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Journal of Translational Medicine Nov 2019Bladder 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...
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogenesis; Gene Expression Regulation, Neoplastic; Inflammation; Male; Mice, Inbred C57BL; Urinary Bladder Neoplasms
PubMed: 31779626
DOI: 10.1186/s12967-019-02146-5 -
Carcinogenesis Oct 2011Thioredoxin-interacting protein (TXNIP), which has a tumor-suppressive function, is underexpressed in some human cancers. The function of TXNIP in vivo in carcinogenesis...
Thioredoxin-interacting protein (TXNIP), which has a tumor-suppressive function, is underexpressed in some human cancers. The function of TXNIP in vivo in carcinogenesis is not fully understood. Here, we show TXNIP to be downregulated in human bladder cancer according to grade and stage and also that loss of TXNIP expression facilitates bladder carcinogenesis using a mouse bladder cancer model. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer was found in 100% of Txnip knockout (KO) mice at week 8 of 0.025% BBN administration but in only 22% of wild-type (WT) mice at the same point. Among growth stimulators, phospho-extracellular signal-regulated kinase (pERK) expression was stronger during bladder carcinogenesis in Txnip-KO mice than in WT mice. We then evaluated TXNIP's effects on ERK activation through various growth stimulators and their receptors. Overexpression of TXNIP in human bladder cancer cells attenuated pERK expression upon stimulation with stromal cell-derived factor-1 (SDF-1) but not with epidermal growth factor or insulin-like growth factor-1. In Txnip-KO mice, immunohistochemical analysis showed enhanced expression of C-X-C chemokine receptor type 4 (CXCR4), the receptor of SDF-1, and of pERK in urothelial cells during BBN-induced bladder carcinogenesis. Finally, subcutaneous injection of CXCR4 antagonist, TF14016, attenuated pERK in urothelial cells and suppressed bladder carcinogenesis. These data indicate that TXNIP negatively regulates bladder carcinogenesis by attenuating SDF-1-CXCR4-induced ERK activation. This signal transduction pathway can be a potent target in preventing or treating bladder cancer.
Topics: Aged; Animals; Blotting, Western; Butylhydroxybutylnitrosamine; Carrier Proteins; Chemokine CXCL12; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Immunoenzyme Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Staging; Prognosis; RNA, Messenger; Receptors, CXCR4; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Survival Rate; Thioredoxins; Urinary Bladder Neoplasms
PubMed: 21771725
DOI: 10.1093/carcin/bgr137 -
Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.Cancer Science Apr 2020Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer....
Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin-1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cell-cycle arrest, p21 upregulation and downregulation of phospho(p)-S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p-S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67-labeling index and p-S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin-3'-O-glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin-3'-glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product-derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling.
Topics: Animals; Apoptosis; Butylhydroxybutylnitrosamine; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Luteolin; Male; Mice; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; TOR Serine-Threonine Kinases; Thioredoxins; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays; rho GTP-Binding Proteins
PubMed: 31994822
DOI: 10.1111/cas.14334 -
Tumour Biology : the Journal of the... Feb 2017Bladder cancer remains a huge concern for the medical community because of its incidence and prevalence rates, as well as high percentage of recurrence and progression....
Bladder cancer remains a huge concern for the medical community because of its incidence and prevalence rates, as well as high percentage of recurrence and progression. Omega-3 polyunsaturated fatty acids and atorvastatin proved anti-inflammatory effects through peroxisome proliferator-activated receptor gamma mechanism. However, their chemopreventive effect still remained to be examined and clarified. In this study, bladder cancer was induced in rats by the chemical carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. Omega-3 polyunsaturated fatty acids (docosahexaenoic acid and eicosapentaenoic acid: 2:3 w/w; 1200 mg/kg) and/or atorvastatin (6 mg/kg) were given orally daily to rats for eight consecutive weeks concomitantly with N-butyl-N-(4-hydroxybutyl)nitrosamine and continued for further 4 weeks after cessation of N-butyl-N-(4-hydroxybutyl)nitrosamine administration. The histopathological examination of rat bladder revealed the presence of tumors and the absence of apoptotic bodies in sections from N-butyl-N-(4-hydroxybutyl)nitrosamine group, while tumors were absent and apoptotic bodies were clearly observed in sections from rat groups treated with omega-3 polyunsaturated fatty acids, atorvastatin, or both drugs. The study of the molecular mechanisms illustrated downregulation of COX-2 and P53 (mutant) genes and suppression of transforming growth factor beta-1 and the lipid peroxidation product malondialdehyde in serum of rats of the three treated groups. This chemopreventive effect was confirmed by and associated with lower level of bladder tumor antigen in urine. However, the combined treatment with both drugs exhibited the major protective effect and nearly corrected the dyslipidemia that has been induced by N-butyl-N-(4-hydroxybutyl)nitrosamine. Collectively, omega-3 polyunsaturated fatty acids and atorvastatin, besides having anti-inflammatory properties, proved a chemopreventive effect against bladder cancer, which nominates them to be used as adjuvant therapy with other chemotherapeutics.
Topics: Animals; Apoptosis; Atorvastatin; Butylhydroxybutylnitrosamine; Cell Proliferation; Docosahexaenoic Acids; Eicosapentaenoic Acid; Male; Oxidative Stress; Random Allocation; Rats; Urinary Bladder Neoplasms
PubMed: 28218036
DOI: 10.1177/1010428317692254 -
Anticancer Research 2006N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced urothelial carcinogenesis is a useful model for studying urothelial carcinogenesis. Here, the DNA content and the...
DNA content analysis, expression of Ki-67 and p53 in rat urothelial lesions induced by N-butyl-N-(4-hydroxybutyl) nitrosamine and treated with mitomycin C and bacillus Calmette-Guérin.
N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced urothelial carcinogenesis is a useful model for studying urothelial carcinogenesis. Here, the DNA content and the expression of Ki-67 and p53 in urothelial lesions induced by BBN and treated with mitomycin C (MMC) and Bacillus Calmette-Guérin (BCG) were investigated. Female Fisher 344 rats were distributed into five groups treated with 0.05% BBN in their drinking water for 20 weeks. Ten animals were used as negative control. Intravesical instillations were performed with MMC, BCG and physiological saline solution (PSS), once per week, for 6 weeks. The animals were sacrificed 1 week after the last intravesical instillation. DNA ploidy analysis was carried out by static cytometry. Ki-67 and p53 were analysed immunohistochemically in paraffin-embedded tissue. The incidence of lesions developed in rats with PSS was greater than in rats instilled with MMC and BCG. The incidence of aneuploidy was lower in tumours treated with MMC and BCG. Low- and high-grade papillary carcinoma treated with MMC and BCG showed a decrease in labelling index and an increase of apoptotic index. The proliferative index was correlated with the apoptotic index (r=0.438, p<0.01). Significant correlations were also found between the proliferative index and lesion, and the apoptotic index and lesion (r=0.425, p<0.01 and r=0.275, p<0.01), respectively. A significant correlation was found between ploidy and the apoptotic index (r=0.245, p<0.05). Our results provide information on the biological behaviour of chemically-induced bladder tumours treated with MMC and BCG.
Topics: Aneuploidy; Animals; Antibiotics, Antineoplastic; BCG Vaccine; Butylhydroxybutylnitrosamine; Carcinogens; DNA, Neoplasm; Female; Immunohistochemistry; Ki-67 Antigen; Mitomycin; Rats; Rats, Inbred F344; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms
PubMed: 16886626
DOI: No ID Found -
PloS One 2018The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For...
The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For non-invasive evaluations in vivo, an anti-CD3 mAb was modified with desferrioxamine (DFO) and radiolabeled with zirconium-89 (Zr-89 or 89Zr). Radiolabeled 89Zr-DFO-anti-CD3 was tested for T cell detection using positron emission tomography (PET) in both healthy mice and mice bearing syngeneic bladder cancer BBN975. In vivo PET/CT and ex vivo biodistribution demonstrated preferential accumulation and visualization of tracer in the spleen, thymus, lymph nodes, and bone marrow. In tumor bearing mice, 89Zr-DFO-anti-CD3 demonstrated an 11.5-fold increase in tumor-to-blood signal compared to isotype control. Immunological profiling demonstrated no significant change to total T cell count, but observed CD4+ T cell depletion and CD8+ T cell expansion to the central and effector memory. This was very encouraging since a high CD8+ to CD4+ T cell ratio has already been associated with better patient prognosis. Ultimately, this anti-CD3 mAb allowed for in vivo imaging of homeostatic T cell distribution, and more specifically tumor-infiltrating T cells. Future applications of this radiolabeled mAb against CD3 could include prediction and monitoring of patient response to immunotherapy.
Topics: Animals; Antibodies; Butylhydroxybutylnitrosamine; CD3 Complex; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Deferoxamine; Flow Cytometry; Lymphocytes, Tumor-Infiltrating; Mice, Inbred C57BL; Neoplasm Transplantation; Positron Emission Tomography Computed Tomography; Radioisotopes; Radiopharmaceuticals; Tissue Distribution; Urinary Bladder Neoplasms; Zirconium
PubMed: 29513764
DOI: 10.1371/journal.pone.0193832 -
PloS One 2016Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a...
Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development.
Topics: Androgens; Animals; Butylhydroxybutylnitrosamine; Carcinoma, Transitional Cell; Cell Division; Cell Transformation, Neoplastic; Drug Implants; Female; Genetic Predisposition to Disease; Humans; Integrases; Male; Mice; Mice, Transgenic; Neoplasms, Hormone-Dependent; Orchiectomy; Promoter Regions, Genetic; Receptors, Androgen; Recombinant Fusion Proteins; Tamoxifen; Testosterone; Transgenes; Urinary Bladder Neoplasms; Uroplakin III
PubMed: 26862755
DOI: 10.1371/journal.pone.0148851 -
Japanese Journal of Cancer Research :... Apr 1988Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5... (Comparative Study)
Comparative Study
Differences in susceptibility of the urinary bladder epithelium to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in various strains were examined. In experiment 1, 5 strains of male rats were given 0.025% BBN in the drinking water for 8 weeks followed by drinking water without BBN for 32 weeks. Analbuminemic rats (NAR) and ACI rats had high incidences of urinary bladder lesions (papillary or nodular hyperplasia, papilloma and carcinoma), F344 and Wistar rats had low incidences, and Sprague-Dawley (SD) rats showed an intermediate incidence. Carcinoma area was largest in NAR rats followed in decreasing order by SD, ACI and F344 rats. The extent of tumor invasion was higher in NAR and ACI rats than in SD rats. In experiment 2, the 5 strains of male rats were administered 0.025% BBN in the drinking water. Some rats from each group were killed after each of weeks 4 and 8. The urinary bladder of ACI and NAR rats given BBN had the most marked lesions observed by scanning electron microscopy, with less marked changes in SD rats. F344 and Wistar rats showed the weakest response. Cytochrome P-450 content of the liver in ACI rats treated with BBN for 4 weeks was significantly higher than those of controls. Cytochrome P-450 and cytochrome b5 contents of the control and BBN-treated rats were significantly higher in ACI and SD rats than in Wistar, F344 or NAR rats. These results indicate that there are strain differences in the urinary bladder response to BBN.
Topics: Animals; Body Weight; Butylhydroxybutylnitrosamine; Cytochrome P-450 Enzyme System; Cytochrome b Group; Cytochromes b5; Disease Susceptibility; Epithelium; Neoplasms, Experimental; Nitrosamines; Organ Size; Rats; Rats, Inbred ACI; Rats, Inbred F344; Rats, Inbred Strains; Species Specificity; Urinary Bladder; Urinary Bladder Neoplasms
PubMed: 3133335
DOI: 10.1111/j.1349-7006.1988.tb01613.x -
Molecular Medicine Reports Mar 2016Bladder cancer frequently shows mutational activation of the oncogene Ras, which is associated with bladder carcinogenesis. However, the signaling pathway downstream of...
Bladder cancer frequently shows mutational activation of the oncogene Ras, which is associated with bladder carcinogenesis. However, the signaling pathway downstream of Ras remains to be fully elucidated. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) is able to induce bladder cancer by driving the clonal expansion of initiated cells carrying the activated form of Ras. Phospholipase Cε (PLCε) is the main target of BBN, while the tumor promoting role of PLCε remains controversial. The present study examined the role of PLCε in BBN‑induced bladder carcinogenesis of mice with genetically inactivated PLCε. Using light and electron microscopy, the present study demonstrated that PLCε(‑/‑) mice were resistant to BBN‑induced bladder carcinogenesis. Furthermore, it was demonstrated that cyclooxygenase 2 and vascular endothelial growth factor‑A were affected by the PLCε background of the mice, suggesting that the role of PLCε in tumor promotion may be ascribed to augmentation of inflammatory responses and angiogenesis. These results indicated that PLCε is crucial for BBN‑induced bladder carcinogenesis as well as signaling downstream of Ras, and that PLCε is a candidate molecular target for the development of anti-cancer drugs.
Topics: Animals; Blotting, Western; Butylhydroxybutylnitrosamine; Carcinogenesis; Cyclooxygenase 2; Fluorescent Antibody Technique; Inflammation; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; Phosphoinositide Phospholipase C; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A
PubMed: 26782701
DOI: 10.3892/mmr.2016.4762 -
EMBO Reports Jun 2005The proapoptotic protein encoded by Par4 (prostate apoptosis response 4) has been implicated in tumour suppression, particularly in the prostate. We report here that... (Comparative Study)
Comparative Study
The proapoptotic protein encoded by Par4 (prostate apoptosis response 4) has been implicated in tumour suppression, particularly in the prostate. We report here that Par4-null mice are prone to develop tumours, both spontaneously and on carcinogenic treatment. The endometrium and prostate of Par4-null mice were particularly sensitive to the development of proliferative lesions. Most (80%) Par4-null females presented endometrial hyperplasia by 9 months of age, and a significant proportion (36%) developed endometrial adenocarcinomas after 1 year of age. Similarly, Par4-null males showed a high incidence of prostate hyperplasia and prostatic intraepithelial neoplasias, and were extraordinarily sensitive to testosterone-induced prostate hyperplasia. Finally, the uterus and prostate of young Par4-null mice have increased levels of the apoptosis inhibitor XIAP (X-chromosome-linked inhibitor of apoptosis), supporting the previously proposed function of Par4 as an inhibitor of the (zeta)PKC (atypical protein kinase)-NF-(kappa)B (nuclear factor-(kappa)B)-XIAP pathway. These data show that Par4 has an important role in tumour suppression, with a particular relevance in the endometrium and prostate.
Topics: Age Factors; Animals; Apoptosis; Butylhydroxybutylnitrosamine; Endometrial Neoplasms; Estradiol; Female; Histological Techniques; Immunoblotting; Male; Mice; Mice, Mutant Strains; Phenotype; Prostatic Neoplasms; Proteins; Receptors, Thrombin; Testosterone; Urinary Bladder Neoplasms; X-Linked Inhibitor of Apoptosis Protein
PubMed: 15877079
DOI: 10.1038/sj.embor.7400421