Bladder Cancer T24 Research Articles

Perfusable Plasma‐Activated Saline for Oxidative Stress Delivery to Induce Tumor Cell Apoptosis in Nonmuscle‐Invasive Bladder Cancer

ABSTRACTNonmuscle‐invasive bladder cancer (NMIBC) is a common urologic malignancy. Currently, conventional postoperative perfusion therapy for NMIBC still has high side effects and recurrence rates. Cold atmospheric plasma is a fledgling reactive oxygen species (ROS)–based therapeutic technique for cancer treatment. In this study, we examined the feasibility of plasma‐activated saline (PAS) perfusion for treating NMIBC. The results show that underwater bubble plasma can generate abundant ROS in PAS. In vitro studies revealed that PAS could trigger apoptosis in bladder cancer T24 cells by activating the ROS‐mediated caspase‐3 cascade reaction. In vivo tests revealed that intravesical PAS perfusion effectively inhibited nonmuscle‐invasive T24 tumor growth without significant side effects. Therefore, our results suggest that intravesical PAS perfusion may be a novel treatment option for NMIBC.

Anticancer transmission effects induced by hybrid plasma bubble-activated medium

As an indirect application of cold atmospheric plasma, the use of plasma-activated solutions has recently attracted significant attention for intracavity tumor perfusion therapy. Here, an underwater plasma-bubble reactor with three discharge modes was applied to activate a cell culture medium, with the aim of inhibiting the growth of bladder cancer T24 cells in vitro. The results showed that although the reactive species in cell culture medium generated by the underwater bubble plasma varied between the dielectric barrier discharge (DBD) mode, spark mode, and hybrid mode, the plasma-activated cell culture medium (PAM) corresponding to all three discharge modes effectively decreased the viability of T24 cells. However, after co-culturing the PAM-pretreated T24 cells with normal T24 cells, it was observed that normal T24 cells were not affected by either the DBD or spark mode; however, the hybrid mode PAM-pretreated T24 cells further induced inactivation and apoptosis of normal T24 cells. Further studies suggested that the aqueous reactive species generated by the underwater bubble plasma in hybrid mode not only induced the apoptosis of T24 cells directly but also triggered PAM-pretreated T24 cell-derived exosome-mediated anticancer activity toward cancer cells. These results may provide a strategy for enhancing the anticancer extent and effect of PAM as well as advancing its clinical application.

Transcriptome Analysis Reveals Anti-Cancer Effects of Isorhapontigenin (ISO) on Highly Invasive Human T24 Bladder Cancer Cells.

Bladder cancer, the most common malignancy of the urinary tract, has a poor overall survival rate when the tumor becomes muscle invasive. The discovery and evaluation of new alternative medications targeting high-grade muscle invasive bladder cancer (MIBC) are of tremendous importance in reducing bladder cancer mortality. Isorhapontigenin (ISO), a stilbene derivative from the Chinese herb Gnetum cleistostachyum, exhibits a strong anti-cancer effect on MIBCs. Here, we report the whole transcriptome profiling of ISO-treated human bladder cancer T24 cells. A total of 1047 differentially expressed genes (DEGs) were identified, including 596 downregulated and 451 upregulated genes. Functional annotation and pathway analysis revealed that ISO treatment induced massive changes in gene expression associated with cell movement, migration, invasion, metabolism, proliferation, and angiogenesis. Additionally, ISO treatment-activated genes involved in the inflammatory response but repressed genes involved in hypoxia signaling, glycolysis, the actin cytoskeleton, and the tumor microenvironment. In summary, our whole transcriptome analysis demonstrated a shift in metabolism and altered actin cytoskeleton in ISO-treated T24 cells, which subsequently contribute to tumor microenvironment remodeling that suppresses tumor growth and progression.

Molecular mechanism of anticancer effect of heat shock protein 90 inhibitor BIIB021 in human bladder cancer cell line

Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity has been a promising pharmacological strategy for blocking of bladder cancer pathogenesis. BIIB021 is a next-generation HSP90 inhibitor which interrupts ATP hydrolysis process of HSP90 and inhibits the stabilization and correct folding of client proteins. In current study, we aimed to investigate the molecular mechanism of the anticancer activity of BIIB021 in human bladder cancer T24 cells. Our results revealed that nanomolar concentration of BIIB021 decreased viability of T24 cell. BIIB021 downregulated HSP90 expression in T24 cells and inhibited the refolding activity of luciferase in the presence of T24 cell lysate. PCR array data indicated a significant alteration in transcript levels of cancer-related genes involved in metastases, apoptotic cell death, cell cycle, cellular senescence, DNA damage and repair mechanisms, epithelial-to-mesenchymal transition, hypoxia, telomeres and telomerase, and cancer metabolism pathways in T24 cells. All findings hypothesize that BIIB021 could exhibit as effective HSP90 inhibitor in the future for treatment of bladder cancer patients.

The expression of SLFN11 is related to the sensitivity of bladder cancer cells to DNA damage agents

BackgroundPutative DNA/RNA helicase Schlafen-11 (SLFN11) was studied for its functions in cancer cells in current years, especially its ability of sensitizing cancer cells to DNA-damaging agents (DDA). However, little is known about the roles it plays in bladder cancer. MethodsBioinformatic tools including GEPIA, UALCAN, MEXPRESS, CTRP, cBioPortal, MethSurv, and Metascape were used. RT-qPCR, CCK-8 assay, and methylation specific PCR were used to verify the results of bioinformatic analysis in bladder cancer cells. ResultsCompared to the normal samples, the expression of SLFN11 was lower in BLCA samples. SLFN11 expression was associated with clinical parameters in BLCA patients, especially histological and molecular subtypes. Besides, its expression correlated to the sensitivity of cancer cells to DDA. Furthermore, the frequency of SLFN11 genetic alterations was low in BLCA patients, and methylationwas significantly negatively related to SLFN11 expression. The mRNA levels of SLFN11 in the bladder cancer cells UMUC-3, T24, and T24-GR were significantly lower than that in the normal SV-HUC-1 cells. SLFN11 expression was positively correlated with bladder cancer cells’ sensitivity to gemcitabine. In addition, partial methylation of SLFN11 was detected in T24-GR, and can be inhibited by 5-Aza. ConclusionsSLFN11 expression was lower in BLCA samples and in vitro bladder cancer cells than in the normal ones. The level of SLFN11 correlated to the sensitivity of cancer cells to DDA. Additionally, the methylation of SLFN11 was negatively related to its expression. It couldbe a potential prognostic biomarker for predicting the therapeutic effect of DNA-damaging agents in patients with bladder cancers.

SIRT1 Promotes Cisplatin Resistance in Bladder Cancer via Beclin1 Deacetylation-Mediated Autophagy.

Autophagy-dependent cisplatin resistance poses a challenge in bladder cancer treatment. SIRT1, a protein deacetylase, is involved in autophagy regulation. However, the precise mechanism through which SIRT1 mediates cisplatin resistance in bladder cancer via autophagy remains unclear. In this study, we developed a cisplatin-resistant T24/DDP cell line to investigate this mechanism. The apoptosis rate and cell viability were assessed using flow cytometry and the CCK8 method. The expression levels of the relevant RNA and protein were determined using RT-qPCR and a Western blot analysis, respectively. Immunoprecipitation was utilized to validate the interaction between SIRT1 and Beclin1, as well as to determine the acetylation level of Beclin1. The findings indicated the successful construction of the T24/DDP cell line, which exhibited autophagy-dependent cisplatin resistance. Inhibiting autophagy significantly reduced the drug resistance index of these cells. The T24/DDP cell line showed a high SIRT1 expression level. The overexpression of SIRT1 activated autophagy, thereby further promoting cisplatin resistance in the T24/DDP cell line. Conversely, inhibiting autophagy counteracted the cisplatin-resistance-promoting effects of SIRT1. Silencing SIRT1 led to increased acetylation of Beclin1, the inhibition of autophagy, and a reduction in the cisplatin resistance of the T24/DDP cell line. Introducing a double mutation (lysine 430 and 437 to arginine, 2KR) in Beclin-1 inhibited acetylation and activated autophagy, effectively reversing the decreased cisplatin resistance resulting from SIRT1 silencing. In summary, our study elucidated that SIRT1 promotes cisplatin resistance in human bladder cancer T24 cells through Beclin1-deacetylation-mediated autophagy activation. These findings suggest a potential new strategy for reversing cisplatin resistance in bladder cancer.

Development of a Screening Nomogram for Regional Lymph Node Metastasis Development in Muscle Invasive Bladder Cancer after Multiagent Systemic Therapy

A reliable method of identifying clinically node negative bladder cancer patients at greater risk of developing lymph node metastasis and may require intensified therapy is needed. The purpose of this study was to create a nomogram to quantify the risk for regional lymphatic involvement in non-metastatic muscle invasive bladder cancer. Usingthe National Cancer Database, patients with clinical T2-4N0M0 urothelial carcinoma of the bladder between the years of 2004 - 2020 were selected. All patients completed multiagent chemotherapy followed by surgery for pathologic nodal staging to determine presence or absence of nodal disease. No neoadjuvant radiation therapy was allowed and patients with history of prior malignancies were excluded. Following a 70:30 training to testing data split, baseline variables were assessed using univariate logistic regression. Variables were selected for inclusion in the multivariate logistic regression model using a combination of previously reported findings in the literature and/or if found to have a p-value < 0.05 on univariate analysis. A nomogram was constructed using this final model with assessment using calibration plots and the area under the receiver operating characteristics curve (AUC), respectively. An empiric cut point was chosen at 95% sensitivity to identify patients at "high" and "low" risk for pathologic nodal disease with overall assessment in both cohorts using chi-square. A total of 6194patients were identified for study with a median age of 65 years (IQR = 58 - 71 years). Most patients were male (68.0%) with T2 disease (81.2%). The final multivariate model included age at time of diagnosis (OR = 0.99; 95% CI = 0.99 - 1.00; p = 0.172), time from diagnosis to initiation of chemotherapy (OR = 1.00; 95% CI = 1.00 - 1.01; p = 0.005), papillary histology (OR = 0.85; 95% CI = 0.72 - 1.01; p = 0.068), and clinical T stage (Table 1). Model calibration demonstrated excellent goodness-of-fit with good discrimination (AUC = 0.644). Within the training data, high risk patients were seen to have a twofold increase in pathologic nodal disease (N = 835/3924, 21.2%) when compared to those identified as low risk (N = 38/347, 9.9%) (p < 0.001). Validation within the testing data set demonstrated similar results with pathologic node rates of 22.8% and 7.8% for high- and low-risk patients, respectively (AUC = 0.645, p < 0.001). This study demonstrates a clinically applicable risk stratification tool for identifying patients at risk for developing lymphadenopathy in T2-4 bladder cancer and may help guide future research in selecting patients eligible for escalation of therapy. Future studies should aim to externally validate this tool within prospective cohorts, and seek to determine if this nomogram may provide further prognostic utility.

Non-enzymatic electrochemical sensor based on Pt/MoSe2 nanomesh for the detection of hydrogen peroxide

The development of diagnostic methods that are sensitive and selective for detection of reactive oxygen species (ROS) is crucial for early diagnosis of bladder cancer. Herein, a new electrochemical sensing platform for hydrogen peroxide (H2O2) was created based on the Pt/MoSe2 nanomesh with more active sites and larger specific surface area. The Pt/MoSe2 nanomesh sensors exhibited highly sensitive and selective for H2O2, wherein the correlation coefficient (R2) and the limit of detection (LOD) were 0.9983 and 2.56 μM obtained at the signal/noise (S/N) ratio of 3, respectively. Additionally, Pt/MoSe2 nanomesh possessed a good anti-interference performance even in the presence of potentially interfering small molecules within cells, such as ascorbic acid (AA), glucose (Glc) and so on. More importantly, the detection of H2O2 in tap, rain Water, urine FBS, human serum and bladder cancer T24 cells could be successfully detected by the Pt/MoSe2 nanomesh electrochemical sensor.

CDK2 and CDK4 targeted liensinine inhibits the growth of bladder cancer T24 cells

Bladder cancer (BCa) is a urinary tumor with limited treatment options and high mortality. Liensinine (LIEN), a natural bisbenzylisoquinoline alkaloid, has shown excellent anti-tumor effects in numerous preclinical studies. However, the anti-BCa effect of LIEN remains unclear. To the best of our knowledge, this is the first study to investigate the molecular mechanism of LIEN in the management of BCa. First, we identified the treatment-related targets of BCa; those that repeatedly occur in more than two databases, including GeneCards, Online Mendelian Inheritance in Man, DisGeNET, Therapeutic Target Database, and Drugbank. The SwissTarget database was used to screen LIEN-related targets, and those with a probability >0 were possible LIEN targets. The prospective targets of LIEN in the treatment of BCa were then determined using a Venn diagram. Second, we discovered that the PI3K/AKT pathway and senescence mediated the anti-BCa action of LIEN by using GO and KEGG enrichment analysis to explore the function of LIEN therapeutic targets. A protein-protein interaction network was created using the String website, and six algorithms of the CytoHubba plug-in were then used in Cytoscape to assess the core targets of LIEN for the therapy of BCa. The outcomes of molecular docking and dynamics simulation demonstrated that CDK2 and CDK4 proteins were the direct targets of LIEN in the management of BCa, among which CDK2 was more stable in binding to LIEN than CDK4. Finally, in vitro experiments showed that LIEN inhibited the activity and proliferation of T24 cells. The expression of p-/AKT, CDK2, and CDK4 proteins progressively decreased, while the expression and fluorescence intensity of the senescence-related protein, γH2AX, gradually increased with increasing LIEN concentration in T24 cells. Therefore, our data suggest that LIEN may promote senescence and inhibit proliferation by inhibiting the CDK2/4 and PI3K/AKT pathways in BCa.

Recognition Imaging of Trace E-cadherins on the Bladder Cancer Cells Surface during Epithelial-Mesenchymal Transition by Force-Distance Curve-Based AFM.

Tumor-associated epithelial-mesenchymal transition (EMT) contains a set of transitional cellular states usually judged by the EMT marker expression. E-cadherin is a down-regulated EMT epithelial marker, and the detection of E-cadherin is challenging on cancer cell surfaces in the middle and late stages of EMT. Here, the trace E-cadherins on the living bladder cancer T24 cell surface during EMT were investigated with force-distance curve-based atomic force microscopy. The results confirmed that T24 cells are still in an intermediate state and can be transferred into the mesenchymal phenotype by long-term TGF-β1 induction. During EMT, E-cadherins on the T24 cell surface gradually decreased and rarely clustered. E-cadherin is not completely missing, even at the end of EMT, but is too sparse to cluster. This work provides us with a visual understanding of the expression and distribution of trace markers during EMT and a deep comprehension of the indispensable significance of E-cadherin in cancer cells.

Tripartite motif–containing 9 promoted proliferation and migration of bladder cancer cells through CEACAM6-Smad2/3 axis

Studies have shown that tripartite motif–containing (TRIM) family proteins function as E3 ubiquitin ligases and play essential roles in cancer biology. In the present study, we validated a contribution of TRIM9 to bladder cancer progression. 296 patients derived from The Cancer Genome Atlas (TCGA) database and 22 clinical specimens were included, in which accumulated TRIM9 correlated with the poor prognosis and higher relapse in bladder patients. In vitro, TRIM9 promoted bladder cancer cells Biu-87 and T24 cell proliferation and migration. Meanwhile, overexpression of TRIM9 reduced the chemosensitivity in Biu-87 and T24 to mitomycin C (MMC) and gemcitabine (GEM). As an underlying mechanism, we found that TRIM9 stimulated carcinoembryonic antigen 6 (CEACAM6) upregulation, which further facilitated Smad2/3-matrix metalloproteinase 2 (MMP2) signaling activation both in vitro and in vivo. Those results indicated that TRIM9 facilitated bladder cancer development and chemoresistance by CEACAM6-Smad2/3 axis. TRIM9 and its associated molecules could be a potential diagnostic indicator and therapeutic target in bladder cancer.Graphic

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway.

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G2 /M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.

Analysis of the Regulatory Effect of ACTG2 on Biological Behavior of Bladder Cancer Cells Based on Database Screening.

To identify genes aberrantly expressed in bladder cancer (BC) in the GEO dataset GSE 52519, and analyze the impact of abnormal Actin Gamma 2, Smooth Muscle (ACTG2) expression on BC cells. GSE 52519, a public dataset in the Gene expression omnibus (GEO) database, was selected for differential expression analysis. Differentially expressed ACTG2 vectors were selected to construct the aberrant expression vectors and transfected into BC T24 and J82 cells. The influences of ACTG2 on BC cell biological behavior were determined by cell cloning, Transwell and flow cytometry, and alterations in cell cycle status were observed. A total of 166 DEGs were found in the GSE 52519 dataset, among which ACTG2 was abnormally low in expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses identified that the keywords involved mainly included "extracellular region", "cytoskeleton", "Vascularsmoothmusclecontraction", "IL-17signalingpathway", etc. Further, through online data analysis, ACTG2 was also found to be under-expressed in neoplastic diseases such as bladder urothelial carcinoma (BLCA) and prostate adenocarcinoma (PRAD). In vitro, ACTG2 presented lower expression in T24 and J82 than in SV-HUC-1 (P<0.05). Enhanced capacities to proliferate and invade and reduced apoptosis of T24 and J82 were found after silencing ACTG2 expression, with shortened G0-G1 phase and prolonged S phase (P<0.05). However, overexpressing ACTG2 came with decreased BC cell activity, enhanced apoptosis, as well as prolonged G0-G1 phase and shortened S phase (P<0.05). In conclusion, low expression of ACTG2 in BC can shorten the G0-G1 phase of BC cells, prolong the S-phase.

Berberine Affects the Proliferation, Migration, Invasion, Cell Cycle, and Apoptosis of Bladder Cancer Cells T24 and 5637 by Down-Regulating the HER2/PI3K/AKT Signaling Pathway.

To assess the anticancer effect, target, and mechanism of berberine on bladder cancer. Bladder cancer T24 and 5637 cells were treated with different concentrations of berberine. Then, cell proliferation was assessed by cell counting kit-8 (CCK8) measure, cell migration and invasion were assessed by transwell method, cell cycle and apoptosis were assessed by flow cytometry, and the expression of human epidermal growth factor receptor-2/PhosphoInositide-3 Kinase/AKT Serine/Threonine Kinase (HER2/PI3K/AKT) proteins were assessed by Western blot. Berberine molecular docking and HER2 target were performed using the AutoDock Tools 1.5.6. Finally, HER2 inhibitors CP-724714 and berberine were used independently or in combination to detect AKT and P-AKT protein downstream changes by Western blot. Berberine inhibited the proliferation of T24 and 5637 bladder cancer cells in a concentration-dependent and time-dependent manner. Berberine can significantly inhibit the migration, invasion, and cell cycle progression of T24 and 5637 bladder cancer cells, promote their apoptosis, and down-regulate the expression of HER2/PI3K/AKT proteins. Berberine showed good docking with HER2 molecular target and had a similar and synergistic effect with HER2 inhibitor in T24 and 5637 bladder cancer cells. Berberine inhibited the proliferation, migration, invasion, and cell cycle progression of T24 and 5637 bladder cancer cells and promoted their apoptosis by down-regulating HER2/PI3K/AKT signaling pathway.

NEAT1 promotes the malignant development of bladder cancer by regulating the miR-101/VEGF-C pathway in vitro and in vivo

BackgroundNEAT1 has been shown to play an oncogenic role in many kinds of cancers. However, detailed roles of NEAT1 in bladder cancer are largely unknown.MethodsIn the present study, the expression of NEAT1, miR-101 and VEGF-C was detected in human bladder cancer samples. The relationship between NEAT1 and the prognosis of patients with bladder cancer was analysed. In vitro experiments explored the effects of NEAT1 on biological behaviours of bladder cancer T24 and 5637 cells. Bioinformatics prediction and luciferase assays were used to assay the regulatory mechanism of action of NEAT1 and miR-101. Loss and gain of the expression of miR-101 and VEGF-C were used to explore the effects of the NEAT1/miR-101/VEGF-C pathway on T24 and 5637 cells. The effect of NEAT1 on the growth of bladder cancer in vivo was explored using an orthotopic tumourigenesis model.ResultsNEAT1 and VEGF-C were significantly upregulated in bladder cancer samples, and miR-101 was significantly downregulated. NEAT1 upregulation was associated with poorer recurrence-free survival of patients with bladder cancer. Overexpression of NEAT1 promoted the proliferation, migration and invasion of bladder cancer cells. The results of the luciferase assay indicated that miR-101 was a target of NEAT1. The promoting effects of NEAT1 on bladder cancer cells were reversed by miR-101 upregulation, and inhibition of miR-101 enhanced the effects of NEAT1. Overexpression of VEGF-C had a clear synergistic effect with the action of NEAT1. Overexpression of NEAT1 increased tumour growth and induced the development of liver metastasis.ConclusionsIn conclusion, our data indicated that NEAT1 was expressed at high levels in bladder cancer patients and correlated with unfavourable prognosis. NEAT1 promoted malignant development of bladder cancer in vitro and in vivo by regulating the miR-101/VEGF-C pathway.

Iron oxide-alginate-berbamine nanocomposites trigger reactive oxygen species-induced apoptosis through down-regulation of PI3K/AKT signaling in human bladder cancer cells

Globally, bladder cancers are the most common cancers with 81,180 new cases in 2022. Being one of the most important urological cancers it has 17,100 estimated deaths in 2022. Unequivocally the best-known treatment so far is radical cystectomy followed by chemotherapy. Despite the treatment strategies including surgical intervention and adjuvant therapy recurrent rate and tumor development prevention remains as a potential problem. Hence targeted treatment strategies gained more interests and the current exploration examines the potential of iron oxide-alginate-berbamine nanocomposites on human bladder cancer T24 cells. The synthesis of iron oxide-alginate-berbamine nanocomposites was with iron (III) nitrate hexahydrate, sodium alginate, and the phytocomponent berbamine. The iron oxide-alginate-berbamine nanocomposites were then treated with human bladder cancer T24 cells. Interestingly, there was a significant dose-dependent reduction in the cell survival rate (2.5–15 µg/ml). Furthermore, the nanocomposites activated caspase-3,8 and -9 almost two-fold. These caspases are the key players in extrinsic and intrinsic apoptosis pathways. The nanocomposites also elevated the ROS status, thereby inactivating the PI3K/Akt signaling pathway. Thus, the current outcomes indicate that iron oxide-alginate-berbamine nanocomposites could effectively inhibit proliferation and induce apoptosis and block the PI3K/Akt pathway in T24 cells.

Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 μM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

Decorin inhibits proliferation and metastasis in human bladder cancer cells by upregulating P21.

Migration of bladder cancer (BC) cells poses a substantial threat to human health. It is critical to elucidate the mechanism of BC invasion and progression for surgical treatment and the prognosis of patients. Decorin is of interest as an anticancer treatment that can play a vital role in regulating tumorigenesis.The effect of decorin expression on survival in clinical patients was screened and analyzed using bladder urothelial carcinoma data from the Cancer Genome Atlas (TCGA) database. The differential expression of transforming growth factor-β1 (TGF-β1) in tumors was compared against that of normal samples to analyze the correlation between them. MTT, flow cytometry, and Wound/Transwell assays were used to detect cell proliferation, cycle arrest, apoptosis, migration, and invasion.Analysis of TCGA data showed that decorin expression was significantly lower in bladder urothelial carcinoma samples than in normal tissues, while TGF-β1 expression did not change significantly. We found that decorin was correlated with TGF-β1 expression in bladder urothelial cancer. In addition, decorin blocked the G1/S phase by upregulating p21 protein and inhibiting the expression of TGF-β1 and MMP2, promoting the occurrence of apoptosis and inhibiting the proliferation of human BC T24 cells. Moreover, decorin increased the adhesion of tumor cells in vitro, and effectively inhibited cell metastasis.Decorin regulated the expression of TGF-β1 and MMP2 through p21 protein, promoted apoptosis and adhesion, and inhibited the proliferation and metastasis of BC cells.

Magnetic multi-enzyme cascade combined with liquid chromatography tandem mass spectrometry for fast DNA digestion and quantitative analysis of 5-hydroxymethylcytosine in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA.

Selective delipidation of Mycobacterium bovis BCG retains antitumor efficacy against non-muscle invasive bladder cancer.

Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment. Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains. Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8+ T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8+ and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro. These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC.