Search

Purpose: T1 high grade (T1HG) bladder cancer (BC) is a type of non-muscle invasive BC (NMIBC) that is recognized as an aggressive subtype with a heightened propensity for progression. Current risk stratification methods for NMIBC rely on clinicopathological indicators; however, these approaches do not adequately capture the aggressive nature of T1HG BC. Thus, new, more accurate biomarkers for T1HG risk stratification are needed. Here, we enrolled three different patient cohorts and investigated expression of collagen type VI alpha 1 ( COL6A1 ), a key component of the extracellular matrix, at different stages and grades of BC, with a specific focus on T1HG BC., Materials and Methods: Samples from 298 BC patients were subjected to RNA sequencing and real-time polymerase chain reaction., Results: We found that T1HG BC and muscle invasive BC (MIBC) exhibited comparable expression of COL6A1 , which was significantly higher than that by other NMIBC subtypes. In particular, T1HG patients who later progressed to MIBC had considerably higher expression of COL6A1 than Ta, T1 low grade patients, and patients that did not progress, highlighting the aggressive nature and higher risk of progression associated with T1HG BC. Moreover, Cox and Kaplan-Meier survival analyses revealed a significant association between elevated expression of COL6A1 and poor progression-free survival of T1HG BC patients (multivariate Cox hazard ratio, 16.812; 95% confidence interval, 3.283-86.095; p=0.001 and p=0.0002 [log-rank test])., Conclusions: These findings suggest that COL6A1 may be a promising biomarker for risk stratification of T1HG BC, offering valuable insight into disease prognosis and guidance of personalized treatment decisions., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association.)

CLPTM1L (Cleft Lip and Palate Transmembrane Protein 1-Like) has previously been implicated in tumorigenesis and drug resistance in cancer. However, the genetic link between CLPTM1L and bladder cancer remains uncertain. In this study, we investigated the genetic association of variable number of tandem repeats (VNTR; minisatellites, MS) regions within CLPTM1L with bladder cancer. We identified four CLPTM1L -MS regions (MS1~MS4) located in intron regions. To evaluate the VNTR polymorphic alleles, we analyzed 441 cancer-free controls and 181 bladder cancer patients. Our analysis revealed a higher frequency of specific repeat sizes within the MS2 region in bladder cancer cases compared to controls. Notably, 25 and 27 repeats were exclusively present in the bladder cancer group. Moreover, rare alleles within the medium-length repeat range (25-29 repeats) were associated with an elevated bladder cancer risk (odds ratio [OR] = 5.78, 95% confidence interval [CI]: 1.49-22.47, p = 0.004). We confirmed that all MS regions followed Mendelian inheritance, and demonstrated that MS2 alleles increased CLPTM1L promoter activity in the UM-UC3 bladder cancer cells through a luciferase assay. Our findings propose the utility of CLPTM1L -MS regions as DNA typing markers, particularly highlighting the potential of middle-length rare alleles within CLPTM1L -MS2 as predictive markers for bladder cancer risk.

Although most human endogenous retroviruses (HERVs) have been silenced and lost their ability to translocate because of accumulated mutations during evolution, they still play important roles in human biology. Several studies have demonstrated that HERVs play pathological roles in numerous human diseases, especially cancer. A few studies have revealed that long non-coding RNAs that are transcribed from HERV sequences affect cancer progression. However, there is no study on microRNAs derived from HERVs related to cancer. In this study, we identified 29 microRNAs (miRNAs) derived from HERV sequences in the human genome. In particular, we discovered that miR-4454, which is HERV-H-derived miRNA, was upregulated in non-muscle-invasive bladder cancer (NMIBC) cells. To figure out the effects of upregulated miR-4454 in NMIBC, genes whose expression was downregulated in NMIBC, as well as tumor suppressor genes, were selected as putative target genes of miR-4454. The dual-luciferase assay was used to determine the negative relationship between miR-4454 and its target genes, DNAJB4 and SASH1 , and they were confirmed to be promising target genes of miR-4454. Taken together, this study suggests that the upregulation of miR-4454 derived from HERV-H in NMIBC reduces the expression of the tumor suppressor genes, DNAJB4 and SASH1 , to promote NMIBC progression.

To evaluate the utility of different risk assessments in non-muscle-invasive bladder cancer (NMIBC) patients, a total of 178 NMIBC patients from Chungbuk National University Hospital (CBNUH) were enrolled, and the predictive value of the molecular signature-based subtype predictor (MSP888) and risk calculators based on clinicopathological factors (EORTC, CUETO and 2021 EAU risk scores) was compared. Of the 178 patients, 49 were newly analyzed by the RNA-sequencing, and their MSP888 subtype was evaluated. The ability of the EORTC, MSP888 and two molecular subtyping systems of bladder cancer (Lund and UROMOL subtypes) to predict progression of 460 NMIBC patients from the UROMOL project was assessed. Cox regression analyses showed that the MSP888 was an independent predictor of NMIBC progression in the CBNUH cohort (p = 0.043). Particularly in patients without an intravesical BCG immunotherapy, MSP888 significantly linked with risk of disease recurrence and progression (both p < 0.05). However, the EORTC, CUETO and 2021 EAU risk scores showed disappointing results with respect to estimating the NMIBC prognosis. In the UROMOL cohort, the MSP888, Lund and UROMOL subtypes demonstrated a similar capacity to predict NMIBC progression (all p < 0.05). Conclusively, the MSP888 is favorable for stratifying patients to facilitate optimal treatment.

Formalin-fixed paraffin-embedded (FFPE) tissue is the most common source of archived material for genomic medicine. However, FFPE tissue is suboptimal for high-throughput analyses, such as RNA sequencing, because the quality of nucleic acids in FFPE tissues is low. We compared RNA-seq with the nCounter system to evaluate use of FFPE tissue for genomic medicine. Twelve fresh frozen bladder cancer samples were analyzed by both RNA sequencing and nCounter, and matched FFPE samples, by nCounter. Gene-expression values obtained by these two platforms were compared by calculating Pearson correlation coefficients for each sample (across the set of matched genes) and for each matched gene (across the set of samples). For each sample, gene-expression levels measured by RNA sequencing highly correlated with those measured by nCounter (all Pearson's R > 0.8, P < 0.0001), as seen by hierarchical clustering. RNA sequencing results for fresh frozen tissues positively correlated with nCounter results for FFPE tissues (R ranged from 0.675 to 0.873, all P < 0.0001). Correlation and hierarchical-clustering analyses of nCounter data from the two specimens demonstrated a strong positive correlation between each group (R ranged from 0.779 to 0.977, all P < 0.0001). Our findings suggest that the nCounter system is useful for assaying archived-FFPE samples and that the gene-expression signatures obtained from FFPE samples represent those from fresh frozen tissues., Competing Interests: Conflict of Interest The author declares that the investigation was conducted without any commercial or financial relationship that could be interpreted as a potential conflict of interest., (Copyright © 2022. Published by Elsevier Inc.)

Platycodin D (PD) is a triterpenoid saponin, a major bioactive constituent of the roots of Platycodon grandiflorum, which is well known for possessing various pharmacological properties. However, the anti-cancer mechanism of PD in bladder cancer cells remains poorly understood. In the current study, we investigated the effect of PD on the growth of human bladder urothelial carcinoma cells. PD treatment significantly reduced the cell survival of bladder cancer cells associated with induction of apoptosis and DNA damage. PD inhibited the expression of inhibitor of apoptosis family members, activated caspases, and induced cleavage of poly (ADP-ribose) polymerase. PD also increased the release of cytochrome c into the cytoplasm by disrupting the mitochondrial membrane potential while upregulating the expression ratio of Bax to Bcl-2. The PD-mediated anti-proliferative effect was significantly inhibited by pre-treatment with a pancaspase inhibitor, but not by an inhibitor of necroptosis. Moreover, PD suppressed the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and the apoptosis-inducing effect of PD was further enhanced by a PI3K inhibitor. In addition, PD increased the accumulation of reactive oxygen species (ROS), whereas N-acetyl cysteine (NAC), an ROS inhibitor, significantly attenuated the growth inhibition and inactivation of the PI3K/Akt/mTOR signaling caused by PD. Furthermore, NAC significantly suppressed apoptosis, DNA damage, and decreased cell viability induced by PD treatment. Collectively, our findings indicated that PD blocked the growth of bladder urothelial carcinoma cells by inducing ROS-mediated inactivation of the PI3K/Akt/mTOR signaling.

The edible Rosa hybrida (RH) petal is utilized in functional foods and cosmetics. Although the biological function of RH petal extract is known, mechanism of action studies involving tumor-associated angiogenesis have not yet been reported. Herein, we investigated the regulatory effect of the ethanol extract of RH petal (EERH) on tumor growth and tumor angiogenesis against bladder cancer. EERH treatment inhibited the bladder carcinoma T24 cell and 5637 cell proliferation because of G 1 -phase cell cycle arrest by inducing p21WAF1 expression and reducing cyclins/CDKs level. EERH regulated signaling pathways differently in both cells. EERH-stimulated suppression of T24 and 5637 cell migration and invasion was associated with the decline in transcription factor-mediated MMP-9 expression. EERH oral administration to xenograft mice reduced tumor growth. Furthermore, no obvious toxicity was observed in acute toxicity test. Decreased CD31 levels in EERH-treated tumor tissues led to examine the angiogenic response. EERH alleviated VEGF-stimulated tube formation and proliferation by downregulating the VEGFR2/eNOS/AKT/ERK1/2 cascade in HUVECs. EERH impeded migration and invasion of VEGF-induced HUVECs, which is attributed to the repressed MMP-2 expression. Suppression of neo-microvessel sprouting, induced by VEGF, was verified by treatment with EERH using the ex vivo aortic ring assay. Finally, kaempferol was identified as the main active compound of EERH. The present study demonstrated that EERH may aid the development of antitumor agents against bladder cancer.

The bladder cancer (BCa) microenvironment comprises heterogeneous tumor cell populations, the surrounding stroma and the extracellular matrix (ECM). Collagen, the scaffold of the tumor microenvironment, regulates ECM remodeling to promote tumor infiltration, angiogenesis, invasion and migration. The present study examined how collagen type VI‑α (COL6A) 1 and 2 function during BCa pathogenesis and progression, with the aim of facilitating the development of precision therapeutics, risk stratification and molecular diagnosis. COL6A1 and COL6A2 mRNA expression in non‑muscle invasive BCa (NMIBC) and MIBC tissue samples was measured using reverse transcription‑quantitative PCR. In addition, the tumor‑suppressive effects of COL6A1 and COL6A2 in human BCa EJ cells (MGH‑U1) were assessed. Compared with normal controls, COL6A1 and COL6A2 mRNA expression was downregulated in both NMIBC and MIBC tissue samples (P<0.05, respectively). COL6A1 and COL6A2 effectively inhibited the proliferation of human BCa EJ cells (MGH‑U1) and induced cell cycle arrest at the G 1 phase. Additionally, COL6A1 and COL6A2 served roles in MAPK and AKT signaling by increasing p38 MAPK phosphorylation and decreasing AKT phosphorylation. Finally, COL6A1 and COL6A2 inhibited wound healing and invasion by suppressing the activity of matrix metalloproteinase (MMP)‑2 and MMP‑9. In conclusion, COL6A1 and COL6A2 may act as classical collagens by forming a physical barrier to inhibit BCa tumor growth and invasion.

Background: ABL1 is primarily known as a leukemia-related oncogene due to translocation, but about 2.2% of ABL1 mutations have been identified in bladder cancer, and high expression in solid cancer has also been detected., Methods: Here, we used the NCBI database, UCSC genome browser gateway and Tandem repeat finder program to investigate the structural characterization of the ABL1 breakpoint region and to identify the variable number of tandem repeats (VNTR). To investigate the relationship between ABL1-MS1 and bladder cancer, a case-controlled study was conducted in 207 controls and 197 bladder cancer patients. We also examined the level of transcription of the reporter gene driven by the ABL1 promoter to determine if the VNTR region affects gene expression., Results: In our study, one VNTR was identified in the breakpoint region, the intron 1 region of ABL1, and was named ABL1-MS1. In the control group, only two common alleles (TR13, TR15) were detected, but an additional two rare alleles (TR14, TR16) were detected in bladder cancer. A statistically significant association was identified between the rare ABL1-MS1 allele and bladder cancer risk: P = 0.013. Investigating the level of transcription of the reporter gene driven by the ABL1 promoter, VNTR showed inhibition of ABL1 expression in non-cancer cells 293 T, but not in bladder cancer cells. In addition, ABL1-MS1 was accurately passed on to offspring according to Mendelian inheritance through meiosis., Conclusions: Therefore, the ABL1-MS1 region can affect ABL1 expression of bladder cancer. This study provides that ABL1-MS1 can be used as a DNA fingerprinting marker. In addition, rare allele detection can predict susceptibility to bladder cancer.

Background: There is an increasing demand for prognostic immune biomarkers of cancer. The prognostic significance of immune markers has been shown for various cancers, but biomarkers of bladder cancer (BCa) have not been fully evaluated. To clarify the role of human leukocyte antigen DR alpha chain (HLA-DRA) in BCa development, we examined expression of HLA-DRA mRNA in tissue samples of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC)., Materials and Methods: Tissues of 96 NMIBC, 43 MIBC and 59 controls comprising noncancerous BCa surrounding tissues were used to examine the expression of HLA-DRA gene by real-time polymerase chain reaction. The expression of up-stream genes regulating HLA-DRA were also measured to explain the role of HLA-DRA in BCa., Results: Patients with high grade NMIBC showed higher expression of HLA-DRA than those with low grade NMIBC (P < 0.05). In addition, NMIBC patients who progressed to MIBC showed high expression of HLA-DRA mRNA. Kaplan-Meier analysis showed that NMIBC patients with low expression of HLA-DRA had better progression-free survival than those with high expression (P = 0.004). Moreover, the expression of genes regulating HLA-DRA varied in NMIBC and MIBC, indicating a different immunoregulation effect of HLA-DRA in both cancers., Conclusions: High expression of HLA-DRA in NMIBC patients has implications for patient stratification strategies, as well as for BCa tumor immunology., Competing Interests: Conflict of interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid and generally found in the bark of birch trees ( Betula sp.). Although several studies have been reported that BA has diverse biological activities, including anti-tumor effects, the underlying anti-cancer mechanism in bladder cancer cells is still lacking. Therefore, this study aims to investigate the anti-proliferative effect of BA in human bladder cancer cell lines T-24, UMUC-3, and 5637, and identify the underlying mechanism. Our results showed that BA induced cell death in bladder cancer cells and that are accompanied by apoptosis, necrosis, and cell cycle arrest. Furthermore, BA decreased the expression of cell cycle regulators, such as cyclin B1, cyclin A, cyclin-dependent kinase (Cdk) 2, cell division cycle (Cdc) 2, and Cdc25c. In addition, BA-induced apoptosis was associated with mitochondrial dysfunction that is caused by loss of mitochondrial membrane potential, which led to the activation of mitochondrial-mediated intrinsic pathway. BA up-regulated the expression of Bcl-2-accociated X protein (Bax) and cleaved poly-ADP ribose polymerase (PARP), and subsequently activated caspase-3, -8, and -9. However, pre-treatment of pan-caspase inhibitor markedly suppressed BA-induced apoptosis. Meanwhile, BA did not affect the levels of intracellular reactive oxygen species (ROS), indicating BA-mediated apoptosis was ROS-independent. Furthermore, we found that BA suppressed the wound healing and invasion ability, and decreased the expression of Snail and Slug in T24 and 5637 cells, and matrix metalloproteinase (MMP)-9 in UMUC-3 cells. Taken together, this is the first study showing that BA suppresses the proliferation of human bladder cancer cells, which is due to induction of apoptosis, necrosis, and cell cycle arrest, and decrease of migration and invasion. Furthermore, BA-induced apoptosis is regulated by caspase-dependent and ROS-independent pathways, and these results provide the underlying anti-proliferative molecular mechanism of BA in human bladder cancer cells.

Non-muscle-invasive bladder cancer (NMIBC) is clinically heterogeneous; thus, many patients fail to respond to treatment and relapse. Here, we identified a molecular signature that is both prognostic and predictive for NMIBC heterogeneity and responses to Bacillus Calmette-Guérin (BCG) therapy. Transcriptomic profiling of 948 NMIBC patients identified a signature-based subtype predictor, MSP888, along with three distinct molecular subtypes: DP.BCG+ (related to progression and response to BCG treatment), REC.BCG+ (related to recurrence and response to BCG treatment), and EP (equivocal prognosis). Patients with the DP.BCG+ subtype showed worse progression-free survival but responded to BCG treatment, whereas those with the REC.BCG+ subtype showed worse recurrence-free survival but responded to BCG treatment. Multivariate analyses revealed that MSP888 showed independent clinical utility for predicting NMIBC prognosis (each p = 0.001 for progression and recurrence, respectively). Comparative analysis of this classifier and previously established molecular subtypes (i.e., Lund taxonomy and UROMOL class) revealed that a great proportion of patients were similar between subtypes; however, the MSP888 predictor better differentiated biological activity or responsiveness to BCG treatment. Our data increase our understanding of the mechanisms underlying the poor prognosis of NMIBC and the effectiveness of BCG therapy, which should improve clinical practice and complement other diagnostic tools.

DNA repair defects are important factors in cancer development. High DNA repair activity can affect cancer progression and chemoresistance. DNA double-strand breaks in cancer cells caused by anticancer agents can be restored by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Our previous study has identified E2F1 as a key gene in bladder cancer progression. In this study, DNA repair genes related to E2F1 were analyzed, and RAD54L involved in HRR was identified. In gene expression analysis of bladder cancer patients, the survival of patients with high RAD54L expression was shorter with cancer progression than in patients with low RAD54L expression. This study also revealed that E2F1 directly binds to the promoter region of RAD54L and regulates the transcription of RAD54L related to the HRR pathway. This study also confirmed that DNA breaks are repaired by RAD54L induced by E2F1 in bladder cancer cells treated with MMC. In summary, RAD54L was identified as a new target directly regulated by E2F1. Our results suggest that, E2F1 and RAD54L could be used as diagnostic markers for bladder cancer progression and represent potential therapeutic targets.

Extra-adrenal pheochromocytoma is called paraganglioma. Paraganglioma near the ureterovesical junction can be confused with urothelial carcinoma in a ureterocele. Urinary metanephrine can be an indicator for bladder paraganglioma. Metaiodobenzylguanidine scintigraphy is an excellent method not only for distinguishing bladder paraganglioma from other submucosal mesenchymal tumors but also for detecting multifocal lesions. In the present case, we did not perform a preoperative metaiodobenzylguanidine scan because the patient was asymptomatic and urinary metanephrine was negative. Partial cystectomy with ureteroneocystostomy was performed for curative treatment because the tumor was very close to the ureteral orifice., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Various physiological benefits have been linked to Hizikia fusiforme (HF), an edible brown seaweed. Here, fucose-containing sulfated polysaccharides were extracted from celluclast-processed HF (SPHF) and their antitumor efficacy against bladder cancer was evaluated in vitro and in vivo. SPHF possesses high sulfated polysaccharide and fucose contents and free radical scavenging activities compared to those of celluclast-processed HF extracts (CHF). SPHF inhibited bladder cancer EJ cell proliferation via G1-phase cell cycle arrest. This was due to the induction of p21WAF1 expression associated with the downregulation of CDKs and cyclins. Moreover, JNK phosphorylation was identified as an SPHF-mediated signaling molecule. SPHF treatment also hindered the migration and invasion of EJ cells by inhibiting MMP-9 expression, which was attributed to the repression of transcriptional binding to NF-κB, AP-1, and Sp-1 in the MMP-9 promoter region. In an animal study, SPHF treatment suppressed EJ tumor growth in xenograft mice similarly to cisplatin. Furthermore, no toxicity signs were found after weight loss assessment, biochemical tests, and organ tissue immunostaining during oral administration of 20-200 mg/kg SPHF for 20 days. Therefore, our study demonstrates the antitumor efficacy of SPHF in vitro and in vivo, thus highlighting its potential for bladder cancer treatment development.

Carnosine, a naturally occurring dipeptide, was recently reported to exhibit anticancer activity; however, the molecular mechanisms and regulators underlying its activity against tumor-associated angiogenesis remain unidentified. In this study, we evaluated the in vitro and in vivo antitumor effects of carnosine in EJ bladder cancer cells and EJ-xenografted BALB/c nude mice, respectively. In addition, in vitro capillary tube formation of HUVECs, ex vivo aortic ring and in vivo Matrigel plug assays were employed to examine the antiangiogenic potential of carnosine. Carnosine significantly inhibited EJ cell proliferation. Flow cytometric and immunoblot analyses indicated that carnosine modulated regulators of the G1 cell cycle phase, including cyclin D1, CDK4 and p21WAF1. The mitogen-activated protein kinases, ERK and p38, but not JNK or AKT, responded to carnosine. Carnosine inhibited the migratory and invasive potential of EJ cells by inhibiting MMP-9 activity, which was associated with suppression of binding activity of NF-κB, SP-1 and AP-1. In xenograft tumors, carnosine exhibited antitumor activity equivalent to cisplatin, but no weight loss occurred in carnosine-treated mice. In HUVECs, carnosine inhibited VEGF-mediated proliferation, colony tube formation, migration and invasion. The antiangiogenic activity of carnosine was partially due to the suppression of VEGFR-2-mediated ERK/AKT/eNOS signaling and MMP-2. Furthermore, using aortic ring and Matrigel plug assays, we confirmed the antiangiogenic activity of carnosine. Given that targeting tumor-associated angiogenesis is a proven effective therapeutic strategy, our results may provide valuable information for the development of preventive or therapeutic agents for bladder cancer patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: A natural compound Jaspine B and its derivative possess potential anti-cancer activities; However, little is known about the underlying mechanism. Here, the role of a new autophagy inducer Jaspine B derivative C-2 in suppressing bladder cancer cells was researched in vitro and in vivo., Methods: The underlying mechanisms and anticancer effect of C-2 in bladder cancer cells were investigated by MTT, western blotting, immunoprecipitation and immunofluorescence assays. The key signaling components were investigated by using pharmacological inhibitors or specific siRNAs. In vivo, we designed a C-2 and SP600125 combination experiment to verify the effectiveness of compound., Results: C-2 exhibits cytotoxic effect on bladder cancer cells, and JNK activated by C-2 triggers autophagy and up-regulates SQSTM1/p62 proteins, contributing to activation of Nrf2 pathway. Utilization of JNK inhibitor SP600125 or knockdown of JNK by siRNA potentiate the cytotoxicity of C-2 through down-regulation of p62 and LC3II proteins and up-regulation of active-Caspase3 proteins, enhance the cell death effect, facilitating the switch from autophagy to apoptosis. In vivo study, C-2 suppresses tumor growth in a xenograft mouse model of EJ cells without observed toxicity. Combined treatment with SP600125 further enhances tumor inhibition of C-2 associated with enhanced activation of caspase3 and reduction of autophagy., Conclusions: It reveals a series of molecular mechanisms about SP600125 potentiate the cytotoxicity and tumor inhibition of C-2 in bladder cancer cells through promoting C-2-induced apoptosis, expecting it provides research basis and theoretical support for new drugs development.

Background: Numerous studies have focused on solvent extracts from locust trees (Gleditsia spp.), which contain diverse bioactive components including saponins, flavonoids, and alkaloids. However, because of the undefined nature of such phytochemicals, their clinical application as chemotherapeutic agents has often been limited., Purpose: This study aimed to evaluate the anti-oncogenic activity of triacanthine, an alkaloid obtained from Gleditsia triacanthos L., Study Design: The anti-oncogenicity of triacanthine in vitro was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell-counting kit-8 assay (CCK-8 assay), flow cytometry, imunoblot, migration and invasion assays, zymography, and electrophoretic mobility shift assay in the human bladder carcinoma cell line EJ. The in vivo efficacy of triacanthine was evaluated via oral administration to EJ-xenografted BALB/c nude mice. To identify the side effects of triacanthine, cisplatin was also administered and an acute toxicity test was performed., Results: Triacanthine significantly inhibited EJ cell proliferation (IC 50 600 µM). Flow cytometry analysis revealed that cells were arrested in the G1 phase, and apoptotic cells accumulated in sub-G1 phase in a dose-dependent manner. Triacanthine inhibited the G1-S transition by deterring complex formation between cyclin-dependent kinases and cyclins, thereby up-regulating cell cycle inhibitors p21WAF1 and p27KIP1. In addition, triacanthine induced a caspase-dependent extrinsic pathway of apoptosis and autophagy. Early responsive kinases, extracellular signal-regulated kinase (ERK) and Janus kinase (JNK) were up-regulated by triacanthine. Triacanthine-mediated inhibition of the migratory and invasive potential of EJ cells was attributed to reduction of matrix metalloproteinase (MMP)-9 due to suppression of binding activities of the transcription factors activator protein (AP)-1, specificity protein (Sp)-1, and nuclear factor (NF)-κB. In an in vivo study, triacanthine significantly limited growth of xenografted tumors. Interestingly, while cisplatin resulted in significant weight loss after a 5-mg/kg dose, triacanthine did not cause weight loss, behavioral abnormalities, altered biochemical parameters, or tissue staining. A single oral dose acute-toxicity test (triacanthine 2,000 mg/kg) produced no adverse cytotoxic effects via blood biochemical tests and tissue-organ staining., Conclusion: To our knowledge, this is the first systematic evaluation of the anti-oncogenic activity of triacanthine. Therefore, we believe that our findings may guide the development of novel chemotherapeutic agents for bladder cancers., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Licochalcone A (LCA) is a chalcone that is predominantly found in the root of Glycyrrhiza species, which is widely used as an herbal medicine. Although previous studies have reported that LCA has a wide range of pharmacological effects, evidence for the underlying molecular mechanism of its anti-cancer efficacy is still lacking. In this study, we investigated the anti-proliferative effect of LCA on human bladder cancer cells, and found that LCA induced cell cycle arrest at G2/M phase and apoptotic cell death. Our data showed that LCA inhibited the expression of cyclin A, cyclin B1, and Wee1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdc2 and Cdk2. LCA activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, LCA increased the Bax/Bcl-2 ratio, and reduced the integrity of mitochondria, which contributed to the discharge of cytochrome c from the mitochondria to the cytoplasm. Moreover, LCA enhanced the intracellular levels of reactive oxygen species (ROS); however, the interruption of ROS generation using ROS scavenger led to escape from LCA-mediated G2/M arrest and apoptosis. Collectively, the present data indicate that LCA can inhibit the proliferation of human bladder cancer cells by inducing ROS-dependent G2/M phase arrest and apoptosis.

Background: Disease monitoring in non-muscle-invasive bladder cancer (NMIBC) patients is crucial for early identification of disease recurrence and progression. High IQGAP3/BMP4 and IQGAP3/FAM107A ratios in urinary cell-free DNA (ucfDNA) are a diagnostic biomarker for bladder cancer. We aimed to investigate whether the levels of these biomarkers in ucfDNA can be used to monitor disease recurrence or progression in patients with NMIBC., Patients and Methods: A total of 103 patients with NMIBC (pTa-pT1) were enrolled. The IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were measured by real-time PCR, and the results were compared with clinical outcome by Kaplan-Meier curves and Cox regression analyses., Results: Overall, 55 patients (53.4%) experienced recurrence and 29 (28.2%) experienced disease progression during a median follow-up of 42.7 months (range, 6.1-172.2 months). Kaplan-Meier analysis revealed that NMIBC patients with a high IQGAP3/BMP4 ratio had worse recurrence-free survival and progression-free survival (PFS) (P = .001 and < .001, respectively), and those with a high IQGAP3/FAM107A ratio had worse PFS (P = .006). Multivariate Cox regression analysis revealed that the IQGAP3/BMP4 ratio was independently associated with recurrence-free survival (hazard ratio, 2.462; P = .003) and PFS (hazard ratio = 3.871; P = .004), whereas the IQGAP3/FAM107A ratio was not an independent factor for PFS (P = .079)., Conclusion: The IQGAP3/BMP4 ratio in ucfDNA might be a valuable novel biomarker for predicting disease recurrence and progression in patients with NMIBC., (Copyright © 2019. Published by Elsevier Inc.)

The most common symptom of bladder cancer (BC) is hematuria. However, not all patients with hematuria are diagnosed with BC. Here, we explored a novel method to discriminate BC from hematuria under nonmalignant conditions by measuring differences in urinary cell-free microRNA (miRNA) expression between patients with BC and those with hematuria. A multicenter study was performed using 543 urine samples obtained from the National Biobank of Korea, including 326 BC, 174 hematuria and 43 pyuria without cancer. The urinary miR-6124 to miR-4511 ratio was considerably higher in BC than in hematuria or pyuria, and enabled the discrimination of BC from patients with hematuria at a sensitivity of >90% (p < 0.001). Conclusively, the proposed noninvasive diagnostic tool based on the expression ratio of urinary cell-free miR-6124 to miR-4511 can reduce unnecessary cystoscopies in patients with hematuria undergoing evaluation for BC, with a minimal loss in sensitivity for detecting cancer., (© 2018 UICC.)

Background: Urinary cell-free DNA (ucfDNA) has great potential as a "liquid biopsy" for use in diagnosis of urological cancers. In this study, we compared ucfDNA gene expression levels between patients with bladder cancer (BC) and those with hematuria, and determined whether they could be used as a noninvasive urine-based marker., Methods: The study cohort of 355 patients included a screening group (40 BC and 41 hematuria controls) and a validation cohort (149 BC and 125 hematuria controls). Expression levels ratios of 1 up-regulated gene (IQGAP3) to those of 7 down-regulated genes were examined in ucfDNA in the screening group to identify ratios that differed significantly between BC and hematuria patients. IQGAP3/BMP4 and IQGAP3/FAM107A ratios were selected and combined to develop a discriminant score (DS) index, which was tested in the validation cohort. Receiver operating characteristic curves and areas under the curve were calculated to evaluate the performance of the DS index., Results: IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were both significantly higher in BC patients than in hematuria patients (both P < 0.001). The DS index had an area under the curve of 0.862, a sensitivity of 71.0%, a specificity of 88.6%, a positive predictive value of 90.3%, and a negative predictive value of 67.2%., Conclusions: Both IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were significantly higher in patients with BC than in those with hematuria. The DS index exhibits good diagnostic performance as a noninvasive biomarker., (Copyright © 2018. Published by Elsevier Inc.)

Cordycepin, a derivative of nucleoside adenosine, is one of the active ingredients extracted from the fungi of genus Cordyceps, which have been used for traditional herbal remedies. In this study, we examined the effect of cordycepin on the proliferation and apoptosis of human bladder cancer T24 cells and its mechanism of action. Cordycepin treatment significantly reduced the cell survival rate of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptosis. Cordycepin activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, cordycepin increased the Bax/Bcl-2 ratio and truncation of Bid, and destroyed the integrity of mitochondria, which contributed to the cytosolic release of cytochrome c. Moreover, cordycepin effectively inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of cordycepin. Cordycepin further enhanced the intracellular levels of reactive oxygen species (ROS), while the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished cordycepin-induced mitochondrial dysfunction and growth inhibition, and also blocked the inactivation of PI3K/Akt signaling pathway. Furthermore, the presence of NAC significantly attenuated the enhanced apoptotic cell death and reduction of cell viability by treatment with cordycepin and LY294002. Collectively, the data indicate that cordycepin induces apoptosis through the activation of extrinsic and intrinsic apoptosis pathways and the ROS-dependent inactivation of PI3K/Akt signaling in human bladder cancer T24 cells.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can preferentially initiate apoptosis in malignant cells with minimal toxicity to normal cells. Unfortunately, many human cancer cells are refractory to TRAIL-induced apoptosis through many unknown mechanisms. Here, we report that TRAIL resistance can be reversed in human bladder cancer cell lines by treatment with sulforaphane (SFN), a well-known chemopreventive isothiocyanate in various cruciferous vegetables. Combined treatment with SFN and TRAIL (SFN/TRAIL) significantly induced apoptosis concomitant with activation of caspases, loss of mitochondrial membrane potential (MMP), Bid truncation, and induction of death receptor 5. Transient knockdown of Bid prevented collapse of MMP induced by SFN/TRAIL, consequently reducing apoptotic effects. Furthermore, SFN increased both the generation of reactive oxygen species (ROS) and the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is an anti-oxidant enzyme. Interestingly, TRAIL effectively suppressed SFN-mediated nuclear translocation of Nrf2, and the period of ROS generation was more extended compared to that of treatment with SFN alone. In addition, silencing of Nrf2 increased apoptosis in cells treated with SFN/TRAIL; however, blockade of ROS generation inhibited apoptotic activity. These data suggest that SFN-induced ROS generation promotes TRAIL sensitivity and SFN can be used for the management of TRAIL-resistant cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Pathologic T1 high-grade (pT1HG) bladder cancer (BC) is characterized by a high progression rate and constitutes an important clinical challenge; however, there is no consensus on the prediction of progression in pT1HG BC. The purpose of this study was to validate previously published molecular progression risk score (MoPRS) for predicting muscle-invasive disease in pT1HG BC., Materials and Methods: The expression of an 8-gene progression-related classifier identified from microarray data was analyzed by real-time PCR, and the MoPRS was calculated in 121 newly recruited patients with pT1HG BC. Progression was defined as muscle invasion or metastasis., Results: Overall, the disease of 28 patients (23.1%) progressed to muscle-invasive BC during the median follow-up of 63.7 (interquartile range, 17.6-96.4) months. The MoPRS was significantly higher in 1973 World Health Organization grade 3 than grade 2 tumors (P = .004). Early development of invasive BC was more prevalent in the highest quartile MoPRS group than in the lowest to 75th percentile MoPRS groups according to Kaplan-Meier analysis. Multivariate Cox regression analysis revealed that the MoPRS was an independent predictor of invasive BC, either as a continuous variable (hazard ratio, 1.624; 95% confidence interval, 1.266-2.082; P < .001) or as a categorical variable (hazard ratio, 3.089; 95% confidence interval, 1.335-7.150; P = .008)., Conclusion: The MoPRS was an independent prognostic factor for identifying patients at high risk of invasive BC in patients with pT1HG BC. This scale may help identify patients who could benefit from more aggressive therapeutic intervention such as early cystectomy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer (BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165 BC patients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20 mg/kg (p<0.05), 200 mg/kg, and 1000 mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36 genes and 37 gene ontologies were mapped to gene networks. Protein kinase A (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity in vivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

Despite the clinical significance of tumorigenesis, little is known about the cellular signaling networks of microRNAs (miRs). Here we report a new finding that mir‑106a regulates the proliferation, migration, and invasion of bladder cancer cells. Basal expression levels of mir‑106a were significantly lower in bladder cancer cells than in normal urothelial cells. Overexpression of mir‑106a suppressed the proliferation of bladder cancer cell line EJ. Transient transfection of mir‑106a into EJ cells led to downregulation of ERK phosphorylation and upregulation of p38 and JNK phosphorylation over their levels in the control. Flow cytometry analysis revealed that mir‑106a-transfected cells accumulated in the G1-phase of the cell cycle, and cyclin D1 and CDK6 were significantly downregulated. This G1-phase cell cycle arrest was due in part to the upregulation of p21CIP1/WAF1. In addition, mir‑106a overexpression blocked the wound-healing migration and invasion of EJ cells. Furthermore, mir‑106a transfection resulted in decreased expression of MMP-2 and diminished binding activity of transcription factor Ets-1 in EJ cells. Collectively, we report the novel mir‑106a-mediated molecular signaling networks that regulate the proliferation, migration, and invasion of bladder cancer cells, suggesting that mir‑106a may be a therapeutic target for treating advanced bladder tumors.

Background: There is growing interest in circulating nucleic acids as cancer detection biomarkers. Therefore, the aim of the present study was to identify a key urinary cell-free RNA marker that may assist in the diagnosis of BC., Results: Five cell-free RNAs were selected as candidate cell-free RNAs from tissue microarray data. An area under the curve (AUC) cut-off value of 0.7 in receiver operating characteristic (ROC) curve analysis identified four urinary cell-free RNAs for further analysis (CDC20, ESM1, UBE2C, and CA9; AUC = 0.716, 0.704, 0.721, and 0.702, respectively). Binary logistic regression analysis revealed that high expression of UBE2C was significantly associated with BC (OR, 1.754; CI, 1.147-2.682; p = 0.010). Analysis of UBE2C expression in urine samples from BC patients and hematuria controls yielded an AUC of 0.839, with a sensitivity of 82.5% and a specificity of 76.2%. UBE2C levels was significantly increased in G2 and G3 tumors compared to normal controls (p <0.001, respectively)., Materials and Methods: Urine samples from 212 BC patients and 106 normal controls (64 healthy individuals and 42 with hematuria) were examined. The candidate cell-free RNAs identified from tissue microarrays derived from BC and normal control tissues was then measured in the urine samples., Conclusions: The levels of urinary UBE2C cell-free RNA were significantly higher in BC samples than in normal and hematuria control samples. The higher levels of urinary UBE2C cell-free RNA in BC might reflect high expression in BC tissues. Therefore, urinary UBE2C cell-free RNA may be a valuable diagnostic marker for BC., Competing Interests: None.

Baicalein is a flavonoid derived originally from the root of Scutellaria baicalensis Georgi, which has been used in Oriental medicines for treating various diseases. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of baicalein on human bladder cancer and its mechanisms of action have not been extensively studied. This study investigated the proapoptotic effects of baicalein in human bladder cancer 5637 cells. For this study, cell viability and apoptosis were evaluated using the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue dye exclusion assay 4,6-diamidino-2-phenylindole staining, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether 5637 cell death occurred by apoptosis. Treatment with baicalein resulted in a concentration-dependent growth inhibition coupled with apoptosis induction, as indicated by the results of nuclei morphology examination and flow cytometry analyses. The induction of the apoptotic cell death of 5637 cells by baicalein exhibited a correlation with the downregulation of members of the inhibitor of apoptosis protein (IAP) family, including cIAP-1 and cIAP-2, and the activation of caspase-9 and -3 accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. The study also showed that baicalein decreases the expression of the proapoptotic protein Bax, increases antiapoptotic Bcl-2 expression, and noticeably aggravates the loss of MMP. Concomitantly, the data showed that baicalein increases the levels of death receptors and their associated ligands and enhances the activation of caspase-8 and truncation of Bid. However, the pan-caspase inhibitor can reverse baicalein-induced apoptosis, demonstrating that it is a caspase-dependent pathway. Moreover, it was found that baicalein can induce the production of reactive oxygen species (ROS) and that pretreatment with the antioxidant N-acetyl-L-cysteine significantly attenuates the baicalein effects on the loss of MMP and activation of caspase. In addition, the blocking of ROS generation decreases the apoptotic activity and antiproliferative effect of baicalein, indicating that baicalein induces apoptosis of 5637 cells through the ROS-dependent activation of caspases.

Purpose: Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome., Materials and Methods: A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed., Results: Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression., Conclusion: Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined.

Purpose: The DHCR24 gene that encodes 3b-hydroxysterol Δ24-reductase, an oxidoreductase involved in cholesterol biosynthesis, has been identified as a progression-related gene based on the quantitative real-time PCR (qPCR) gene signature. Here, the functional role of DHCR24 and its clinical relevance in non-muscle-invasive urothelial carcinoma (NMIUC) were investigated., Methods: Primary NMIUC tissue specimens (n = 162) were analyzed by qPCR. Immunohistochemical staining was also performed on 63 subsets of NMIUC tissues. The present study was also undertaken in order to verify the effect of DHCR24 on human urothelial carcinoma cells., Results: The mRNA expression levels of DHCR24 were significantly higher for patients in with higher grades of tumors than for those with lower grades of tumors (P = 0.003). Kaplan-Meier estimates revealed significant differences in the time to progression between low- and high-mRNA expression groups (log-rank test, P < 0.001). Multivariate Cox regression analysis revealed that the level of DHCR24 expression is an independent predictor of progression (hazard ratio, 5.464; 95 % confidence interval, 1.746-17.099; P = 0.004). The results of immunohistochemical staining were generally concordant with mRNA expression levels. Enforced expression of DHCR24 caused proliferation, adhesion, and migration, while DHCR24 loss resulted in slower proliferation and a reduction in cell viabilities compared with control cells., Conclusions: DHCR24 was found to be closely associated with progression among patients with NMIUC and showed aggressive properties in human UC cells.

Sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, has received a great deal of attention because of its ability to inhibit cell proliferation and induce apoptosis in cancer cells. In this study, we investigated the anticancer activity of sulforaphane in the T24 human bladder cancer line, and explored its molecular mechanism of action. Our results showed that treatment with sulforaphane inhibited cell viability and induced apoptosis in T24 cells in a concentration-dependent manner. Sulforaphane-induced apoptosis was associated with mitochondria dysfunction, cytochrome c release and Bcl-2/Bax dysregulation. Furthermore, the increased activity of caspase-9 and -3, but not caspase-8, was accompanied by the cleavage of poly ADP-ribose polymerase, indicating the involvement of the mitochondria-mediated intrinsic apoptotic pathway. Concomitant with these changes, sulforaphane triggered reactive oxygen species (ROS) generation, which, along with the blockage of sulforaphane-induced loss of mitochondrial membrane potential and apoptosis, was strongly attenuated by the ROS scavenger N-acetyl-L-cysteine. Furthermore, sulforaphane was observed to activate endoplasmic reticulum (ER) stress and the nuclear factor-E2-related factor-2 (Nrf2) signaling pathway, as demonstrated by the upregulation of ER stress‑related proteins, including glucose-regulated protein 78 and C/EBP-homologous protein, and the accumulation of phosphorylated Nrf2 proteins in the nucleus and induction of heme oxygenase-1 expression, respectively. Taken together, these results demonstrate that sulforaphane has antitumor effects against bladder cancer cells through an ROS-mediated intrinsic apoptotic pathway, and suggest that ER stress and Nrf2 may represent strategic targets for sulforaphane-induced apoptosis.

Tianeptine sodium salt (TSS) is a selective facilitator of serotonin, but there are no reports regarding anti-invasive effects of TSS. Therefore, we investigated the effect of TSS on the expression of matrix metalloproteinase-9 (MMP-9) and invasion in three different human carcinoma cell lines. Our findings showed that MMP-9 activity was significantly increased in response to tumor necrosis factor-α (TNF-α), and that TSS reduced TNF-α-induced MMP-9 activity in a dose-dependent manner. TSS also downregulated both MMP-9 expression and TNF-α-induced MMP-9 promoter activity. Using a matrigel invasion assay, we showed that TSS significantly attenuated invasive rates in TNF-α-stimulated LNCaP prostate carcinoma cells. Furthermore, TSS suppressed TNF-α-induced NF-κB activity, which is a potential transcriptional factor for regulating many invasive genes, including MMP-9, by suppressing IκB degradation and nuclear translocation of NF-κB subunits in LNCaP prostate carcinoma cells. TSS also downregulated TNF-α-induced phosphorylation of phosphatidyl-inositol 3 kinase (PI3K) and Akt, and a selective PI3K/Akt inhibitor, LY294002, diminished TNF-α-induced NF-κB activation followed by levels of MMP-9, suggesting that TSS also reduces MMP-9 expression by inhibiting the PI3K/Akt-mediated NF-κB pathway. These results indicate that TSS is a potential anti-invasive agent by suppression of TNF-α-induced MMP-9 expression via inhibition of PI3K/Akt-mediated NF-κB activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although fucoidan has been shown to exert anticancer activity against several types of cancer cell lines, no reports have explored fucoidan-affected cell growth in human urinary bladder cancer cells. In this study, we investigated the anti-proliferative effects of fucoidan in human bladder cancer T24 cells. Our results indicated that fucoidan decreased the viability of T24 cells through the induction of G1 arrest and apoptosis. Fucoidan-induced G1 arrest is associated with the enhanced expression of the Cdk inhibitor p21WAF1/CIP1 and dephosphorylation of the pRB along with enhanced binding of p21 to Cdk4/6 as well as pRB to the transcription factor E2Fs. Further investigations showed the loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol, proving mitochondrial dysfunction upon fucoidan treatment with a corresponding increase in the Bax/Bcl-2 expression ratio. Fucoidan-triggered apoptosis was also accompanied by the up-regulation of Fas and truncated Bid as well as the sequential activation of caspase-8. Furthermore, a significant increased activation of caspase-9/-3 was detected in response to fucoidan treatment with the decreased expression of IAPs and degradation of PARP, whereas a pan-caspase inhibitor significantly suppressed apoptosis and rescued the cell viability reduction. In conclusion, these observations suggest that fucoidan attenuates G1-S phase cell cycle progression and serves as an important mediator of crosstalk between caspase-dependent intrinsic and extrinsic apoptotic pathways in T24 cells.

Introduction: We evaluated the predictive value of glutathione S transferase mu (GSTM1) and theta (GSTT1) polymorphisms in early response to bacillus Calmette-Guérin (BCG) induction therapy in patients with primary non-muscle invasive bladder cancer., Methods: GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction using blood genomic DNA from 135 patients with primary non-muscle invasive bladder cancer who were being treated with a single induction course of BCG. BCG nonresponsiveness (early BCG failure) was defined as a tumor recurrence or progression within 12 months after BCG induction therapy. The predictive value of GST polymorphisms was evaluated by Kaplan-Meier analysis and multivariate logistic regression models., Results: Patients carrying a GSTT1-positive genotype demonstrated a higher likelihood of early BCG failure regardless of cigarette smoking. After stratification based on the tumor stage and grade, the high-risk group (T1G3) with a GSTT1-positive genotype showed a 14-fold higher risk of early BCG failure compared with those with a GSTT1-null genotype. In a combined analysis of 2 genes, the GSTT1-positive/GSTM1-null genotype had a higher risk of BCG nonresponsiveness compared with the GSTT1-null/GSTM1-null genotype (odds ratio = 4.17, 95% CI: 1.54-11.26). By multivariate logistic regression analysis, the GSTT1-positive genotype was an independent predictor of early BCG failure (odds ratio = 3.67, 95% CI: 1.61-8.38). Kaplan-Meier estimates revealed a significant difference in disease-free survival depending on the GSTT1 genotype (log rank test, P = 0.038)., Conclusions: The results of this study suggest that the GSTT1-positive genotype is an independent predictor of early BCG failure. These results can help determine whether patients would benefit from adjuvant BCG treatment or may require more aggressive alternative therapies., (© 2013 Published by Elsevier Inc.)

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.

Human urinary bladder cancer is the fifth most common cancer, with a worldwide estimate of about two million patients. Recurrence after complete transurethral prostatic resection is the most important problem in therapy. Combination therapy is a new approach in the treatment of cancers that do not respond to current therapies. These therapies have many advantages over conventional therapies, such as fewer side-effects and greater efficiency. Research efforts using natural compounds for the elimination or growth suppression of the cancer arise from studies on methylsulfonylmethane (MSM). MSM is a natural sulfur compound with no side-effects. AG490 is a tyrosine kinase inhibitor that has been extensively used for inhibiting Jak2 in vitro and in vivo. In our study, the combinatorial effect of these two agents on human bladder cancer cell lines and xenografts was analyzed. We observed that the combination of AG490 and MSM inhibited cancer cell viability and cell migration in vitro. This combination inhibited VEGF mRNA expression in bladder cancer cell lines. In vivo experiments showed that oral administration of AG490 and MSM combination significantly inhibited the growth of tumor xenografts in mice. Our study clearly demonstrates that the predominant effect of this combination is the reduction of signaling molecules including STAT3, STAT5b, IGF-1R, VEGF and VEGF-R2 which are involved in the growth, progression and metastasis of human bladder cancer. The anti-metastatic ability of this drug combination is confirmed using metastatic animal models. Therefore, this combination could have the effect of genesistasis and powerful anticancer effects against bladder cancer.

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Diallyl trisulfide (DATS), an organosulfur compound in garlic, possesses pronounced anti-cancer potential. However, the anti-invasive mechanism of this compound in human bladder carcinoma is not fully understood. In this study, we evaluated the anti-invasive effects of DATS on a human bladder carcinoma (5637) cell line and investigated the underlying mechanism. The results indicated that DATS suppressed migration and invasion of 5637 cells by reducing the activities and expression of matrix metalloproteinase (MMP)-2 and MMP-9 at both the protein and mRNA levels. DATS treatment up-regulated expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in 5637 cells. The inhibitory effects of DATS on invasiveness were associated with an increase in transepithelial electrical resistance and repression of the levels of claudin family members. Although further studies are needed, our data demonstrate that DATS exhibits anti-invasive effects in 5637 cells by down-regulating the activity of tight junctions and MMPs. DATS may have future utility in clinical applications for treating bladder cancer.

Inflammatory cytokines may be a critical component of epithelial cancer progression. We examined the role of interleukin (IL)-5 in the migration of bladder cancer cells. The expression of IL-5 and its receptor IL-5Rα was enhanced in patients with muscle invasive bladder cancers (MIBC), and then it was detected in bladder cancer cell lines 5637 and T-24. IL-5 increased migration and MMP-9 expression via activation of transcription factors NF-κB and AP-1, and induced activation of ERK1/2 and Jak-Stat signaling in both cells. Treatment with ERK1/2 inhibitor U0126 significantly inhibited induction of migration, MMP-9 expression, and activation of NF-κB and AP-1 in IL-5-treated cells. However, none of the Jak inhibitors affected the IL-5-induced migration of bladder cancer cells. Moreover, gene knockdown for IL-5Rα, using siRNA transfection, suppressed migration, ERK1/2 activation, MMP-9 expression, as well as the binding activation of NF-κB and AP-1 in IL-5-treated bladder cancer cells. Similar results were observed in βc siRNA (si-βc) transfected cells. Unexpectedly, IL-5 treatment resulted in significant induction of p21WAF1 in both cell lines. The p21WAF1-specific small interfering RNA inhibited IL-5-induced cell migration, ERK activity, MMP-9 expression, and activation of NF-κB and AP-1 in bladder cancer cells. The effects of IL-5-induced cell responses were confirmed by transfection of IL-5 gene, which demonstrated that p21WAF1 participates in the induction of cell migration, leading to an increase in ERK1/2-mediated MMP-9 expression through activation of NF-κB and AP-1 in IL-5-treated bladder cancer cells. These unexpected results provide a theoretical basis for the therapeutic targeting of IL-5 in bladder cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: We investigated whether genetic polymorphisms in the glutathione S transferase mu (GSTM1) and theta (GSTT1) genes modulated risk, disease progression and survival in primary muscle invasive bladder cancer (MIBC)., Methods: GSTM1 and GSTT1 polymorphisms were analysed by multiplex polymerase chain reaction (PCR) using blood genomic DNA in 110 MIBC patients and 220 gender- and age-matched healthy controls. The influence of the genetic polymorphisms on patient survival was evaluated by Kaplan-Meier survival curves and Cox Proportional Hazard models. We also evaluated whether cigarette smoking and treatment modality modified the association between genotype and prognosis., Results: GSTM1-null individuals exhibited increased risk for MIBC and an association with cigarette smoking. GSTT1-null subjects showed significant disease progression and cancer-specific death. In the combined analysis, GSTT1-null genotype was an independent risk factor for disease progression and cancer specific death regardless of GSTM1 genotype. Significant differences in progression-free survival (PFS) and cancer-specific survival (CSS) were seen based on GSTT1 genotype. The survival impact of the GSTT1 genotype was only valid for smokers. The GSTT1-null genotype was an independent prognostic factor for shorter PFS in patients who received chemotherapy and those who did not undergo radical cystectomy. By multivariate Cox regression analysis, GSTT1-null genotype was a predictive factor for disease progression and cancer specific survival regardless of treatment modality., Conclusions: The GSTM1-null genotype plays an important role in genetic susceptibility to MIBC and the GSTT1-null genotype is associated with disease progression and shorter survival in MIBC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although the effects of sanguinarine, a benzophenanthridine alkaloid, on the inhibition of some kinds of cancer cell growth have been established, the underlying mechanisms are not completely understood. This study investigated possible mechanisms by which sanguinarine exerts its anticancer action in cultured human bladder cancer cell lines (T24, EJ, and 5637). Sanguinarine treatment resulted in concentration-response growth inhibition of the bladder cancer cells by inducing apoptosis. Sanguinarine-induced apoptosis was correlated with the up-regulation of Bax, the down-regulation of Bid and XIAP, the activation of caspases (-3, -8, and -9), and the generation of increased reactive oxygen species (ROS). The ROS scavenger N-acetyl cysteine (NAC) completely reversed the sanguinarine-triggered apoptotic events. In addition, sanguinarine effectively increased the activation of the c-Jun N-terminal kinase (JNK) and the expression of the early growth response gene-1 (Egr-1), which was recovered by pretreatment with NAC. Furthermore, knockdown of Egr-1 expression by small interfering RNA attenuated sanguinarine-induced apoptosis, but not the JNK inhibitor, indicating that the interception of ROS generation blocked the sanguinarine-induced apoptotic effects via deregulation of the expression of Egr-1 proteins. Taken together, the data provide evidence that sanguinarine is a potent anticancer agent, which inhibits the growth of bladder cancer cells and induces their apoptosis through the generation of free radicals.

The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21(WAF1) was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21(WAF1) function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21(WAF1) is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.

Bufalin, a cardiotonic steroid extracted from toad venom, has generally been known to possess a range of biological activities; however, only a few studies have reported the anti-metastatic activity of bufalin. In the present study, we investigated the inhibitory effects of bufalin on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer cell line, T24. Within the concentration range that was not cytotoxic, bufalin markedly inhibited the cell motility and invasiveness of T24 cells. The inhibitory effects of bufalin on cell invasiveness were associated with the tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Bufalin treatment also repressed the levels of claudin proteins (claudin-2, -3 and -4) and the major components of TJs that play key roles in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)‑2 and -9 in T24 cells were dose‑dependently inhibited by treatment with bufalin and this also correlated with a decrease in their mRNA and protein expression levels; however, the mRNA and protein levels of the tissue inhibitor of metalloproteinase (TIMP)‑1 and -2 were increased. In addition, these effects were related to the increased phosphorylation of the extracellular signal-regulated protein kinase (ERK) pathway. The inhibition of ERK (PD98059) significantly prevented the bufalin‑induced suppression of T24 cell migration. These findings suggest that bufalin inhibits the migration and invasion of T24 cells by modulating the activity of TJs and MMPs, possibly in association with the activation of ERK.

miRNAs are small, non-coding RNAs that play important roles in various biological processes. The aims of our study were to investigate whether cell-free miRNAs can be measured in urine samples and might be an accurate biomarker of bladder cancer. Datasets of GSE20418 and GSE19717 were used for analysis, and two miRNAs, miR-145 and miR-200a, were selected for study. A total of 207 patients with primary transitional cell carcinoma of the urinary bladder and 144 healthy normal controls were enrolled. Using quantitative PCR, the levels of miR-145 and miR-200a in urine were measured and compared with the clinicopathological features of bladder cancer. According to our experiments, cell-free miRNAs were present in urine and were stable. Assessment of miR-145 levels was able to distinguish bladder cancer patients from non-cancer controls (77.8% sensitivity and 61.1% specificity for NMIBC, AUC 0.729; 84.1 and 61.1% for MIBC, respectively, AUC 0.790) and showed good correlation with grade (p=0.048). In addition, miR-200a was shown to be an independent predictor of NMIBC recurrence by multivariate analysis (OR 0.449, 95% CI 0.239‑0.842, p=0.013). A higher risk of recurrence was observed among patients with a lower miR-200a level compared to patients with higher miR-200a levels (log-rank test, p=0.040). Urinary cell-free miRNAs show promise as noninvasive biomarkers for diagnosis and recurrence of bladder cancer.

Objective: DNA damage repair mechanisms are a source of genetic mutation and are believed to play an important role in human cancer. Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is involved in the recognition and repair of DNA damage. The value of the hOGG1 genotype as a prognostic indicator for bladder cancer (BC) was assessed using a novel technological approach., Materials and Methods: The association between genetic polymorphisms of hOGG1 codon 326 and clinicopathologic characteristics of 337 patients with BC was analyzed using peptide nucleic acid (PNA)-mediated real-time PCR clamping., Results: Tumor grade and size were significantly associated with the hOGG1 codon 326 genotype in non-muscle-invasive bladder cancer (NMIBC). The Cys326Cys polymorphism was significantly associated with progression and cancer specific survival in patients with muscle-invasive bladder cancer (MIBC). Multivariate Cox regression analysis indicated that the hOGG1 Cys326Cys polymorphism is associated with a protective effect on progression and a more dominant survival benefit than the Ser326Ser polymorphism in MIBC (hazard ratio 0.284 and 0.305, respectively)., Conclusions: Analysis of genotypes and clinical data for 337 BC patients indicates that the hOGG1 genotype may be a useful prognostic genetic marker for MIBC., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Interleukin (IL)-28A, also called IFN-λ2, is a member of the classIIcytokine family and has demonstrated anti-proliferative and anti-viral signals. The present study demonstrated migration inducement of IL-28A-treated bladder cancer cells - a novel function. RNA microarray analysis showed an enhanced expression of IL-28A and its receptor IL-28AR1 in muscle invasive urothelial carcinoma in a human bladder. Strong expression of IL-28A and IL-28AR1 was detected in bladder cancer tissues and cell lines (5637, T-24, and HT1376 cells), as determined by real-time PCR and immunoblot analysis. IL-28A treatment induced migration of bladder cancer cells, independent of the cell growth. IL-28AR1-specific small interfering RNA (si-IL-28AR1) inhibited the induction of migration in IL-28A-treated cells. IL-28A treatment stimulated the expression of matrix metalloproteinases-9 (MMP-9) via activation of transcription factor NF-κB. Gene knockdown for MMP-9 and the p65 subunit of NF-κB, using siRNA transfection, suppressed wound healing migration in IL-28A-treated bladder cancer cells. Also, treatment with IL-28A induced activation of mitogen-activated protein kinase (MAPK) in bladder cancer cells. MAPK function blockage by a MAPK-specific inhibitor showed that MAPK phosphorylation is required for IL-28A-induced MMP-9 expression through activation of NF-κB. The transient transfection of bladder cancer cells with ERK1/2 knock down (si-ERK1/2) and dominant negative p38 (DNp38) suppressed IL-28A-induced migration. IL-28A-induced induction of MMP-9 expression, MAPK activation, and DNA binding activity of NF-κB was abolished in the presence of IL-28A neutralizing antibody or by transfection of si-IL-28AR1. These results show that IL-28A/IL-28AR1 dyad-induced wound healing migration requires NF-κB-mediated MMP-9 expression by MAPK activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To identify prognostic markers in nonmuscle invasive bladder cancer (NMIBC), the combined effect of RUNX3 and MGC17624 for predicting NMIBC progression was assessed. RUNX3 promoter methylation was examined using methylation specific-polymerase chain reaction (MS-PCR). MGC17624 mRNA expression was evaluated by real-time PCR. Patients were divided into three groups according to the status of the two genes and the prognostic effects of these markers were evaluated. The median follow-up period was 57.8 months (range, 9.1-189.7). The mRNA expression level of MGC17624 was significantly lower in patients with positive RUNX3 methylation than in those with negative methylation (p = 0.047). Kaplan-Meier estimates showed significant differences in time-to-progression between the genetic combination predictors (log-rank test; p < 0.001). Patients with a poor predictive combination were at a significantly higher risk for progression [Hazard ratio (HR), 22.579] than patients with a good predictive combination in multivariate Cox regression analysis. In the subgroup analysis, a poor predictive combination accurately estimated progression in patients with intravesical therapy (HR, 20.081) and in those who experienced recurrence (HR, 54.233). Assessment of the status of RUNX3 and MGC17624 in combination was identified as a reliable method for predicting NMIBC progression., (Copyright © 2011 UICC.)