Cells Of Urinary Bladder Research Articles

ROLE OF HAEMATOLOGY BIOMARKERS IN URINARY BLADDER AND RENAL CELL CANCER PATIENTS TREATED WITH IMMUNE-ONCOLOGICAL DRUGS

Background: Urinary bladder (UB) and Renal Cell Cancer (RCC) are common in men than women with poor outcome. The novel immunotherapy drugs like Immune Check Point Inhibitors (ICIs) are effective but expensive. However, a cost-effective and reliable biomarker to predict response and clinical outcomes is lacking. Objective: To study the association of pre-treatment haematological parameters with clinical outcome in urinary bladder and renal cell cancer patients treated with ICIs. Method: In a retrospective study, we included 52 patients with urinary bladder and RCC treated with ICIs from Jan 2008 to Dec 2019. Clinical evaluation and laboratory investigations were performed as a part of standard protocol. CBC parameters such as WBC, TLC, DLC, Hb, Platelet, Neutrophil:Lymplocyte Ratio(NLR), Platelet:Lymphocyte Ratio (PLR), Lymphocyte:Monocyte Ratio(LMR) were studied at the time of TMC enrolment(first) and before the start (pre-treatment) of the ICIs therapy(io). Results: Amongst the CBC parameters, WBC count, when categorized based on cut off from ROC at the time of TMC enrolment(first) and the pre-treatment of the ICIs therapy(io) were found to be significantly associated with progression of disease with p- value 0.012 with Hazard Ratio (HR) - 2.52 (95% C.I. - 1.2-5.31) & p- value 0.060 with Hazard Ratio (HR) – 1.99 (95% C.I. – 0.96-4.12) respectively. The ROC - based cut off for WBC at the time of TMC enrolment(first) and the pre-treatment of the ICIs therapy(io) were found to be 7.16 X10e3/µL (AUC of 72%) with 71% sensitivity and 72% specificity and 6.48 X10e3/µL (AUC of 74%) with 67% sensitivity and 72% specificity respectively to predict progressed cases with respect to non-progressed cases. Along with WBC, the pre-treatment Absolute Neutrophil Count (ANC) (>3.67)& Absolute Monocyte count (AMC)(>0.42) pre-treatment of the ICIs therapy(io)) showed a significance with Hazard Ratio (HR) – 3.09(95% C.I.– 1.26-7.6) with p-value 0.010, Hazard Ratio (HR) – 3.48(95% C.I. - 1.6-7.57)and p-value <0.001 respectively. The ROC- based cut off for ANC at the pre-treatment of the ICIs therapy(io) were found to be 3.67 X10e3/µL (AUC of 68.4%) with 82.4% sensitivity and 50% specificity. Similarly The ROC- based cut off for AEC at the time of TMC enrolment(first) and AMC at the pre- treatment of the ICIs therapy(io) were found to be 0.22 X10e3/µL (AUC of 69.6%) with 55.9% sensitivity and 82.3% specificity and 0.42 X10e3/µL (AUC of 74.7%) with 72.7% sensitivity and 72.2% specificity respectively to predict progressed cases compared to non-progressed cases. Of 52 patients, 34 (65.4%) had progression. The median (m) PFS was 8.67 months (95% CI 2.26 - 15.09).The one year PFS rate was 46.5 (95% CI 31.2 -60.5). The mPFS in WBC <=7.16 was 26.48 months (95% CI 0 - 53.08) higher than the mPFS of 5.75 (95% CI 0.21 - 11.29) in WBC >7.16 with P value 0.012 at the time of TMC enrolment. Similarly, the mPFS in WBC pre- treatment of the ICIs therapy(io))<=6.48 was13.11 months (95% CI 0 – 28.95) higher than the mPFS of 6.18 (95% CI 2.67-9.68) in WBC >6.48 with P value 0.060.Along with WBC, the mPFS in ANC at the pre- treatment of the ICIs therapy(io)<=3.67 was 27.4(95% CI 0-55.21) higher than the mPFS of 6.18(95% CI 1.47-10.88)in ANC >3.67 with p value 0.010. The mPFS in AMC at the pre-treatment of the ICIs therapy(io)< =0.415 was 27.4 (95% CI 9.41-45.39) higher than the mPFS of 3.91 (95% CI 0.06-7.76) in AMC >0.415with p- value <0.001.We observed mPFS in immunotherapy cycles >=6 to be 15.9 (95% CI 6.98-24.82) was higher than the mPFS of 2.23 (95% CI 0.99-3.481) in immunotherapy cycles<6 with p value 0.025. The patients were followed for a period of 40.35 months (95% CI 26.98 - 53.71). Of 52 patients, 32(61.5%) patients deceased with median OS of 18.46 months (95% CI 10.12 - 26.80) and the one year OS rate was 57.6 (95% CI 41.6 - 70.6). Conclusion: Simple, routinely available and cost effective WBC (TLC), Absolute Neutrophil Count (ANC) ,Absolute Monocyte Count (AMC) and Absolute Eosinophil Count (AEC) from CBC test has a great potential to be used as a biomarker to predict response and clinical outcomes in patients with urinary bladder cancer and RCC patients treated with ICIs.

Polo-like kinase 1 inhibition modulates urinary tract smooth muscle contraction and bladder cell transcriptional programs.

The serine/threonine kinase polo-like kinase 1 (PLK1) is a master regulator of cell proliferation and contraction, but its physiological role in the lower urinary tract is unknown. We utilized transcriptomic programs of human bladder smooth muscle cells (hBSMCs), 3D bladder spheroid viability assays, and human ureterovesical junction contractility measurements to elucidate the impacts of PLK1 inhibition. This work reveals PLK1 reduction with the selective inhibitor TAK-960 (500 nM) suppresses high K+-evoked contractions of human urinary smooth muscle ex vivo while decreasing urothelial cell viability. Transcriptomic analysis of hBSMCs treated with TAK-960 shows modulation of cell cycle and contraction pathways, specifically through altered expression of Cys2/His2-type zinc finger transcription factors. In bladder spheroids, PLK1 inhibition also suppresses smooth muscle contraction protein filamin. Taken together, these findings establish PLK1 is a critical governor of urinary smooth muscle contraction and urothelial proliferation with implications for lower urinary tract disorders. Targeting PLK1 pharmacologically may therefore offer therapeutic potential to ameliorate hypercontractility and aberrant growth. Further elucidation of PLK1 signaling networks promises new insights into pathogenesis and much needed treatment advances for debilitating urinary symptoms.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, −34b-3p, −212-3p, −449a-5p, −21a-3p, −376b-3p, −376b-5p and − 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-β1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-β, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Dimethylarsinic acid induces bladder carcinogenesis via the amphiregulin pathway

Dimethylarsinic acid (DMA) is a major metabolite in the urine of humans and rats exposed to inorganic arsenicals, and is reported to induce rat bladder carcinogenesis. In the present study, we focused on early pathways of carcinogenesis triggered by DMA that were also active in tumors. RNA expression in the bladder urothelium of rats treated with 0 and 200 ppm DMA in the drinking water for 4 weeks and in bladder tumors of rats treated with 200 ppm DMA for 2 years was initially examined using microarray analysis and Ingenuity Pathway Analysis (IPA). Expression of 160 genes was altered in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. IPA associated 36 of these genes with liver tumor diseases. IPA identified the amphiregulin (Areg)-regulated pathway as a Top Regulator Effects Network. Therefore, we focused on Areg and 6 of its target genes: cyclin A2, centromere protein F, marker of proliferation Ki-67, protein regulator of cytokinesis 1, ribonucleotide reductase M2, and topoisomerase II alpha. We confirmed high mRNA expression of Areg and its 6 target genes in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. RNA interference of human amphiregulin (AREG) expression in human urinary bladder cell lines T24 and UMUC3 decreased expression of AREG and its 6 target genes and decreased cell proliferation. These data suggest that Areg has an important role in DMA-induced rat bladder carcinogenesis.

Analysis of the distal urinary tract in larval and adult zebrafish reveals homology to the human system.

Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we have undertaken multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a (ukp1a), uroplakin 2 (upk2) and uroplakin 3b (upk3b) genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated ukp1a expression in the zebrafish pronephros and cloaca from 96 h post-fertilization. Haematoxylin and Eosin staining of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder that leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a, Uroplakin 2 and GATA3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage and intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans, and offer the former as a model system to study disease.

Histopathological Study on Patients with Transitional Cell Carcinoma of Urinary Bladder in Al-Diwaniyah City/ Iraq

This study conducted on inpatients admitted to Al-Diwaniyah teaching hospital at period from January to June 2010 to recognize the histopathological changes of tumor cells of urinary bladder in comparison with normal cells. The histopathological examination of urinary tract infections (UTIs) showed normal thickness of urothelium, in addition, the submucosa and the muscular tissue were congested and oedematous. The present study showed that the transitional cell carcinoma of the bladder divided into three major grades: Grade І , Grade ІІ, Grade ІІІ. In this study, the immunehistochemical expression of cytokeratin 7 used to confirm the diagnosis of urinary bladder cancer. The results revealed a positive and negative reaction between tumor cells and cytokeratin7 antibody.

Coinfection with canine distemper virus and canine circovirus in a dog in Brazil.

Canine distemper virus (CDV) and canine circovirus (CanineCV) have been described worldwide in multi-systemic disease in dogs. Both agents may be occasionally associated with other viral pathogens, but reports of coinfection by CDV and CanineCV associated with disease are rare. In this article, we report a coinfection between CDV and CanineCV detected during an investigation of viral agents involved in multisystemic disease in dogs in Southern Brazil. Molecular testing by PCR in lungs and intestines of 77 dogs necropsied in pathology services (2015-2020) revealed several single and mixed viral infections, including a CDV/CanineCV coinfection. In the case reported here, gross and histological findings were compatible with CDV pathology (bronchointerstitial pneumonia and viral intracytoplasmatic inclusions in pneumocytes and transitional epithelial cells of urinary bladder). CanineCV DNA and CDV antigens were detected in lung and intestine fragments by PCR and immunohistochemistry, respectively. CanineCV PCR amplicons subjected to nucleotide sequencing showed > 98.6% nucleotide identity with CanineCV sequences from GenBank. Although the role of CanineCV in the pathogenesis of the reported case could not be determined, our results show that CanineCV may be associated with other viral infections in cases of multisystemic disease in dogs. These results reinforce the circulation of CanineCV in dogs in Brazil and highlight the importance of including this virus in the list of differential diagnoses of respiratory and gastroenteric infectious diseases in dogs.

Detection of Epithelial-Mesenchymal Transition Markers in High Grade Bladder Cancer and Special Variants of Urothelial Carcinoma.

Transitional cell carcinoma is considered the most predominant type of bladder cancer. Bladder can cer can also be found as squamous cell carcinoma that accounts for 5% of the total bladder cancer due to its etiology. The biomarkers associated with grade, prognosis, and stage of the disease are not well proved and known however, many studies have pointed to the association between SNAL/SLUG and Twist2 to the overall survival in patients with bladder cancer. These biomarkers were found to have a crucial role in inhibiting cadherin mediators specifically E-cadherin which are found normally in high level to integrate cell adhesion and normal function of the bladder. This research aims to detect SNAL/SLUG and Twist2 biomarkers in specimens of patients with bladder cancer and to detect their impact on E-cadherin, a tumor suppressor mediator responsible for improving survival and prevent metastasis. Materials and Methods:Using 150 archival tissue blocks from human bladder cancer cases to detect expression of SNAIL/SLUG and Twist2 in relation to loss of E-cadherin by immunohistochemical method. Results:Our results have revealed that in squamous cell carcinoma 40 specimens showed marked Twist 2 expression, and 30 specimens showed marked snail/slug biomarkers expression while poorly differentiated cancer cases showed marked expression of Twist 2 in 60 specimens and marked expression of Snail/slug marked expression in 50 specimens. Both were associated with E-cadherin loss. Among the 100 specimens with transitional cell carcinoma, 70 specimens showed divergent differentiation with 7 subtypes each showed different medium to high expression of Snail/Slug and Twist 2 biomarkers with the loss of E-cadherin. E-cadherin was strongly associated with the inverse increase in SNAL/SLUG and Twist2 biomarkers in urothelial carcinoma. Conclusion:Detection of SNAIL/SLUG and Twist 2 biomarkers in urothelial cancer is an important predictor for the loss of E-cadherin, a cornerstone in urinary bladder cell adhesion and its loss in urothelial carcinoma may contribute to cancer invasion and poor prognosis.

Discovery and characterization of a potent activator of the BKCa channel that relives overactive bladder syndrome in rats

The large-conductance Ca2+-activated K+ channel (BKCa channel) is involved in repolarizing the membrane potential and has a variety of cellular functions. The BKCa channel is highly expressed in bladder smooth muscle and mediates muscle relaxation. Compounds that activate the BKCa channel have therapeutic potential against pathological symptoms associated with the overactivity of bladder smooth muscle. In this regard, we screened a chemical library of 9938 compounds to identify novel BKCa channel activators. A cell-based fluorescence assay identified a structural family of compounds containing a common tricyclic quinazoline ring that activated the BKCa channel. The most potent compound TTQC-1 (7-bromo-N-(3-methylphenyl)-5-oxo-1-thioxo-4,5-dihydro[1,3]thiazolo[3,4-a]quinazoline-3-carboxamide) directly and reversibly activated the macroscopic current of BKCa channels expressed in Xenopus oocytes from both sides of the cellular membrane. TTQC-1 increased the maximum conductance and shifted the half activation voltage to the left. The apparent half-maximal effective concentration and dissociation constant were 2.8 μM and 7.95 μM, respectively. TTQC-1 delayed the kinetics of channel deactivation without affecting channel activation. The activation effects were observed over a wide range of intracellular Ca2+ concentrations and dependent on the co-expression of β1 and β4 auxiliary subunits, which are highly expressed in urinary bladder. In the isolated smooth muscle cells of rat urinary bladder, TTQC-1 increased the K+ currents which can be blocked by iberiotoxin. Finally, oral administration of TTQC-1 to hypertensive rats decreased the urination frequency. Therefore, TTQC-1 is a BKCa channel activator with a novel structure that is a potential therapeutic candidate for BKCa channel-related diseases, such as overactive bladder syndrome.

Proteomes of Uropathogenic Escherichia coli Growing in Human Urine and in J82 Urinary Bladder Cells.

Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, they undergo metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. To understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of the UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, most prominently arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were likewise upregulated together with proteins involved in sulfur compound turnover. Ribosomal proteins were downregulated relative to growth in MOPS in this environment. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.

Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation

Ethnopharmacological relevanceArctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. Aim of the studyThis study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. Materials and methodsQuantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. ResultsThrough in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 μg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. ConclusionsDespite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

Single-Cell RNA Sequencing of Urinary Cells Reveals Distinct Cellular Diversity in COVID-19-Associated AKI.

AKI is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized patients with COVID-19 with (n=5) or without AKI (n=3) as well as four patients with non-COVID-19 AKI (n=4) to assess differences in cellular composition and gene expression during AKI. Analysis of 30,076 cells revealed a diverse array of cell types, most of which were kidney, urothelial, and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared with those without AKI. ACE2 and TMPRSS2 expression was highest in urothelial cells among cell types recovered. Notably, in one patient, we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with antiviral and anti-inflammatory pathways. We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally, we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases.

Prevention of urinary infection through the incorporation of silver-ricinoleic acid-polystyrene nanoparticles on the catheter surface.

Nosocominal infections associated with biofilm formation on urinary catheters cause serious complications. The aim of this study was to investigate the feasibility of the polyurethane (PU) catheter modified with tetracycline hydrochloride (TCH) attached Ag nanoparticles embedded PolyRicinoleic acid-Polystyrene Nanoparticles (PU-TCH-AgNPs-PRici-PS NPs) and the influence on antimicrobial and antibiofilm activity of urinary catheters infected by Escherichia coli and Staphylococcus aureus. For this purpose, AgNPs embedded PRici graft PS graft copolymers (AgNPs-PRici-g-PS) were synthesized via free radical polymerization and characterized by FTIR, HNMR and DSC. AgNPs-PRici-PS NPs were prepared and optimized by the different parameters and the optimized size of nanoparticle was found as about 150 ± 1 nm. The characterization of the nanoparticles and the morphological evaluation were carried out by FTIR and SEM. Short term stability of nanoparticles was realised at 4°C for 30 days. In vitro release profiles of TCH and Ag NPs were also investigated. The formation of biofilm on PU modified TCH-Ag NPs-PRici-PS NPs, was evaluated and the biocompatibility test of the nanoparticles was realized via the mouse fibroblast (L929) and mouse urinary bladder cells (G/G An1). This is the first time that TCH-AgNPs-PRici-PS NPs used in the modification of PU catheter demonstrated high antimicrobial and antibiofilm activities against the urinary tract infection.

EGFR activity addiction facilitates anti-ERBB based combination treatment of squamous bladder cancer

Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation (Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA (n = 125) on genetic and protein expression level, and performed functional pathway and drug-response analyses with cell line models and isolated primary SCC (p-SCC) cells of the human urinary bladder. We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activating EGFR mutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative “Achilles heel” of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression.

Cytotoxicity of pristine and functionalized tungsten disulfide particles in the urinary system

Tungsten disulfide (WS2) particles have excellent lubrication properties and low surface energy. These features are exploited for numerous applications in the biomedical field, from the development of low-friction orthodontic wires to urinary encrustation-resisting coatings. Nevertheless, the cytotoxicity of spherical WS2 particles still remains unclear. We report the biocompatibility of pristine WS2 particles in human urinary bladder cells after the exposure to a wide range of concentrations (from 0 to 100 μg/mL). Based on the cell viability assessments using trypan blue exclusion assay and the PrestoBlue Cell viability reagent after 24, 48, and 72 h exposure, it can be concluded that these particle aggregates, with a diameter of ~ 500 nm, do not show any evidence of alterations or toxicity in the cells, even with high concentrations there exist a 87% of cell viability. Furthermore, due to the predisposition to agglomerate of this material and the possible use as a coating in urinary devices, we functionalized these particles with carboxylic groups. Aggregates with a diameter of ~ 370 nm were obtained. Zeta potential measurements highlighted that carboxylation correctly occurred, showing a switch from ~ + 4 for pristine particles to ~ − 35 for COOH-functionalized ones. Cytotoxic studies with these functionalized WS2 particles also showed good biocompatibility on human urinary bladder cells, with an 86% of viability after incubation with 100 μg/mL of particles over 72 h. These results highlight a good potential of WS2 particles for a future safe application as components of implantable medical devices.

Mitigation of aflatoxin B1- and sodium arsenite-induced cytotoxicities in HUC-PC urinary bladder cells by curcumin and Khaya senegalensis.

Background Concomitant exposure to environmental/occupational toxicants such as aflatoxin B1 (AFB1) and arsenic in some regions of the world has been well reported. Therefore, this calls for the assessment of the efficacy of agents such as phytochemicals, which are already known for their ethno-medicinal uses in prophylaxis/remediation. We investigated the possible cytotoxic bio-interactions between AFB1 and sodium arsenite (SA) in urinary bladder cells. We also assessed the cytoprotective effects of curcumin and the ethanol stem bark extract of Khaya senegalensis (K2S). Methods The cells were exposed to graded levels of AFB1, SA, curcumin, and K2S for 24, 48, and 72 h. Subsequently, using optimum toxic concentrations of AFB1 and SA, respectively, the influence of non-toxic levels of curcumin and/or K2S was tested on exposure of the cells to AFB1 and/or SA. Hoechst 33342/propidium iodide staining technique was used to determine the end-points due to cytotoxicity with changes in adenosine triphosphate (ATP) levels determined using Promega's CellTiter-Glo luminescent assay. Results Co-treatment of the cells with AFB1 and SA resulted in synergy in cytotoxic effects. Cytotoxicity was reduced by 3.5- and 2.9-fold by pre-treatment of the cells with curcumin and K2S before treatment with AFB1, while post-treatment resulted in 1.1- and 2.6-fold reduction, respectively. Pre-exposure of the cells with curcumin and K2S before treatment with SA ameliorated cytotoxicity by 3.8- and 3.0-fold, but post-treatment caused a 1.2- and 1.3-fold reduction, respectively. Conclusions Pre-treatment of the cells with either curcumin or K2S exhibited cytoprotective effects by ameliorating AFB1- and SA-induced cytotoxicity with inferred tendencies to prevent carcinogenesis.

Stem cell antigen/Ly6a protects against bladder fibrosis in mice.

We have defined a population of stem cell antigen (Sca)-1+/CD34+/lin- mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34+/lin-, collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34+/lin- cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.

Properties of SK3 channel-expressing PDGFRα (+) cells in the rodent urinary bladder

Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 μM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100 nM), ADP (100 μM) or ATP (100 μM) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.

Abstract 1867: Liver and urinary bladder cancers: The modifying role of aqueous leaf extract of Terminalia glaucescens Planch. ex Benth

Abstract Background: The aggressive and asymptomatic nature of liver cancer etiology in blacks is well documented. Furthermore, in Egypt, bladder cancer accounts for as many as 31% of all cancer cases, while in the US, the incidence in whites is higher than in blacks. There has been an increased search for phytochemicals, such as capsaicin (a bioactive component of hot peppers), which are easily available, and possess chemopreventive and chemotherapeutic activities. We therefore investigated the potential scientific relevance of aqueous leaf extract of Terminalia glaucescens (ALET) in cancer management. Terminalia glaucescens has been ethnomedicinally associated with various biological and therapeutic properties. Methods: Total flavonoid and phenolic contents of ALET were assessed spectrophotometrically using gallic acid and quercetin as standards, in addition to its free-radical-scavenging activity involving 2,2-diphenyl-1-picrylhydrazyl assay with butylated hydroxytoluene and vitamin C as reference compounds. The hydroxyl-radical-scavenging and reducing-power activities were correspondingly assessed. In addition, antiproliferative activities of ALET in comparison with capsaicin on normal and cancer cell lines of human liver (THLE-3 and HepG2) and urinary bladder (HUC-PC and MC-T11) were evaluated using fluorometry. Luminometry was used to determine ATP concentrations, caspase 3/7 activities, glutathione status, and mitochondrial functions. Results: Quantitative phytochemical assessments indicate the predominance of phenolic compounds (599.61 ± 6.14 µg gallic acid equivalents per mg of ALET) as compared with flavonoids (144.27 ± 3.44 µg quercetin equivalents per mg of ALET). ALET possesses comparable free-radical-scavenging, antioxidant and reducing-power activities in comparison to the standards. Treatment of the four cell lines with ALET for three days results in the following percent total cells: THLE-3 (30.13% at 24 h; 5.65% at 48 h; 5.43% at 72 h), HepG2 (31.09% at 24 h; 4.75% at 48 h; 1.41% at 72 h), HUC-PC (7.33% at 24 h; 2.35% at 48 h; 1.74% at 72 h), and MC-T11 (19.79% at 24 h; 9.20% at 48 h; 0.29% at 72 h). Assessment of the ATP levels after 24 h treatment with ALET resulted in a concentration-dependent depletion with a remarkable effect on HUC-PC and MC-T11 urinary bladder cells. ALET specifically caused a concentration-dependent decrease in caspase 3/7 activities and glutathione levels in HepG2 cells. ALET seems more toxic to the mitochondria at higher concentrations as compared with capsaicin. Conclusions: ALET could be a natural source of mitocans for the treatment of cancers. Citation Format: Jeremiah Olorunjuwon Olugbami, Robert Damoiseaux, James Kazemier Gimzewski, Oyeronke Adunni Odunola. Liver and urinary bladder cancers: The modifying role of aqueous leaf extract of Terminalia glaucescens Planch. ex Benth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1867.

Mesenchymal stromal cells modulate the molecular pattern of healing process in tissue-engineered urinary bladder: the microarray data

BackgroundMolecular mechanisms underlying the regenerative process induced by stem cells in tissue-engineered urinary bladder are poorly explained. The study was performed to explore the pathways associated with regeneration process in the urinary bladder reconstructed with adipose tissue-derived mesenchymal stromal cells (ASCs).MethodsRat urinary bladders were reconstructed with bladder acellular matrix (BAM) (n = 52) or BAM seeded with adipose tissue-derived mesenchymal stromal cells (ASCs) (n = 52). The process of bladder healing was analyzed at 7, 30, 90, and 180 days postoperatively using macroscopic histologic and molecular techniques. Gene expression was analyzed by microarrays and confirmed by real-time PCR.ResultsNumerous differentially expressed genes (DEGs) were identified between the bladders augmented with BAM seeded with ASCs or BAM only. Pathway analysis of DEGs allows to discover numerous pathways among them Hedgehog, TGF-β, Jak-STAT, PI3-Akt, and Hippo modulated by ASCs during the healing process of tissue-engineered urinary bladder. Real-time PCR analysis confirmed upregulation of genes involved in the Hedgehog signaling pathway including Shh, Gli1, Smo, Bmp2, Bmp4, Wnt2, Wnt2b, Wnt4, Wnt5a, and Wnt10 in urinary bladders reconstructed with ASC-seeded grafts.ConclusionThe study provided the unequivocal evidence that ASCs change the molecular pattern of healing in tissue-engineered urinary bladder and indicated which signaling pathways triggered by ASCs can be associated with the regenerative process. These pathways can be used as targets in the future studies on induced urinary bladder regeneration. Of particular interest is the Hedgehog signaling pathway that has been upregulated by ASCs during healing of tissue-engineered urinary bladder.