Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin

Summary We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGU-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of ∼100 μm/d in multiwell dishes, and necrosis occurred only in spheroids of diameter >1 mm. Spheroids placed in spinner culture at a higher concentration (∼1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at ∼190 μm over a wide range of spheroid diameters (400 to 1000 μm). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids. Supported in part by a grant from the National Cancer Institute of Canada and by grant CA29526 NCI through the National Bladder Cancer Project, U.S.A.     

TURis-Bt术前膀胱灌注表柔比星治疗NMIUC的临床疗效观察

目的:探讨TURis-Bt术前膀胱灌注表柔比星治疗非肌层浸润性膀胱癌(NMIUC)的临床疗效。方法:选取2008年9月至2012年1月潍坊市中医院和上海交通大学附属第一人民医院泌尿外科收治的76例NMIUC患者,并将其随机分为观察组(TURis-Bt术前膀胱灌注表柔比星+术后常规灌注组)41例和对照组(TURis-Bt术后常规表柔比星膀胱灌注组)35例。灌注前将50mg表柔比星溶解于50ml 5%葡萄糖注射液,术前膀胱保留灌注30分钟后膀胱镜观察肿瘤组织及周围膀胱粘膜染色情况,将表柔比星橙染的膀胱粘膜活检并行TURis-Bt;对照组取瘤旁2 cm处及其他部位膀胱粘膜多点活检。比较两组的原位癌(CIS)、非典型性增生及腺性膀胱炎等病变检出率和术后肿瘤的复发率。结果:观察组患者瘤旁膀胱粘膜橙染56处,其中7处病理证实为膀胱原位癌、5处为非典型性增生、11处为腺性膀胱炎;对照组患者膀胱原位癌1处、非典型性增生3处、腺性膀胱炎2处,两组病变阳性率分别为41.1%(23/56)和13.4%(17/127),差异有显著统计学意义(P0.01)。观察组与对照组术后2年内肿瘤复发率分别为10.3%(4/39)和35.3%(12/34),差异有统计学意义(P0.05)。结论:TURis-Bt术前膀胱灌注表柔比星能提高NMIUC病变的早期检出率并降低肿瘤术后复发率。     

Hypermethylation-mediated partial transcriptional silencing of DAP-kinase gene in bladder cancer

Death-associated protein kinase (DAP-kinase) is a novel serine/threonine kinase whose expression is required for interferon-γ-induced apoptosis. This study evaluated the methylation pattern and its impact on the expression of the DAP-kinase gene in transitional cell carcinoma of the bladder as hypermethylation is one of the earliest and most frequent alterations leading to cancer. The frequency of hypermethylation of the gene promoter was 37.8%. On correlation with clinicopathological features, methylation was seen mostly in superficial tumours in the group aged?>?60 years (42.9 vs 33.3% of those?≤?60 years) and in smokers (48.1 vs 27.4% of non-smokers). The increased risk of bladder cancer was 6.70-fold (95% confidence interval (CI) 2.09–23.87; p?=?0.000) in those carrying methylated DAP-kinase and it was elevated in patients who smoked (odds ratio 7.87; 95% CI 1.50–54.96; p?=?0.007). This study demonstrated that methylation in the gene promoter on its own could significantly decrease the mRNA expression level of DAP-kinase by 27.68%. Interestingly, patients within the group aged?>?60 years and with a smoking habit showed increased downregulation of mRNA compared with non-smokers of this age group (similar pattern of methylation). Hypermethylation can decrease the expression of DAP-kinase and may be one of the reasons for conversion of normal cells to malignant cells, as the frequency of methylation at the early stage (superficial) of tumours was elevated. Methylation of DAP-kinase can be considered as one of the prognosis indicators for progression and development of bladder cancer.     

Discovery of urine biomarkers for bladder cancer via global metabolomics

Bladder cancer (BC) is latent in its early stage and lethal in its late stage. Therefore, early diagnosis and intervention are essential for successful BC treatment. Considering the limitations of current diagnostic tools, noninvasive biomarkers that are both highly sensitive and specific are needed to improve the overall survival and quality of life of patients. With the advent of systems biology, “-omics” technologies have been developed over the past few decades. As a promising member, global metabolomics has increasingly been found to have clear potential for biomarker discovery. However, urinary metabolomics studies related to BC have lagged behind those of other urinary cancers, and major findings have not been systematically reported. The objective of this review is to comprehensively list the currently identified potential urinary metabolite biomarkers for BC.     

Pro-apoptotic effect of a nitric oxide-donating NSAID,NCX 4040, on bladder carcinoma cells

Nitric oxide-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs) are a promising class of compounds that cause cell cycle perturbations and induce apoptosis in cell lines from different tumors. We investigated the activity of a recently developed NO-NSAID (NCX 4040) in bladder cancer cell lines (HT1376 and MCR). Cells were treated with different drug concentrations for different exposure times. Cytostatic and cytocidal activity was tested by SRB assay and apoptosis was evaluated by TUNEL analysis, ANNEXIN V assay and fluorescence microscopy. To further investigate the cell death-inducing mechanisms of NCX 4040, we analyzed gp-170, caspase expression and mitochondrial membrane potential (Δ Ψ) depolarization. NCX 4040 showed a striking cytocidal activity in both cell lines, reaching LC₅₀ at a 10-μ M and 50-μ M concentrations in HT1376 and in MCR cells, respectively, after an exposure of only 6 h followed by an 18-h washout. Apoptosis was triggered in up to 90% of cells and was associated with active caspase-3 expression and Δ Ψ depolarization in both cell lines after a 6-h exposure. In conclusion, NCX 4040, which probably causes apoptosis via a mitochondrial-dependent mechanism, could prove to be a useful agent for improving bladder cancer treatment.This study was supported by Istituto Oncologico Romagnolo, Forlì, and by the Italian Ministry of Health, 2002.     

TUR-BT术后膀胱灌注A群链球菌联合丝裂霉素治疗高危非肌层浸润性膀胱癌的疗效观察

目的：总结经尿道膀胱肿瘤电切（TUR-BT）术后辅以沙培林（注射用A群链球菌）联合丝裂霉素（MMC）膀胱内灌注治疗高危膀胱癌的疗效。方法：回顾性研究2009年1月~2012年8月我院收治的符合高危非肌层浸润性膀胱癌患64例,观察组：TUR-BT术后行沙培林联合MMC膀胱内灌注化疗32例,对照组;MMC单药灌注32例。结果：观察组患者,平均年龄63.7岁,治疗随访时间为6~54个月,中位时间27.3个月。治疗随访期间有3例患者出现膀胱内肿瘤复发（9.3%）,1例患者疾病进展,发展为肌层浸润性膀胱癌,于术后7个月死亡（3.1%）。与对照组患者相比,疾病复发率及进展率均明显改善。结论：高危非肌层浸润膀胱癌临床复发率、进展率高,TUR-BT术后沙培林联合MMC膀胱内灌注通过局部化疗及免疫治疗联合,可有效控制疾病的复发和进展,降低患者接受膀胱部分切除或膀胱全切手术的机率,值得推广。     

高强度聚焦超声对人膀胱癌细胞的生长抑制作用及机理研究

研究高强度聚焦超声（high intensity focused ultrasound,HIFU）对人膀胱癌细胞的杀伤作用,并探索其作用机理.用不同强度的HIFU处理人膀胱癌细胞T24,用台盼蓝排斥法染色、细胞增殖试验（MTT法）研究HIFU对癌细胞的杀伤和生长抑制作用;流式细胞术检测其对癌细胞周期、凋亡及相关基因表达的影响.HIFU处理后,癌细胞死亡率升高,增殖活性降低,G0/G1期细胞数增加,S期细胞数减少,凋亡指数升高,与对照组比较,差异有显著性意义（P＜0.05）;P53、BCL-2和FAS表达阳性细胞数与对照组比较,无显著性意义（P＞0.05）;HIFU强度为600 W/cm^2时,HSP70表达阳性细胞数升高,与对照组比较差异有显著性意义（P＜0.05）.HIFU对人膀胱癌细胞T24具有明显的杀伤及抑制作用,其机理与抑制DNA合成有关;HIFU具有诱导癌细胞凋亡的作用,其诱导凋亡作用可能与P53、BCL-2和FAS无关.     

miR‐222 attenuates cisplatin‐induced cell death by targeting the PPP2R2A/Akt/mTOR Axis in bladder cancer cells

Increased miR‐222 levels are associated with a poor prognosis in patients with bladder cancer. However, the role of miR‐222 remains unclear. In the present study, we found that miR‐222 enhanced the proliferation of both the T24 and the 5637 bladder cancer cell lines. Overexpression of miR‐222 attenuated cisplatin‐induced cell death in bladder cancer cells. miR‐222 activated the Akt/mTOR pathway and inhibited cisplatin‐induced autophagy in bladder cancer cells by directly targeting protein phosphatase 2A subunit B (PPP2R2A). Blocking the activation of Akt with LY294002 or mTOR with rapamycin significantly prevented miR‐222‐induced proliferation and restored the sensitivity of bladder cancer cells to cisplatin. These findings demonstrate that miR‐222 modulates the PPP2R2A/Akt/mTOR axis and thus plays a critical role in regulating proliferation and chemotherapeutic drug resistance. Therefore, miR‐222 may be a novel therapeutic target for bladder cancer.     

TURBT术后即刻膀胱灌注不同浓度吡柔比星对浅表性膀胱癌患者免疫功能及生活质量的影响

目的:研究经尿道膀胱肿瘤电切术(TURBT)术后即刻膀胱灌注不同浓度吡柔比星对浅表性膀胱癌(SBC)患者免疫功能及生活质量的影响。方法:选择从2015年6月到2018年6月在我院治疗的SBC患者126例作为此次研究对象。依据随机数字法将患者分成观察组以及对照组,每组各63例,两组患者均常规给予TURBT治疗,手术完成后即刻,观察组患者采用30 mg的吡柔比星~+30 m L浓度为5%的葡萄糖液行胱灌注化疗,对照组采用30 mg的吡柔比星~+50 m L浓度为5%的葡萄糖液行胱灌注化疗。治疗1年后对比两组复发情况、免疫功能指标、生活质量以及不良反应。结果:观察组的复发率明显低于对照组,且复发时间明显长于对照组,差异均有统计学意义(P0.05)。两组治疗后的CD3~+、CD4~+、CD8~+水平及生活质量各方面评分均明显高于治疗前,差异有统计学意义(P0.05);两组治疗前及治疗后的CD3~+、CD4~+、CD8~+水平及生活质量各方面评分相比差异无统计学意义(P0.05)。观察组各种不良反应及其总发生率与对照组相比差异无统计学意义(P0.05)。结论:SBC患者在TURBT术后即刻实施吡柔比星膀胱灌注能够提升其免疫功能及生活质量,但30 mg的吡柔比星~+30 m L浓度为5%的葡萄糖液的膀胱灌注用药方案能够获得更低的术后复发率,复发时间也随之延长。     

Urinary cytology and the Paris system for reporting urinary cytology: Implications for urological management

By reducing the rate of indeterminate (atypical) diagnoses and standardising reporting terminology, The Paris System for Reporting Urine Cytology helps focus the application of cytology towards the detection primarily of high‐grade urothelial carcinoma. We present a urology‐based perspective of how the new system has influenced clinical decision‐making.     

Cancer-associated fibroblasts–derived exosomes-mediated transfer of LINC00355 regulates bladder cancer cell proliferation and invasion

The aim of this study was to investigate the regulatory mechanism of cancer-associated fibroblasts (CAFs) exosome in bladder cancer (BC) cell proliferation and invasion. CAFs and normal fibroblasts (NFs) were isolated from tumor tissues and adjacent normal tissues of BC patients, and examined by immunocytochemistry for the expression of fibroblast activation protein alpha (FAP) and α-smooth muscle actin (α-SMA). Exosomes were extracted from CAFs and NFs and observed under a transmission electron microscope, and expression of the exosome markers CD9 and CD63 was confirmed by western blotting. The distribution and intensity of fluorescence were observed by confocal laser microscopy to analyze exosomes uptake by BC cell lines T24 or 5367. BC cell proliferation and invasion were detected by MTT and Transwell assays, respectively. LINC00355 levels in CAFs, NFs, CAFs exosome, NFs exosome, and BC cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that CAFs exosome significantly promoted BC cell proliferation and invasion relative to NFs exosome. LINC00355 expression was significantly elevated in CAFs exosome when compared with that in NFs-exosome. Up-regulated LINC00355 expression was observed both in T24 and 5367 cells co-incubated with CAFs exosome. Exosomes derived from LINC00355 siRNA-transfected CAFs observably repressed BC cell proliferation and invasion when compared with control siRNA-CAFs exosome. In conclusion, CAFs exosome–mediated transfer of LINC00355 regulates BC cell proliferation and invasion. Significance of the study. In this study, our data suggest that the exosomes released from CAFs promote BC cell proliferation and invasion. The mechanism of this effect is, at least in part, related to the increased LINC00355. Regulation of LINC00355 expression in exosomes released from CAFs might be a putative therapeutic strategy against the pathogenesis of BC.     

非肌层侵润性膀胱癌的治疗

膀胱癌是一种全球性疾病。在我国泌尿外科肿瘤中的发病率和死亡率均占首位,非肌层侵润性膀胱癌占初发膀胱肿瘤的70%。对膀胱癌的研究已成为目前学术界的热点话题。目前学界对于非肌层侵润性膀胱癌主要采用以外科手术为主的综合治疗方案。为探讨该类肿瘤的治疗方法,本文就近年来对非肌层侵润性膀胱癌的各种治疗措施进行了比较系统的阐述。我们希望能尽可能的找到高效低风险并且经济的方法,为膀胱癌的诊断和治疗提供新途径。     

The role of c-FLIP splice variants in urothelial tumours

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin''s lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP_long (c-FLIP_L) and c-FLIP_short (c-FLIP_S), which can have opposing functions. We observed diminished expression of the c-FLIP_L isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP_S was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP_L to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP_L isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.     

USP24-GSDMB complex promotes bladder cancer proliferation via activation of the STAT3 pathway

Background: Bladder cancer is the fourth and tenth most common malignancy in men and women worldwide, respectively. One of the main reasons for the unsatisfactory therapeutic control of bladder cancer is that the molecular biological mechanism of bladder cancer is complex. Gasdermin B (GSDMB) is one member of the gasdermin family and participates in the regulation of cell pyroptosis. The role of GSDMB in bladder cancer has not been studied to date.Methods: TCGA database was used to exam the clinical relevance of GSDMB. Functional assays such as MTT assay, Celigo fluorescent cell-counting assay, Annexin V-APC assay and xenografts were used to evaluate the biological role of GSDMB in bladder cancer. Mass spectrometry and immunoprecipitation were used to detect the protein interaction between GSDMB and STAT3, or GSDMB and USP24. Western blot and immunohistochemistry were used to study the relationship between USP24, GSDMB and STAT3.Results: In this study, bioinformatics analysis indicated that the mRNA expression level of GSDMB in bladder cancer tissues was higher than that in adjacent normal tissues. Then, we showed that GSDMB promoted bladder cancer progression. Furthermore, we demonstrated that GSDMB interacted with STAT3 to increase the phosphorylation of STAT3 and modulate the glucose metabolism and promote tumor growth in bladder cancer cells. Besides, we also showed that USP24 stabilized GSDMB to activate STAT3 signaling, which was blocked by the USP24 inhibitor.Conclusions: We suggested that aberrantly up-regulated GSDMB was responsible for enhancing the growth and invasion ability of bladder cancer cells. Then, we showed that GSDMB could bind to STAT3 and activate STAT3 signaling in bladder cancer. Furthermore, we also demonstrated that USP24 interacted with GSDMB and prevented GSDMB from degradation in bladder cancer cells. Therefore, the USP24/GSDMB/STAT3 axis may be a new targetable signaling pathway for bladder cancer treatment.     

How Creditable are Cancer Risk Estimates from the S.W. Taiwan Database for Arsenic in Drinking Water?

A database of cancer mortality and arsenic concentrations in village wells in an arseniasis-endemic area of southwestern Taiwan has been the predominant source of information for risk assessments of U.S. Environmental Protection Agency and two National Research Council reports on arsenic and drinking water. A limitation of the data, however, is that exposure is ecological, that is, cancer mortality cannot be matched with arsenic exposure on an individual basis, just grouped by village. The resultant potential for bias is examined by comparing dose-response analyses of villages divided into two groups, those with well concentrations in a narrow range and the remainder. The narrow range group suggests a flat or downward change in risk in the low dose range, whereas the other group indicates increasing risk. More disturbingly, however, the dose-response data are highly dispersed for both groups and the dose-response curve predicts background bladder/lung cancer levels much higher than a southwestern Taiwan comparison population. This may be due to a large variability between villages of the study area in bladder/lung cancer not directly attributable to arsenic. Including the comparison population in the dose-response analysis artificially anchors the dose-response curve at a background level inconsistent with the study population and likely just biases the slope factor upward.