Chemokine receptor 7 contributes to T- and B-cell filtering in ageing bladder, cystitis and bladder cancer.

BACKGROUND: Research has suggested significant correlations among ageing, immune microenvironment, inflammation and tumours. However, the relationships among ageing, immune microenvironment, cystitis and bladder urothelial carcinoma (BLCA) in the bladder have rarely been reported. METHODS: Bladder single-cell and transcriptomic data from young and old mice were used for immune landscape analysis. Transcriptome, single-cell and The Cancer Genome Atlas Program datasets of BLCA and interstitial cystitis/bladder pain syndrome (IC/BPS) were used to analyse immune cell infiltration and molecular expression. Bladder tissues from mice, IC/BPS and BLCA were collected to validate the results. RESULTS: Eight types of immune cells (macrophages, B-cells, dendritic cells, T-cells, monocytes, natural killer cells, γδ T-cells and ILC2) were identified in the bladder of mice. Aged mice bladder tissues had a significantly higher number of T-cells, γδ T-cells, ILC2 and B-cells than those in the young group (P < 0.05). Three types of T-cells (NK T-cells, γδ T-cells and naïve T-cells) and three types of B-cells (follicular B-cells, plasma and memory B-cells) were identified in aged mice bladder. Chemokine receptor 7 (CCR7) is highly expressed in aged bladder, IC/BPS and BLCA (P < 0.05). CCR7 is likely to be involved in T- and B-cell infiltration in aged bladder, IC/BPS and BLCA. Interestingly, the high CCR7 expression on BLCA cell membranes was a prognostic protective factor. CONCLUSIONS: In this study, we characterised the expression profiles of immune cells in bladder tissues of aged and young mice and demonstrated that CCR7-mediated T- and B-cell filtration contributes to the development of bladder ageing, IC/BPS and BLCA.

Role of extracellular vesicles in castration-resistant prostate cancer.

Prostate cancer (PCa) is a common health threat to men worldwide, and castration-resistant PCa (CRPC) is the leading cause of PCa-related deaths. Extracellular vesicles (EVs) are lipid bilayer compartments secreted by living cells that are important mediators of intercellular communication. EVs regulate the biological processes of recipient cells by transmitting heterogeneous cargoes, contributing to CRPC occurrence, progression, and drug resistance. These EVs originate not only from malignant cells, but also from various cell types within the tumor microenvironment. EVs are widely dispersed throughout diverse biological fluids and are attractive biomarkers derived from noninvasive liquid biopsy techniques. EV quantities and cargoes have been tested as potential biomarkers for CRPC diagnosis, progression, drug resistance, and prognosis; however, technical barriers to their clinical application continue to exist. Furthermore, exogenous EVs may provide tools for new therapies for CRPC. This review summarizes the current evidence on the role of EVs in CRPC.

The expression and distribution of TACAN in human and rat bladders.

OBJECTIVES: A lot of ion channels participate in the regulation of bladder function. TACAN, a new mechanosensitive ion channel, was first discovered in 2020. TACAN has been found to be expressed in many tissues, such as the dorsal root ganglia (DRG) and adipose tissue. However, it is unclear whether or not TACAN is expressed in the bladder. In this work, we decided to study the expression and distribution of TACAN in human and rat bladders. Meanwhile, the expression of TACAN in the rat model of interstitial cystitis/bladder pain syndrome (IC/BPS) was studied. METHODS: Human bladder tissues were obtained from female patients. Cyclophosphamide (CYP) was used to build the rat model of IC/BPS. Real-time polymerase chain reaction, agarose gel electrophoresis, and western blotting were used to assess the expression of TACAN in human and rat bladders. Immunohistochemistry and immunofluorescence were used to observe the distribution of TACAN in human and rat bladders. Hematoxylin-eosin stain, withdrawal threshold, and micturition interval were used to evaluate animal models. RESULTS: The results of agarose gel electrophoresis and western blotting suggested that TACAN was expressed in human and rat bladders. Immunohistochemical results suggested that TACAN showed positive immunoreaction in the urothelial and detrusor layers. The immunofluorescence results indicated that TACAN was co-stained with UPKIII, α-SMA, and PGP9.5. The IC/BPS model was successfully established with CYP. The mRNA and protein expression of TACAN was upregulated in the CYP-induced rat model of IC/BPS. CONCLUSIONS: TACAN was found in human and rat bladders. TACAN was mainly distributed in the urothelial and detrusor layers and bladder nerves. The expression of TACAN was upregulated in the CYP-induced rat model of IC/BPS. This new discovery will provide a theoretical basis for future research on the function of TACAN in the bladder and a potential therapeutic target for IC/BPS.

TrkA inhibition alleviates bladder overactivity in cyclophosphamide-induced cystitis by targeting hyperpolarization-activated cyclic nucleotide-gated channels.

Objectives: To investigate the potential of Tropomyosin receptor kinase A (TrkA) for the treatment of interstitial cystitis/ bladder pain syndrome (IC/BPS). Materials and Methods: Sixty-four female rats were randomly assigned to the control and cyclophosphamide (CYP) groups. Quantitative reverse transcription polymerase chain reaction was utilized to detect the mRNA level of TrkA. Western blot analysis was used to measure the protein levels of TNF-α, IL-6, and TrkA. Immunostaining was used to detect the expression of TrkA in bladder sections. Contractility studies and urodynamic measurements were utilized to test the spontaneous contractions of detrusor muscle strips and the global bladder activity, respectively. Results: Rat models of chronic cystitis were successfully established. The mRNA and protein levels of TrkA were significantly increased in the bladders of CYP-treated rats. Also, results of immunohistochemical staining and immunofluorescence staining showed that increased TrkA expression in the CYP group was mainly observed in the urothelium layer and bladder interstitial Cajal-like cells (ICC-LCs) but not in the detrusor smooth muscle cells. The specific inhibitor of TrkA, GW441756 (10 µM), significantly suppressed the robust spontaneous contractions of detrusor muscle strips in the CYP group and alleviated the overall bladder overactivity of CYP-treated rats. However, the inhibitory effects of GW441756 (10 µM) on the spontaneous contractions of detrusor muscle strips and the overall bladder activity were eliminated after pretreatments with the specific blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ZD7288 (50 µM). Conclusion: Our results suggested that increased TrkA expression during chronic cystitis promotes the development of bladder overactivity by targeting the HCN channels.

Decreased autophagic activity of detrusor cells is involved in the inflammatory response of interstitial cystitis/bladder pain syndrome.

INTRODUCTION AND HYPOTHESIS: Genome-wide association studies suggest that autophagy plays an important regulatory role in inflammatory and autoimmune diseases. Inflammation and immune regulation disorders are involved in the occurrence and development of interstitial cystitis/bladder pain syndrome (IC/BPS). However, the changes and roles of autophagy in IC/BPS have not been reported. Therefore, this study aimed to investigate bladder autophagy and inflammation changes in patients with IC/BPS. METHODS: Bladder specimens (n = 5) from patients with cystectomy due to end-stage IC/BPS were collected. The bladder samples of the control group (n = 5) were derived from the normal area bladder tissue after radical cystectomy. H&E and toluidine blue staining were used for histological evaluation. The co-location of LC3, alpha-smooth muscle actin (α-SMA), and autophagosome was investigated with double-labeled immunofluorescence and transmission electron microscopy (TEM). The expression of IL-6, TNF-α, Bax, caspase-3, and BCL-2 in the detrusor layer was analyzed using immunohistochemistry (IHC) and Western blot (WB). RESULTS: Compared with the control group, bladder tissue from IC/BPS patients revealed thinner and edematous epithelium with many mast cells (P < 0.05) infiltrating into the muscle layer. By using TEM (P < 0.05), double-labeled immunofluorescence (P < 0.05), and Western blot (P < 0.05) in IC/BPS patients, autophagy was also found and was significantly increased in detrusor myocytes. IHC and WB indicate the expression of BCL-2 (P < 0.05) was decreased, while IL-6, TNF-α, Bax, and caspase-3 expression was elevated (P < 0.05). CONCLUSIONS: The number of autophagosomes in detrusor cells was increased in IC/BPS. However, autophagy of detrusor muscle cells may not have sufficient phagocytic activity to effectively remove damaged proteins and mitochondria, which may lead to the continued deterioration of IC/BPS inflammation and apoptosis.

Alteration of the gut microbiota profile in children with autism spectrum disorder in China.

Background: Autism spectrum disorder (ASD) is associated with alterations in the gut microbiome. However, there are few studies on gut microbiota of children with ASD in China, and there is a lack of consensus on the changes of bacterial species. Purpose: Autism spectrum disorder (ASD) is associated with alterations in the gut microbiome. However, there are few studies on gut microbiota of children with ASD in China, and there is a lack of consensus on the changes of bacterial species. Methods: We used 16S rRNA sequencing to analyze ASD children (2 to 12 years), HC (2 to 12 years). Results: Our findings showed that the α-diversity, composition, and relative abundance of gut microbiota in the ASD group were significantly different from those in the HC groups. Compared with the HC group, the α-diversity in the ASD group was significantly decreased. At the genus level, the relative abundance of g_Faecalibacterium, g_Blautia, g_Eubacterium_eligens_group, g_Parasutterella, g_Lachnospiraceae_NK4A136_group and g_Veillonella in ASD group was significantly increased than that in HC groups, while the relative abundance of g_Prevotella 9 and g_Agathobacter was significantly decreased than that in HC groups. In addition, KEGG pathway analysis showed that the microbial functional abnormalities in ASD patients were mainly concentrated in metabolic pathways related to fatty acid, amino acid metabolism and aromatic compound metabolism, and were partially involved in neurotransmitter metabolism. Conclusion: This study revealed the characteristics of gut microbiota of Chinese children with ASD and provided further evidence of gut microbial dysbiosis in ASD.

Inhibition of U-II/UT signaling ameliorates cystitis-associated bladder hyperactivity by targeting the RhoA/Rho-kinase pathway.

Urotensin II (U-II) and its receptor (UT) are involved in the pathogenesis of various diseases; however, their association with the development of cystitis has not been elucidated. The present study was designed to investigate the functional role of U-II/UT signaling in cyclophosphamide (CYP)-induced cystitis. A total of 60 female rats were randomly divided into the control and CYP-treated groups. Intraperitoneal injection of CYP successfully induced cystitis in rats of the CYP-treated group. The protein and mRNA expression levels of U-II and UT were significantly enhanced in rat bladder tissues of the CYP-treated group. Furthermore, the results of the immunofluorescence staining analysis demonstrated that CYP treatment apparently increased the expression levels of UT in the urothelium layer, detrusor smooth muscle, and bladder interstitial Cajal-like cells. The selective antagonist of UT, SB657510 (10 µm), significantly suppressed the CYP-induced increase in the spontaneous contractions of muscle strips and ameliorated the bladder hyperactivity of CYP-treated rats. Moreover, CYP treatment significantly increased the protein expression levels of Ras homolog family member (Rho) A and Rho-associated protein kinase 2 in rat bladder tissues. Following pretreatment with the Rho-kinase inhibitor Y-27632 (10 µm), the inhibitory effects of SB657510 (10 µm) on the spontaneous contractions of muscle strips were eliminated. In conclusion, the results of the present study suggested that activation of U-II/UT signaling promoted the development of cystitis-associated-bladder hyperactivity by targeting the RhoA/Rho-kinase pathway, indicating that the U-II/UT signaling could serve as a novel target for the treatment of interstitial cystitis/bladder pain syndrome.

Activation of CXCL13/CXCR5 axis aggravates experimental autoimmune cystitis and interstitial cystitis/bladder pain syndrome.

The abnormal CXCL13/CXCR5 axis is involved in many inflammatory diseases and its selective inhibitor, TAK-799 has exhibited strong anti-inflammatory potency. The sequencing of clinical specimens from interstitial cystitis/bladder pain syndrome (IC/BPS) has shown that CXCL13 and CXCR5 are highly expressed, but the role of CXCL13/CXCR5 axis in IC/BPS has not been rarely reported. Therefore, in this study, we analyzed the GSE11783 sequencing data of IC/BPS patients and investigate the role and mechanism of CXCL13/CXCR5 axis and TAK-779 in the mouse model of experimental autoimmune cystitis (EAC). We verified that CXCL13 and CXCR5 were significantly up-regulated in EAC model. EAC mice exhibited increased bladder inflammatory factors (IL-6, TNF-α, IL-1ß), apoptosis-related proteins (Bax, Caspase-3, Caspase-8), frequency of voiding. Using TAK779 to block CXCL13/CXCR5 axis significantly attenuated these inflammatory damages and efficiently improved bladder function (significant reduction in micturition frequency, significant prolongation of inter-contraction interval). Further investigation showed that inhibiton of JNK and NF-kappaB activation, the bioinformatics analysis-indicated downstream signaling of CXCL13/CXCR5 axis, is responsible for the protective effect of TAK779. Taken together, we demonstrate that activation of the CXCL13/CXCR5 axis is involved in the pathophysiology of IC/BPS and EAC. Blocking CXCL13/CXCR5 axis activation by TAK-779 reduces bladder inflammation and improves bladder function in EAC mice.

Human Urine-Derived Stem Cells Improve Partial Bladder Outlet Obstruction in Rats: Preliminary Data and microRNA-mRNA Expression Profile.

Partial bladder outlet obstruction (pBOO) often results in bladder tissue inflammation and remodeling. As human urine-derived stem cells (USCs) have demonstrated therapeutic benefits, we used a rat model to investigate the effect of USCs on bladder function and explore the miRNA and gene expression profiles in bladder tissue using RNA sequencing. Eighteen rats were assigned to a sham surgery group, pBOO group, and pBOO+USC group (six biweekly treatments). Routine urodynamic monitoring, analysis of detrusor muscle strips, and pathophysiology assessments were conducted. Finally, altered miRNA and mRNA expression profiles of bladder tissue were examined using RNA sequencing and bioinformatics analysis. After USC treatment, elevated bladder compliance and maximal voiding pressure, declined end filling pressure and voided volume, and improved detrusor muscle contractility and carbachol sensitivity were found. Histology and TUNEL assay revealed reduced collagen deposition and muscle cell apoptosis in bladder tissue. The differential expression of eight miRNAs was reversed by USC treatment. Two large nodes (miR-142 and miR-9a) were identified in the miRNA-gene interaction network in the USC-treated group. The Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of multiple significant pathways, including those involved in necroptosis and cytokine-cytokine receptor interactions. This is the first study to demonstrate the protective effect of USCs on bladder function and remodeling in pBOO rats. The miRNA and mRNA expression levels differed in the bladder of pBOO rats with and without USC treatment. Although the mechanism underlying these effects has not been fully elucidated, necroptosis and cytokine-cytokine receptor interaction-related pathways may be involved.

Increased transient receptor potential canonical 3 activity is involved in the pathogenesis of detrusor overactivity by dynamic interaction with Na+/Ca2+ exchanger 1.

Transient receptor potential canonical 3 (TRPC3) is a nonselective cation channel, and its dysfunction is the basis of many clinical diseases. However, little is known about its possible role in the bladder. The purpose of this study was to explore the function and mechanism of TRPC3 in partial bladder outlet obstruction (PBOO)-induced detrusor overactivity (DO). We studied 31 adult female rats with DO induced by PBOO (the DO group) and 40 sham-operated rats (the control group). Here we report that the expression of TRPC3 in the bladder of DO rats increased significantly. Furthermore, PYR10, which can selectively inhibit the TRPC3 channel, significantly reduced bladder excitability in DO and control rats, but the decrease of the bladder excitability of DO rats was more obvious. PYR10 significantly reduced the intracellular calcium concentration in smooth muscle cells (SMCs) in DO and control rats. Finally, Na+/Ca2+ exchanger 1 (NCX1) colocalizes with TRPC3 and affects its expression and function. Collectively, these results indicate that TRPC3 plays an important role in the pathogenesis of DO through a synergistic effect with NCX1. TRPC3 and NCX1 may be new therapeutic targets for DO.

Luteolin Improves Cyclophosphamide-Induced Cystitis through TXNIP/NLRP3 and NF-κB Pathways.

Hemorrhagic cystitis is an important complication of cyclophosphamide chemotherapy, and current therapies for the disease are limited. The natural flavonoid luteolin (LUT) has significant anti-inflammatory and antioxidant properties, but its protective effect on cyclophosphamide (CYP)-induced bladder toxicity has yet to be evaluated. This study aims to explore the protective effect of LUT on CYP-induced acute cystitis in rats. Female Sprague-Dawley rats were randomly assigned to the control (CON) group, CON + LUT group, CYP group, and CYP + LUT group. A single intraperitoneal injection of CYP was administered to establish an acute hemorrhagic cystitis model. HE staining was performed to detect the degree of bladder tissue damage, and TUNEL staining was performed to count apoptotic cells. Oxidative stress indicators were measured using commercial kits, and bladder surgery was performed to assess urinary function. The levels of inflammatory cytokines, apoptosis-related indicators, TXNIP/NLRP3 pathway, and NF-κB pathway were detected by western blot. We found that LUT treatment reduced bladder bleeding, congestion, and edema caused by CYP. Compared with the CYP + LUT group, the level of apoptosis was more highly expressed in the CYP group. We also found that caspase-3, caspase-8, and Bax were significantly upregulated and Bcl-2 was downregulated after LUT treatment. In addition, LUT inhibited the activation of NF-κB signal pathway in the rat bladder tissue after CYP exposure. LUT treatment can also reduce the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) and TXNIP in the bladder. Finally, LUT can reduce the increase in the urination frequency and maximum urination pressure caused by cystitis. These results indicate that LUT displays effective anti-inflammatory, antioxidant, and antiapoptotic properties in CYP-induced acute hemorrhagic cystitis rats by inhibiting the TXNIP/NLRP3 and NF-κB pathways. LUT may be a potent therapeutic agent for the prevention and treatment of hemorrhagic cystitis.

Corrigendum to "Therapeutic Effects of Human Urine-Derived Stem Cells in a Rat Model of Cisplatin-Induced Acute Kidney Injury In Vivo and In Vitro".

[This corrects the article DOI: 10.1155/2019/8035076.].

Suppression of adenosine A2a receptors alleviates bladder overactivity and hyperalgesia in cyclophosphamide-induced cystitis by inhibiting TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a type of chronic bladder inflammation characterized by increased voiding frequency, urgency and pelvic pain. The sensitization of bladder afferents is widely regarded as one of the pathophysiological changes in the development of IC/BPS. There is evidence that adenosine A2a receptors are involved in regulating the sensitization of sensory afferents. However, the effect of adenosine A2a receptors on cystitis remains unknown. In the present study, a rat model of chronic cystitis was established by intraperitoneal injection with cyclophosphamide (CYP). Cystometry and behavioral tests were performed to investigate bladder micturition function and nociceptive pain. The rats with chronic cystitis showed symptoms of bladder overactivity, characterized by an increase in bladder voiding frequency and voiding pressure. CYP treatment significantly increased the expression of the A2a receptor in bladder afferent fibers and dorsal root ganglion (DRG) neurons. The A2a receptor antagonist ZM241385 prevented bladder overactivity and hyperalgesia elicited by CYP-induced cystitis. In addition, the A2a receptor and TRPV1 were coexpressed on DRG neurons. The TRPV1 antagonist capsazepine blocked bladder overactivity induced by the A2a receptor agonist CGS21680. In contrast, ZM241385 significantly inhibited the capsaicin-induced increase in intracellular calcium concentration in DRG neurons. These results suggest that suppression of adenosine A2a receptors in bladder afferents alleviates bladder overactivity and hyperalgesia elicited by CYP-induced cystitis in rats by inhibiting TRPV1, indicating that the adenosine A2a receptor in bladder afferents is a potential therapeutic target for the treatment of IC/BPS.

Differentiation of human urine-derived stem cells into interstitial cells of Cajal-like cells by exogenous gene modification: A preliminary study.

Human urine-derived stem cells (hUSCs) show multipotential differentiation ability and can differentiate into mesodermal cell lineages. Interstitial cells of Cajal-like cells (ICC-LCs) are crucial for the pace-making function of spontaneous contraction in the bladder. However, the mechanisms by which hUSCs generate ICC-LCs have not been elucidated. In this study, we developed a strategy for directional differentiation of hUSCs into ICC-LCs. hUSCs were transfected with lentiviral vectors encoding c-Kit, stem cell factor (SCF), hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4), and 5-azacytidine induced 2 (AZI2) genes, and the cells were cultured for an additional 7 days in specific medium. The expression of the surface marker c-Kit on ICC-LCs was determined at 7 days after transfection. hUSCs were successfully expanded and transfected with the four lentiviral vectors. hUSCs transfected with lentiviral-c-Kit, lentiviral-HCN4, and lentiviral-AZI2 showed higher expression of c-Kit 7 days after transfection, but only the lentiviral-HCN4-transfected cells showed morphological alterations in ICC-LCs. These cells also displayed visible HCN current amplitude and density. This approach may provide a new strategy for the treatment of underactive bladder.

Therapeutic Effects of Human Urine-Derived Stem Cells in a Rat Model of Cisplatin-Induced Acute Kidney Injury In Vivo and In Vitro.

Acute kidney injury (AKI) is an extremely dangerous clinical syndrome with high morbidity and mortality. Stem cell-based therapies have shown great promise for AKI treatment. Urine-derived stem cells (USCs) are a novel cell source in tissue engineering and cell therapy which provide advantages of simple, noninvasive, and low-cost harvest methods, efficient proliferation, and multi-differentiation potential. Here, we described the therapeutic effects of USCs in a rat model of cisplatin-induced AKI as a novel therapy. In vivo, the intravenous administration of USCs alleviated the renal functional damage in AKI rats, for the levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were significantly decreased. The USCs-treated group also exhibited improved histological and ultrastructural changes, promoted proliferation, and inhibited apoptosis in renal tissues. After the USC therapy, the expression levels of proinflammatory cytokines (TNF-α and IL-6) and apoptosis-related proteins (BAX and cleaved caspase-3) were downregulated. In addition, the presence of a few GFP-labeled USCs was confirmed in rat renal tissues. In vitro, rat tubular epithelial (NRK-52E) cells were incubated with cisplatin to induce cell damage and then cocultured with USCs. After coculture with USCs, the cisplatin-induced NRK-52E cells showed higher cell viability and a lower apoptosis ratio than those of the control group, and cell cycle arrest was improved. In conclusion, our results demonstrated that USC therapy significantly improved the renal function and histological damage, inhibited the inflammation and apoptosis processes in the kidney, and promoted tubular epithelial proliferation. Our study exhibited the potential of USCs in the treatment of AKI, representing a new clinical therapeutic strategy.

Increased Piezo1 channel activity in interstitial Cajal-like cells induces bladder hyperactivity by functionally interacting with NCX1 in rats with cyclophosphamide-induced cystitis.

The Piezo1 channel is a mechanotransduction mediator, and Piezo1 abnormalities have been linked to several clinical disorders. However, the role of the Piezo1 channel in cystitis-associated bladder dysfunction has not been documented. The current study aimed to discover the functional role of this channel in regulating bladder activity during cyclophosphamide (CYP)-induced cystitis. One hundred four female rats were randomly assigned to the control, CYP-4h, CYP-48h and CYP-8d groups. CYP successfully induced acute or chronic cystitis in these rats. CYP treatment for 48h or 8d significantly increased Piezo1 channel expression in bladder interstitial Cajal-like cells (ICC-LCs), and the increase in CYP-8d rats was more prominent. In addition, 2.5 µM Grammostola spatulata mechanotoxin 4 (GsMTx4) significantly attenuated bladder hyperactivity in CYP-8d rats by inhibiting the Piezo1 channel in bladder ICC-LCs. Furthermore, by using GsMTx4 and siRNA targeting the Piezo1 channel, we demonstrated that hypotonic stress-induced Piezo1 channel activation significantly triggered Ca2+ and Na+ influx into bladder ICC-LCs during CYP-induced chronic cystitis. In addition, the Piezo1 channel functionally interacted with the relatively activated reverse mode of Na+/Ca2+ exchanger 1 (NCX1) in bladder ICC-LCs from CYP-8d rats. In conclusion, we suggest that the functional role of the Piezo1 channel in CYP-induced chronic cystitis is based on its synergistic effects with NCX1, which can significantly enhance [Ca2+]i and result in Ca2+ overload in bladder ICC-LCs, indicating that the Piezo1 channel and NCX1 are potential novel therapeutic targets for chronic cystitis-associated bladder hyperactivity.

Cyanidin Curtails Renal Cell Carcinoma Tumorigenesis.

BACKGROUND/AIMS: Cyanidin is an anthocyanin found in many foods. Although its variable antioxidant levels are well-documented, little is known about its effects on renal cell carcinoma (RCC) tumorigenesis. This study, therefore, investigated the effects of cyanidin on the proliferation, migration, and invasion of renal cell carcinoma lines and demonstrated, for the first time, significant inhibitory effects of cyanidin on RCC tumorigenesis. METHODS: RCC cells were treated with different doses of cyanidin and the effects were tested by Cell Counting Kit-8 reagent, clone formation assay, transwell assay, and flow cytometry. Moreover, the cyanidin-mediated mechanism that curtailed tumorigenesis was analyzed by RNA sequencing (RNA-seq). Sequencing data from The Cancer Genome Atlas (TCGA) were used to compare the expression of both early growth response protein 1 (EGR1) and selenoprotein W (SEPW1) in RCC and tumor-free adjacent normal tissue samples. Real-time PCR (RT-PCR) and/or western blot were used to assess the expression of E-cadherin, cleaved-caspase3, Bcl2, p62, and ATG4. RESULTS: We found significantly greater induction of cell-cycle arrest, apoptosis, and suppression of RCC cell invasion and migration at concentrations of 25 µM and 100 µM than at a concentration of 50 µM. It was also discovered, first through RNA-seq then confirmed by RT-PCR, that cyanidin (100 µM) inhibited RCC carcinogenesis through EGR1 and SEPW1. TCGA data indicated that the expression level of EGR1 was lower and that of SEPW1 was higher in RCC tumor tissue than in normal tissues. Moreover, western blot and/or RT-PCR indicated that cleaved-caspase3 was enhanced and E-cadherin was inhibited by cyanidin treatment. Furthermore, western blot and RT-PCR also showed regulation of p62 and ATG4, which are associated with autophagy. Cyanidin in vivo significantly inhibited the growth of xenografts in nude mice. CONCLUSIONS: The results of this study showed the therapeutic potential of cyanidin for the treatment of RCC and the prevention of recurrence and metastasis.

Interleukin-6/signal transducer and activator of transcription 3 promotes prostate cancer resistance to androgen deprivation therapy via regulating pituitary tumor transforming gene 1 expression.

Prostate cancer can progress from androgen dependence to androgen deprivation resistance with some unknown mechanisms. The current study aims to explore the possible role of pituitary tumor transforming gene1 (PTTG1) in castration-resistant prostate cancer (CRPC). Initially, we found that PTTG1 expression was significantly increased in androgen-independent prostate cancer cell lines PC3, DU145 and CRPC specimens compared with that in androgen-dependent prostate cancer cell line LNCaP and initial prostate cancer specimens. PTTG1 overexpression significantly enhanced the cell survival rate, clonality and tumorigenicity in LNCaP cells upon androgen-deprivation therapy (ADT). While knockdown of PTTG1 expression significantly elevated the sensitivity of DU145 cells to ADT. The effects of PTTG1 overexpression on LNCaP cells may be ascribed to the induced EMT and increased CD44+ CD24- cancer stem cell population. Furthermore, we detected that PTTG1 expression was regulated by interleukin-6 via activated signal transducer and activator of transcription 3 (STAT3) directly binding to the region -500 to +1 of PTTG1 promoter in LNCaP cells. In conclusion, our results elucidate that interleukin-6/STAT3 activation can increase PTTG1 expression and, consequently, promote the resistance to ADT in CRPC by inducing EMT and increasing the cancer stem cell population, suggesting that PTTG1 may be a novel therapeutic target for CRPC.

PDE1A polymorphism contributes to the susceptibility of nephrolithiasis.

BACKGROUND: Previous studies have confirmed a family risk of nephrolithiasis (NL), but only 15% of all cases are associated with an identified monogenic factor. In clinical practice, our group encountered a patient with NL combined with cystic kidney disease that had 3 affected family members. No known mutations association with NL was detected in this family, and thus further investigation of the molecular cause of NL was deemed to be necessary. RESULTS: Quality analysis from the sequencing stage showed a more than 80-fold average depth and 95% coverage for each sample, and six mutations within six genes were chosen as candidate variants for further validation. Genotyping of rs182089527in the phosphodiesterase 1A (PDE1A) gene in the validation cohort indicated that the alternative allele was present in 15 patients with heterozygosity and in 1 patient with homozygosity, and exhibited significant enrichment in NL patients (Fisher's exact test, adjusted p = 0.0042) and kidney cystic patients (Fisher's exact test, adjusted p = 0.067) compared to controls. In addition, function analysis displayed a significant decrease in the protein and mRNA expression levels resulting from the rs182089527 mutant sequence compared with the wild-type sequence. Moreover, patients with this mutation displayed a high level of creatinine and urea in urinalysis. CONCLUSIONS: Our study provides genetic evidence that the rs182089527 mutation in PDE1A is involved in the development of NL and kidney cysts, which should help to improve personalized medicine for diagnosis and treatment.

Large conductance voltage and Ca2+-activated K+ channels affect the physiological characteristics of human urine-derived stem cells.

We investigated the current characteristics of large conductance voltage and Ca2+-activated K+ (BK) channels in human urine-derived stem cells (hUSCs) and the effect of BK channels on proliferation and differentiation of hUSCs. Fresh human urine (n=6) was collected from healthy donors to isolate hUSCs. Human KCNMA1 gene silencing U6 shRNA was used to down regulate the expression of BK in hUSCs. IBTX (BK channel antagonist) and NS1619 (BK channel agonist) were used to examine the effect of BK channels on hUSCs. Whole cell patch-clamping was employed to detect the current of BK channels. Flow cytometry, immunofluorescence, and western blotting were used to analyze the cell cycle and related protein levels. The results showed that the activities of BK channels were significantly decreased in P5, P7 and induced hUSCs (endothelial, urothelial and smooth muscle cells) compared with P3 hUSCs when normalized to the cell capacitance. In addition, the average BK channel current density of hUSCs was significantly decreased upon silencing BK channel expression by hnRNA. Apoptosis rates of hUSCs in iberiotoxin (IBTX) and hnRNA treatment groups were significantly increased compared with the control group, whereas treatment with BK agonist NS1619 decreased apoptosis rates. Compared with the control group, hUSCs in S phase were significantly decreased in IBTX and hnRNA treatment groups. In conclusion, BK channels play an important role in maintaining the proliferation and differentiation of hUSCs. Overexpression of BK channels in hUSCs be provide a basis for future clinical application to an overactive bladder.