine tumors displayed an upregulated expression of immunoinhibitory molecules such as CTLA-4, PD-L1, and IDO1 which can lead to cytotoxic resistance and tumor escape.
Despite the recent advances in bladder cancer therapy which include the use of checkpoint inhibitors, the treatment options for patients with locally advanced and metastatic BC remain limited. BBN-induced BC in mice displays an immunological profile which shares similarities with human MIBC thus representing an optimal model for preclinical studies on immunomodulation in management of BC.
•Nicotine induced urothelial cytotoxicity with regenerative proliferation.•Nicotine promoted BBN-induced rat urothelial carcinogenesis.•nAChR inhibitors inhibited proliferation and simple hyperplasia ...induced by nicotine.•nAChR inhibitors didn’t inhibit cytotoxicity of nicotine.•The cytotoxic concentration of nicotine was > 1 mM in vitro.
Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.
Dapagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, is indicated to improve glycaemic control in adults of type 2 diabetes. In nonclinical studies, dapagliflozin was neither genotoxic ...nor carcinogenic. However, in some clinical studies, an increased incidence of bladder cancer was observed in the dapagliflozin group vs. the placebo. Therefore, this study was undertaken to determine if dapagliflozin can act as a promoter in a 2-stage bladder cancer model in rats induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Rats given BBN (100 or 400 mg/kg, po) twice weekly for 6 weeks in Phase 1 were assigned in Phase 2 to receive daily dose of vehicle, dapagliflozin (0.5 mg/kg, po) or uracil (positive control, 3% in diet) from weeks 8–34. All bladders were evaluated by histopathology. Verifying the validity of the model, uracil increased the incidence of bladder cancer, while dapagliflozin had no effect on the incidence or invasiveness of transitional cell carcinoma. The exposure of dapagliflozin at 0.5 mg/kg/day in rats was 7 times the clinical exposure at maximal therapeutic dose (10 mg). In conclusion, dapagliflozin does not act as promoter or progressor of bladder cancer in a validated bladder cancer model in rats.
•Some clinical studies with dapagliflozin had increased incidence of bladder tumours compared to placebo controls.•There were 4 cases remaining after excluding patients in with less than 1 year drug exposure at diagnosis of bladder cancer.•A 2-stage bladder cancer model with BBN as an initiator was used to determine potential effect with dapagliflozin.•No effects with dapagliflozin on incidence/invasiveness of bladder transitional cell carcinoma were observed in rats.•This shows that dapagliflozin does not act as a promoter or progressor of bladder cancer in the presence of glucosuria.
Bladder-sparing options are being developed for muscle-invasive bladder cancer in place of radical cystectomy, including the combination of chemotherapy and radiation therapy. We reasoned that ...improving the radiotherapy component of chemoradiation could improve the control of locally advanced disease. Previously, we showed that gold nanoparticles (AuNPs) are potent enhancers of radiation therapy. We hypothesized that if AuNPs were to preferentially localize to bladder tumors, they may be used to enhance the radiation component of muscle-invasive bladder tumor therapy. Mice were treated with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 17, 20, and 22 weeks - long enough to induce muscle-invasive tumors. Mice were then anesthetized and injected intravenously with 1.9 nm AuNPs of which most were rapidly cleared from the blood and excreted after a 30-50 minute residence time in the bladder. We found AuNPs distributed throughout the bladder wall, but most of the AuNPs were associated with the stroma surrounding the tumor cells or extracellular keratin produced by the tumor cells. There were relatively few AuNPs in the tumor cells themselves. The AuNPs therefore localized to tumor-associated stroma and this tumor specificity might be useful for specific X-ray dose enhancement therapy of muscle-invasive bladder carcinomas.
Introduction:
Chemoprevention may involve perturbation of a variety of steps in tumor initiation, promotion, and progression.
Objective:
To investigate the antiproliferative and anti-inflammatory ...potential effects of diindolylmethane (DIM) and lupeol on experimental bladder carcinogenesis.
Methods:
Sixty healthy male Wistar rats were selected and randomly divided into six groups, with 10 rats in each group. Group I: control; group II: N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN; 150 mg/gavage/twice a week) for 8 weeks, and then they were given 100 ppm concentrations of dimethylarsenic acid (DMA) in the drinking water for 28 weeks; group III: BBN + DMA + DIM (5 mg/kg body weight (b.w.)/day) treatment was started after BBN treatment, and it was orally administered for 28 weeks); group IV: BBN + DMA + lupeol (50 mg/kg b.w./day) treatment was started after BBN treatment, and it was orally administered for 28 weeks); and groups V and VI: DIM and lupeol treatment alone for 36 weeks. Bladder tissues were collected after 36th week study protocol for further analysis.
Results:
Our results revealed that DIM and lupeol treatment showed inhibition of tumor growth in the bladder by histopathological confirmations as well as significantly (p < 0.001) increased the expression of phosphotensin (PTEN) and significantly (p < 0.001) decreased the expression of tumor necrosis factor α, nuclear factor κβ (p65) were quantified using Western blot analysis. DIM and lupeol treatment significantly (p < 0.001) decreased the levels of Cox-2 in bladder tissue samples and NMP 22 in urine samples were quantified using enzyme-linked immunosorbent assay method.
Conclusion:
Preventive DIM and lupeol administration act as potent Cox-2 inhibitors, which activates the tumor suppressor protein PTEN against experimental bladder carcinogenesis by antiproliferative and anti-inflammatory properties.
Effects of various chemicals on the development of urinary bladder tumors in mouse treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) were examined, Male C3H/He mice received 0.05% BBN in ...drinking water for 8 weeks, and were subsequently treated with basal diet containing trisodium nitrilotriacetate monohydrate (Na3NTA·H2O), nitrilotriacetic acid (H3NTA), sodium saccharin, and DL-tryptophan for 12 weeks. DL-tryptophan tended to induce a more significant increase in neoplastic and dysplastic lesions of the urinary bladder as compared to Na3NTA·H2O, H3NTA and sodium saccharin. These data showed that DL-tryptophan was more effective than Na3NTA·H2O, H3NTA and sodium saccharin with regard to promoting potential on the development of urinary bladder tumors in mouse treated with BBN.
Administrations of 0.01% and 0.005% BBN were undertaken in female Sprague Dawley rats (aged 12 weeks) and female Wistar rats (aged 16 weeks) for 4 and 8 weeks, respectively. Escherichia coli isolated ...from the urine of patients with urinary tract infections, which invade the rat tunica mucosa vesicae urinariae to varying degrees, were transurethrally inoculated into the bladder. When 0.1ml of 103/ml bacteria of a highly invasive strain was inoculated twice at intervals of 2 weeks before, during and after the administration of 0.01% BBN, the incidences of tumors were 64.3% (9/14), 57.1% (8/14) and 46.7% (7/15), respectively. Administration of BBN alone resulted in a tumor incidence of 33.3% (5/15). Transitional cell carcinoma was observed in only two animals in which inflammation was induced after BBN administration. Among the animals given 0.005% BBN after the induction of inflammation, 69.2% (9/13) of those inoculated twice with 0.1ml of 103/ml bacteria of the highly invasive strain at intervals of 4 weeks had tumors (papilloma in one and hyperplasia in eight). The incidence of tumors in animals which had the combined treatment was lower than the 77.8% (14/18) found in the animals which received only a single administration of BBN (hyperplasia in 14), but the tumors were larger in the latter group. The incidence was highest in animals inoculated four times with 0.1ml of 105/ml bacteria of a moderately invasive strain at intervals of three weeks, 87.6% (14/16). Half of them had transitional cell carcinoma or squamous cell carcinoma. There were significant differences (p<0.01) in both the number of tumors occurring in one animal and the sizes of the tumors in the single administration group and the combined treatment group. In contrast, the incidence was lowest, 43.8% (7/16), in animals inoculated four times with 0.1ml of 108/ml bacteria of a moderately invasive strain at intervals of three weeks. All the animals with tumors showed simple hyperplasia of less than 1mm in diameter, indicating a decrease in carcinogenesis. From these results, it was concluded that tissues with inflammatory changes are more likely to be initiated by a carcinogenic substance than are normal tissues, in so far as the inflammatory changes induce no separation or destruction of basal cells even if superficial cells are separated. When inflammatory changes occurred in the tissues during the promotive stage, the inflammation stimuli also exerted a promoting effect on tumor formation.
The effect of cyclophosphamide (CP) on the urinary bladder epithelium of rats treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was examined histologically to detect preneoplastic papillary ...or nodular hyperplasia. The urinary bladder epithelium of rats injected intraperitoneally with 40 or 80mg/kg body weight of CP after treatment with 0.025% BBN for 2 weeks showed extensive epithelial erosion and then simple, or papillary or nodular hyperplasia, but this soon almost disappeared. In contrast, rats given BBN alone showed papillary or nodular hyperplasia later. These findings indicate that epithelial damage by CP inhibited the early neoplastic process of BBN.
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