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Scientific Reports Nov 2021Bladder cancer (BCa) is the most common malignancy of the urinary system with increasing incidence, mortality, and limited treatment options. Therefore, it is imperative...
Bladder cancer (BCa) is the most common malignancy of the urinary system with increasing incidence, mortality, and limited treatment options. Therefore, it is imperative to validate preclinical models that faithfully represent BCa cellular, molecular, and metabolic heterogeneity to develop new therapeutics. We performed metabolomic profiling of premalignant and non-muscle invasive bladder cancer (NMIBC) that ensued in the chemical carcinogenesis N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model. We identified the enriched metabolic signatures that associate with premalignant and NMIBC. We found that enrichment of lipid metabolism is the forerunner of carcinogen-induced premalignant and NMIBC lesions. Cross-species analysis revealed the prognostic value of the enzymes associated with carcinogen-induced enriched metabolic in human disease. To date, this is the first study describing the global metabolomic profiles associated with early premalignant and NMIBC and provide evidence that these metabolomic signatures can be used for prognostication of human disease.
Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogens; Cell Line; Humans; Male; Metabolome; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Urinary Bladder Neoplasms; Urothelium
PubMed: 34764423
DOI: 10.1038/s41598-021-99746-3 -
BioMed Research International 2022The effects of thymoquinone (TQ) in a carcinogen-based models of urinary bladder cancer were evaluated, using 45 male rats in five groups. In negative control ( = 10),...
The effects of thymoquinone (TQ) in a carcinogen-based models of urinary bladder cancer were evaluated, using 45 male rats in five groups. In negative control ( = 10), only tap water was given. In positive control ( = 10), the rats received 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in drinking water for 9 weeks. In preventive groups with 25 mg/kg ( = 10) and 50 mg/kg ( = 10), oral TQ was concurrently given with 0.05% BBN for 9 weeks and continued for one more week after cessation of BBN. Preventive-treatment group ( = 5) received 50 mg/kg TQ orally for 20 weeks. Five rats from each group were sequentially sacrificed in two phases: the induction phase at 12th week (except the last group) and the rest in postinduction phase at 20th week. The bladders were examined macroscopically for lesion formation, and the masses were submitted for histopathological evaluation. Markers for total oxidant status (TOS), inflammation (nuclear factor kappa B (NF-B)), and angiogenesis (vascular endothelial growth factor (VEGF)) were also assessed. There was a reduced number of bladder lesions in the TQ groups versus the carcinogen group at both phases. Histopathological findings demonstrated a significant improvement in the abnormal morphological changes in the urothelium of the TQ-treated groups. Thymoquinone exerted a significant antioxidant and anti-inflammatory effect by a decrease in serum level of TOS and NF-B at week 12 which was maintained low in phase two at week 20. The serum level of VEGF was also alleviated in the induction phase at week 12 and maintained low in postinduction period. In TQ preventive-treatment approach, a nonsignificant elevation of serum level of TOS and NF-B and slight reduction in VEGF were observed at the end of the experiment. These data suggest that TQ may be effective in preventing bladder carcinogenesis, and the suggested mechanisms might be related to antioxidant, prooxidant, and anti-inflammatory properties of TQ.
Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Benzoquinones; Butylhydroxybutylnitrosamine; Carcinogenesis; Carcinogens; Drinking Water; Male; NF-kappa B; Oxidants; Rats; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A
PubMed: 36158887
DOI: 10.1155/2022/6276768 -
JCI Insight Jun 2021Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but...
Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer, and this phosphorylation corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model, we show that this single posttranslational modification is critical for bladder cancer initiation and progression, despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical-grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis, and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.
Topics: Animals; Butylhydroxybutylnitrosamine; Carcinoma, Transitional Cell; Cell Transformation, Neoplastic; Drug Screening Assays, Antitumor; Eukaryotic Initiation Factor-4E; Gene Knock-In Techniques; Homeostasis; Humans; Mice; Neoplasm Transplantation; Protein Biosynthesis; Protein Kinase Inhibitors; Pyridines; Pyrimidines; RNA, Messenger; Ribosomal Proteins; Urinary Bladder Neoplasms; Urothelium
PubMed: 34032633
DOI: 10.1172/jci.insight.144920 -
Biology of Sex Differences May 2022Sex and age associated differences in the tumor immune microenvironment of non-muscle invasive bladder (NMIBC) cancer and associated clinical outcomes are emerging...
Sex and age associated differences in the tumor immune microenvironment of non-muscle invasive bladder (NMIBC) cancer and associated clinical outcomes are emerging indicators of treatment outcomes. The incidence of urothelial carcinoma of the bladder is four times higher in males than females; however, females tend to present with a more aggressive disease, a poorer response to immunotherapy and suffer worse clinical outcomes. Recent findings have demonstrated sex differences in the tumor immune microenvironment of non-muscle invasive and muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches to more precisely recapitulate these differences towards improved therapeutic design. Given the similarities in mucosal immune physiology between humans and mice, we evaluated the sex and age-related immune alterations in healthy murine bladders. Bulk-RNA sequencing and multiplex immunofluorescence-based spatial immune profiling of healthy murine bladders from male and female mice of age groups spanning young to old showed a highly altered immune landscape that exhibited sex and age associated differences, particularly in the context of B cell mediated responses. Spatial profiling of healthy bladders, using markers specific to macrophages, T cells, B cells, activated dendritic cells, high endothelial venules, myeloid cells and the PD-L1 immune checkpoint showed sex and age associated differences. Bladders from healthy older female mice also showed a higher presence of tertiary lymphoid structures (TLSs) compared to both young female and male equivalents. Spatial immune profiling of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen exposed male and female bladders from young and old mice revealed a similar frequency of TLS formation, sex differences in the bladder immune microenvironment and, age associated differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of bladder cancer and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes.
Topics: Aging; Animals; Butylhydroxybutylnitrosamine; Carcinogens; Carcinoma, Transitional Cell; Female; Humans; Male; Mice; Sex Characteristics; Tumor Microenvironment; Urinary Bladder; Urinary Bladder Neoplasms
PubMed: 35505436
DOI: 10.1186/s13293-022-00428-0 -
Cancer Science Aug 2022Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and...
Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2-overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN-induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.
Topics: Acetazolamide; Animals; Butylhydroxybutylnitrosamine; Carbonic Anhydrase Inhibitors; Carcinoma, Transitional Cell; Humans; Male; Mice; Rats; Urinary Bladder Neoplasms; beta Catenin
PubMed: 35723039
DOI: 10.1111/cas.15467 -
Scientific Reports Nov 2020Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with...
Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.
Topics: Animals; Butylhydroxybutylnitrosamine; Disease Models, Animal; Male; Muscle Contraction; Rats; Rats, Wistar; TRPV Cation Channels; Urinary Bladder; Urinary Bladder Neoplasms; Urinary Incontinence
PubMed: 33184390
DOI: 10.1038/s41598-020-76653-7 -
International Journal of Molecular... Jan 2020This study aims to evaluate the potency of cisplatin (Cispt)-loaded liposome (LCispt) and PEGylated liposome (PLCispt) as therapeutic nanoformulations in the treatment...
This study aims to evaluate the potency of cisplatin (Cispt)-loaded liposome (LCispt) and PEGylated liposome (PLCispt) as therapeutic nanoformulations in the treatment of bladder cancer (BC). Cispt was loaded into liposomes using reverse-phase evaporation method, and the formulations were characterized using dynamic light scattering, scanning electron microscopy, dialysis membrane, and Fourier-transform infrared spectroscopy (FTIR) methods. The results showed that the particles were formed in spherical monodispersed shapes with a nanoscale size (221-274 nm) and controlled drug release profile. The cytotoxicity effects of LCispt and PLCispt were assessed in an in vitro environment, and the results demonstrated that PLCispt caused a 2.4- and 1.9-fold increase in the cytotoxicity effects of Cispt after 24 and 48 h, respectively. The therapeutic and toxicity effects of the formulations were also assessed on BC-bearing rats. The results showed that PLCispt caused a 4.8-fold increase in the drug efficacy (tumor volume of 11 ± 0.5 and 2.3 ± 0.1 mm in Cispt and PLCispt receiver rats, respectively) and a 3.3-fold decrease in the toxicity effects of the drug (bodyweight gains of 3% and 10% in Cispt and PLCispt receiver rats, respectively). The results of toxicity were also confirmed by histopathological studies. Overall, this study suggests that the PEGylation of LCispt is a promising approach to achieve a nanoformulation with enhanced anticancer effects and reduced toxicity compared to Cispt for the treatment of BC.
Topics: Animals; Antineoplastic Agents; Butylhydroxybutylnitrosamine; Cell Line, Tumor; Cell Survival; Cisplatin; Drug Liberation; Female; Humans; Liposomes; Microscopy, Electron, Scanning; Particle Size; Polyethylene Glycols; Rats, Wistar; Treatment Outcome; Tumor Burden; Urinary Bladder Neoplasms
PubMed: 31952316
DOI: 10.3390/ijms21020559 -
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 -
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 -
PloS One 2021Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic...
Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations.
Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.
Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogens; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Longitudinal Studies; Male; Mice; Mice, Inbred C57BL; Polymerase Chain Reaction; Proteinuria; Proteomics; Sequence Analysis, RNA; Single-Cell Analysis; Urinary Bladder; Urinary Bladder Calculi; Urothelium
PubMed: 34232958
DOI: 10.1371/journal.pone.0253178