Dictamnine Inhibits the Adhesion to and Invasion of Uropathogenic Escherichia Coli (UPEC) to Urothelial Cells.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

Estrogen inhibits bladder overactivity in rats with cyclophosphamide-induced cystitis via downregulating the expression of P2X3 receptors in bladder epithelium cells.

AIMS: The therapeutic effect of estrogen on interstitial cystitis/bladder pain syndrome is unclear. We aim to explore the effect of estrogen on bladder overactivity in rats with cyclophosphamide-induced cystitis and its underlying mechanism. METHODS: In vivo cystometry was used to determine the effect of estrogen on bladder excitability. The effect of estrogen on the expression of P2X3 receptors in bladder epithelium was detected by real-time polymerase chain reaction and western blot. Effect of P2X3 receptors in bladder urothelium on stretch-released adenosine triphosphate was performed by a Flexcell FX5000 Compression system and an Enzyme-Linked Immunosorbent Assay Kit. RESULTS: Estrogen deprivation significantly increased the urinary frequency, while supplementation with diarylpropionitrile (DPN), an estrogen receptor ß (ERß) agonist, alleviated the urinary frequency. 17ß-Estradiol and DPN decreased the expression of P2X3 receptors in urothelium cells which was partially inhibited by ERß antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Meanwhile, inhibiting the expression of P2X3 receptors by ERß agonist or antagonizing the function of P2X3 receptors by selective P2X3 receptor antagonist AF-353 or A-317491 significantly reduced the stretch-released ATP from urothelium cells. CONCLUSIONS: Estrogen has a direct effect on the regulation of bladder overactivity in rats with cyclophosphamide-induced cystitis by downregulating the expression of bladder epithelial P2X3 receptors through ERß and reducing the adenosine triphosphate released from urothelium during bladder filling, thereby inhibiting the generation of the micturition reflex.

Curcumin May Prevent Basement Membrane Disassembly by Matrix Metalloproteinases and Progression of the Bladder Cancer.

Authors present a review of crucial mechanisms contributing to the invasion of the basement membrane (BM) of the urothelium by cancer cells and to the progression of bladder cancer (BC). The breeching of the urothelial BM, facilitated by an aberrant activation of matrix metalloproteinases (MMP) is particularly perilous. Inhibition of activation of these proteinases constitutes a logic opportunity to restrain progression. Because of limited efficacy of current therapeutic methods, the search for the development of alternative approaches constitutes "the hot spot" of modern oncology. Recent studies revealed significant anticancer potential of natural phytochemicals. Especially, curcumin has emerged as a one of the most promising phytochemicals and showed its efficacy in several human malignancies. Therefore, this article addresses experimental and clinical data indicating multi-directional inhibitory effect of curcumin on the growth of bladder cancer. We particularly concentrate on the mechanisms, by which curcumin inhibits the MMP's activities, thereby securing BM integrity and alleviating the eventual cancer invasion into the bladder muscles. Authors review the recently accumulating data, that curcumin constitutes a potent factor contributing to the more effective treatment of the bladder cancer.

Shionone alleviates NLRP3 inflammasome mediated pyroptosis in interstitial cystitis injury.

Shionone is a triterpenoid component derived from the herbal medicine Aster tataricus, and it has been reported to possess marked anti-inflammatory properties. The activation of NLRP3 inflammasome plays an important role in cystitis, and the effect of Shionone on NLRP3 inflammasome-dependent pyroptosis remains unclear. In this study, we established an interstitial cystitis (IC) rat model and SV-HUC-1 cell model with CYP or LPS + ATP treatment to mimic inflammation response and induce NLRP3 inflammasome activation. Shionone treatment significantly attenuated the bladder wet weight, score of edema and hemorrhage, enhanced the viability of SV-HUC-1 cell, decreased the rate of pyroptosis. Moreover, Shionone reduced the expression of NF-κB, NLRP3, ASC, Pro-caspase-1, Caspase-1, GSDMD, GSDMD-N at the mRNA and protein levels both in rat and SV-HUC-1 cell model, demonstrating NLRP3 inflammasome pathway was blocked and pyroptosis degree was reduced. These results indicated that Shionone could alleviate interstitial cystitis in Rat model and enhancing the viability of SV-HUC-1 cells via NF-κB/NLRP3/GSDMD-N pathway, which illustrated that Shionone could be used as a drug candidate for the treatment of interstitial cystitis.

Toward clinical understanding of aristolochic acid upper-tract urothelial carcinoma.

A cluster of patients poisoned by herbal medicine in the 1990s revealed that aristolochic acid (AA) causes kidney failure and upper tract urothelial carcinoma (UTUC). Recent research demonstrated that this was not an isolated incident; on the contrary, AA exposure is widespread in East Asia. This editorial highlights research by Lu and colleagues that investigates clinical characteristics of AA and non-AA UTUCs from 90 patients in Beijing based on the AA mutational signature. The study also detected AA mutations in non-tumor tissue of AA exposed patients and showed that AA mutations can be detected in urine, which might form the basis for non-invasive tests for AA exposure.

Aristolochic Acid Affects Upper Tract Urothelial Cancer Behavior through the MAPK Pathway.

The prevalence of upper tract urothelial carcinoma (UTUC) in Taiwan is relatively higher than thatin Western countries. Aristolochic acid (AA), which is widely used in traditional Chinese herbology, is now recognized to be one of the carcinogens for UTUC. Numerous UTUC patients have chronic kidney diseases or end-stage renal diseases; however, little literature hasreported on theoncogenic pathway of AA-related UTUC. The aim of our study was to identify the potential target treatment for AA-related UTUC. Here, we established an AA pre-exposure followed bya 3-methylcholanthrene (MCA) stimulus tumorigenic cell model. We not only demonstrated that AA pre-exposure MCA stimulus tumorigenic cells have more behaviors of cell migration and invasion by enhancing the metalloproteinases (MMP) activity, which is compatible with clinical findings of AA-related UTUC, but we also validated that AA pre-exposure MCA stimulus tumorigeniccells could be activated through the mitogen-activated protein kinases (MAPK) pathway. We further dissected the route of the MAPK pathway and found that the p38 and extracellular signal regulated kinases (ERK) sub-pathways might play essential roles in AA pre-exposure urothelial cancer cell lines. This consequence was also corroborated with a tissue study in AA-exposed patients.

Ba-Wei-Die-Huang-Wan (Hachimi-jio-gan) can ameliorate ketamine-induced cystitis by modulating neuroreceptors, inflammatory mediators, and fibrogenesis in a rat model.

AIM: We investigated the effects of Ba-Wei-Die-Huang-Wan (BWDHW) on ketamine-induced cystitis (KIC) in a rat model. METHODS: Female Sprague-Dawley rats were distributed into three groups: control (saline), ketamine (25 mg/kg/day for 28 days), or ketamine (25 mg/kg/day for 28 days) plus BWDHW (90 mg/kg/day, started from day 14). Functional magnetic resonance imaging (fMRI), metabolic cage study, and cystometry were evaluated. Bladder histology was evaluated. Western blots of the bladder proteins were carried out. RESULTS: Compared with controls, ketamine-treated rats showed stronger fMRI intensity in the periaqueductal gray area and bladder overactivity in the bladder functional study, but the ketamine/BWDHW-treated rats did not. Furthermore, ketamine breached the uroplakin III membrane at the apical surface of the urothelium, enhanced substance P spread over the urothelium, induced suburothelial hemorrhage and monocyte/macrophage infiltration, and caused interstitial fibrosis deposition. By contrast, the BWDHW-treated rats exhibited less substance P spread, lower suburothelial monocyte/macrophage infiltration, and lower interstitial fibrosis deposition. The ketamine group showed significant overexpression of neuroreceptors in the bladder mucosa (the transient receptor potential vanilloid 1 and M2 - and M3 -muscarinic receptors) and detrusor (M2 - and M3 -muscarinic receptors); inflammatory mediators in the detrusor (interleukin-1ß [IL-1ß], IL-6, tumor necrosis factor-α, nuclear factor-κB, cyclooxygenase-2, and intercellular adhesion molecule-1); and fibrogenesis molecules in the detrusor (transforming growth factor-ß1, collagen I, collagen III, and fibronectin). However, no significant changes were noted between the ketamine/BWDHW and control groups. CONCLUSION: BWDHW could exert therapeutic effects by inhibiting the upregulation of neuroreceptors, modulating inflammatory mediators, suppressing fibrogenesis, and ameliorating bladder overactivity in rats with KIC.

Alleviation of Cyclophosphamide-induced Hemorrhagic Cystitis by Dietary Pomegranate: A Comparative Experimental Study With Mesna.

In this study, we investigated the effects of pomegranate on alleviating cyclophosphamide-induced hemorrhagic cystitis (HC). Initially, 16 Sprague-Dawley rats were allocated into 4 groups: group 1 (control), group 2 (CP) in which HC was induced by cyclophosphamide; group 3 (CP+M), HC-induced rats that received Mesna regimen, and group 4 (CP+P), which compromised rats that had been on a 14-day diet of pomegranate juice before HC induction. Cystometry was performed a few hours before euthanasia; after euthanasia, aortic blood samples and bladder tissue samples were obtained to perform TUNEL assay, and histopathologic and biochemical assessments. Urodynamic findings revealed that mean detrusor pressure in CP+P was significantly lower compared with that in CP and CP+M (P<0.05). Histopathologically, urothelium destruction and inflammation were lower in CP+P and CP+M compared with that in CP. Collagen destruction was less prominent in CP+P compared with that in CP and CP+M. Tissue and plasma levels of malondialdehyde were significantly lower in CP+P versus CP (P<0.05). Catalase activity and total protein thiol group levels in plasma and bladder tissue were higher in CP+P versus CP (P<0.05). The TUNEL positivity in CP+P was significantly weaker than that in CP, indicating less DNA fragmentation and apoptosis. Pomegranate's characteristics could significantly affect the inflammatory and destructive process of hemorrhagic cystitis.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.

The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules. These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium.

Cigarette smoke extract induces the proliferation of normal human urothelial cells through the NF-κB pathway.

Bladder cancer is a common genitourinary malignant disease worldwide. Convincing evidence shows that cigarette smoke (CS) is a crucial risk factor for bladder cancer, yet the role of the NF-κB signaling pathway in the development of CS-associated bladder cancer has not been fully elucidated. In the present study, we found that exposure to cigarette smoke extract (CSE) induced proliferation and triggered the transition of normal human urothelial cells from G1 to S phase. Moreover, CSE exposure enhanced the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA) and decreased the expression of p21 in SV-HUC-1 cells. Furthermore, the levels of nuclear NF-κB p65/p50 were significantly elevated by CSE. Pre-treatment with the NF-κB inhibitor (PDTC) reversed CSE-triggered cell proliferation. Taken together, our study revealed that CSE induced proliferation of normal human urothelial cells through the NF-κB pathway, and these data enhance our understanding of the CSE-related carcinogenesis of bladder cancer.

Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA­containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA­induced upper urothelial tract cancer (AAN­UUC) and non­AAN­UUC tissues. Patients with AAN­UUC and non­AAN­UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN­UUC and non­AAN­UUC tissues (P<0.05). TargenScan and Gene ontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa­miR­4795­5p, Hsa­miR­488, Hsa­miR­4784, Hsa­miR­330, Hsa­miR­3916, Hsa­miR­4274 and Hsa­miR­181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN­UUC and non­AAN­UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively.

Effect of testosterone therapy on the urinary bladder in experimental hypogonadism of rats.

Testosterone (T) deficiency is prevalent particularly in elderly men and lead to physical and sexual morbidities. Although low levels of T are associated with low urinary tract symptoms, the correlation between T deficiency and bladder dysfunction is not clearly identified. The aim of this study was to investigate the effect of high dose testosterone replacement therapy (TRT) on the histological structure of the UB in castrated rats. Twenty-five adult male rats were divided into three groups: control, castrated and castrated + TRT. T was administrated in high dose (100 mg/kg) two intramuscular injections/week for 60 days. UB sections were prepared and stained with H&E, Masson's trichrome and immunohistochemical detection of Cytokeratin 20 (Ck20). All data were morphometrically and statistically analyzed. In castrated group, significant atrophy of the urothelium (P < 0.001) accompanied with widening of the corium were observed. The smooth muscle appeared thin with marked increase in the collagen fibers. On treating the castrated group with TRT, atypical Ck20 expression as well as significant increase in urothelial thickness (P < 0.05) and smooth muscle/collagen ratio (P < 0.001) were detected. In castrated rat model, high dose TRT has a positive effect on the UB smooth muscle rather than the urothelium which acquired atypical patterns.

Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue.

The time needed to produce engineered tissue is critical. A self-assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L-arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis.

Decoctions from Citrus reticulata Blanco seeds protect the uroepithelium against Escherichia coli invasion.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional usage suggests Citrus reticulata Blanco seeds have beneficial effects against infection. The purpose of this study was to investigate the effect of Citrus reticulata on the uroepithelium and to determine the mechanisms responsible for protection against urinary tract infection (UTI). MATERIALS AND METHODS: Human bladder cell lines T24 and 5637 were employed in a cell culture infection model to determine the effects of Citrus reticulata treatment on Escherichia coli adherence and invasion of the uroepithelium. ß1 integrin and caveolin-1 mRNA expression was assessed using RT real-time PCR. ß1 integrin protein expression was confirmed by Western Blot. The effect of Citrus reticulata on bacteria was investigated using antibacterial sensitivity, yeast agglutination and biofilm assays. RESULTS: Citrus reticulata treatment decreased ß1 integrin expression and reduced bacterial invasion while adhesion of uroepithelial cells was not affected. Caveolin-1 expression was not influenced either and Citrus reticulata did neither exhibit any direct antimicrobial effect nor interfered with type 1 fimbriae binding. CONCLUSIONS: Our results show that Citrus reticulata has a protective effect on the uroepithelium as seen by reduced bacterial invasion of uroepithelial cells. These properties suggest that seeds from Citrus reticulata may have therapeutic potential in preventing UTI.

Toll-like receptors in urothelial cells--targets for cancer immunotherapy.

Toll-like receptors (TLRs) have an important role in the activation of both innate and adaptive immunity in response to pathogens and danger signals. These receptors are expressed in immune cells and in some epithelia. They are expressed in the epithelium of the urinary bladder, where they actively participate in the fight against infection by uropathogens. TLR expression is decreased (although still evident) in bladder tumours, especially in non-muscle-invasive tumours. Intravesical immunotherapy with BCG to prevent recurrence of these tumours has been shown to involve the participation of three different TLRs (TLR2, TLR4, and TLR9). However, alternative therapies are needed as BCG fails in some patients and can sometimes cause severe adverse effects that are difficult to tolerate. In recent years, TLR2, TLR4, TLR7, and TLR9 agonists have been tested in vitro and in vivo for their ability to activate an antitumour immune response against bladder cancer. Promising results from these studies have led to the testing of TLR7 and TLR9 agonists in clinical trials.

Painful bladder syndrome and interstitial cystitis: treatment options.

Painful bladder syndrome (PBS) and interstitial cystitis (IC) are associated with bladder pain, increased urinary frequency, urgency and reduced quality of life. The cause is still unknown, although there are several possible hypotheses. PBS/IC may significantly impact all areas of a patient's life as they try to manage their symptoms. Treatment options usually involve diet and fluid modifications, oral medications, intravesical therapy or as a last resort, surgery. The bladder is protected by a mucous layer known as the GAG layer and is thought to protect the underlying urothelium from coming into contact with potassium ions and other irritants contained in urine. Due to research suggesting that sufferers of PBS/IC have dysfunctional GAG layers, there is rationale for intravesical GAG replacement therapy as a treatment option in reducing PBS/IC symptoms such as pain, frequency and urgency. Early diagnosis and treatment may lead to better long-term outcomes.

L-Leucine and L-isoleucine enhance growth of BBN-induced urothelial tumors in the rat bladder by modulating expression of amino acid transporters and tumorigenesis-associated genes.

We investigated the underlying mechanisms of L-leucine and L-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks and then fed AIN-93G basal diet or diet supplemented with L-leucine or L-isoleucine for 8 weeks followed by the basal diet for another 8 weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. L-Leucine and L-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, ß-catenin, p53, p21(Cip1/WAF1), cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes.

A novel p53 mutant found in iatrogenic urothelial cancers is dysfunctional and can be rescued by a second-site global suppressor mutation.

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.

Adjuvant methods to improve results of local bladder irrigations by chemotherapy for NMIBC.

Non-muscle-invasive bladder cancer is characterized by frequent recurrences requiring repeated transurethral resections and carries a risk of progression to muscle-invasive disease. The routinely used, passive diffusion irrigation of the bladder with mitomycin C has achieved limited success in avoiding these events. We review two methods that may improve penetration of mitomycin C into the bladder wall, thus leading to better results in terms of recurrence rate and bladder preservation.