The green tea compound, (-)-epigallocatechin-3-gallate downregulates N-cadherin and suppresses migration of bladder carcinoma cells

Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P 相似文献   

MicroRNA-576-3p Inhibits Proliferation in Bladder Cancer Cells by Targeting Cyclin D1

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3′-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3′-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.     

Limiting processes for perennial plant reintroduction to restore dry grasslands

In restored grasslands of southern Europe, perennial plants remain highly underrepresented compared with the reference ecosystems. We tested various treatments to reintroduce common perennial plant species (Brachypodium retusum, Poaceae, and Thymus vulgaris, Lamiaceae), which are usually not or poorly reintroduced via soil and hay transfer. Treatments included microenvironmental manipulations (rock cover and plant interactions) and two grazing intensities. Target perennial species were transplanted in 2002 in the reference grassland ecosystem (intact grassland area used as a control) and in two abandoned fields. Survival was assessed in June 2003 and June 2004. Target species shoot and root biomass were measured in June 2004. Grazing greatly reduced the survival and biomass of both target species and its effects were reinforced by summer drought: plants that did not establish well enough during the autumn and spring did not survive summer. The restored rock cover had a mild positive effect, particularly on B. retusum. There were no negative or positive plant neighbor interactions in the steppe, while there was competition in both abandoned fields. Competition was particularly intense in the abandoned melon field, composed of a dense sward of annual grasses (Bromus sp.). In order to reintroduce perennial species to dry grasslands, the ideal combination of treatments is to exclude or reduce grazing during the first year to allow seedlings to establish and to recreate adequate microenvironmental conditions. Reducing competition from arable weeds may help but is not essential in such dry grasslands.     

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies

Background

Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.

Results

Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.

Conclusions

Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1450-3) contains supplementary material, which is available to authorized users.     

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells

The expression of N‐cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N‐cadherin expressed in various, but cancer‐related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N‐cadherin molecule, probed with the same antibody both on a surface of living non‐malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N‐cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non‐malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N‐cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non‐malignant cells reveal less stable N‐cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer‐related changes in a structure of the binding site of the antibody, located at the extracellular domain of N‐cadherin. Copyright © 2011 John Wiley & Sons, Ltd.     

The RNA binding protein Musashi1 regulates apoptosis,gene expression and stress granule formation in urothelial carcinoma cells

The RNA‐binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected MSI1 mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of MSI1 expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after MSI1 knockdown significantly up‐regulated 735 genes, but down‐regulated only 31. Up‐regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21^CIP1 and Numb proteins as targets of Musashi1, suggesting additionally p27^KIP1 in cell‐cycle regulation and Jagged‐1 in Notch signalling. A significant number of up‐regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down‐regulation. Our data show that ectopic MSI1 expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation.     

Involvement of the PLCε/PKCα pathway in human BIU-87 bladder cancer cell proliferation

PLCε (phospholipase Cε), one of effectors belonging to the small GTPase superfamily, has been suggested to play a crucial role in carcinogenesis. However, its bio-function in bladder cancer has never been demonstrated. In our previous study, we found that PLCε mRNA was highly expressed in bladder cancer tissues. In the present study, we silenced the PLCε gene by shRNA (small-hairpin RNA) in the bladder cancer cell line BIU-87. The results showed that it significantly inhibited cell proliferation and arrested the cell cycle at G0/G1-phase. The regulation of cell characteristics has been related to PKCα (protein kinase Cα) activity. Further study showed that knockdown of the PLCε gene down-regulated oncogenes c-fos and c-jun. These results indicate that PLCε plays a crucial role in bladder cancer, and PLCε may be a key molecule regulating the signal pathway of bladder cancer proliferation.     

Ras mutation cooperates with ��-catenin activation to drive bladder tumourigenesis

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras^Q61L or K-Ras^G12D) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.     

神经节苷脂联合α-硫辛酸治疗糖尿病神经源性膀胱临床观察

目的:观察α-硫辛酸、神经节苷脂联合治疗糖尿病神经源性膀胱的临床疗效。方法:60例2型糖尿病神经源性膀胱患者,随机分为α-硫辛酸治疗对照组、神经节苷脂治疗对照组,联合治疗组,疗程30d,观察症状体征及治疗前后B超膀胱残余尿量症状的变化,比较不同方案的疗效。结果:α-硫辛酸治疗对照组有效率90%,神经节苷脂治疗对照组有效率90%,联合治疗组有效率95%两组间无统计学差异(P>0.05),但3组的治愈率分别为90.0%、50.0%、60.0%,治疗组的治愈率高于两个对照组,有统计学差异(P<0.05)。结论:α-硫辛酸和神经节苷脂联合治疗糖尿病神经源性膀胱有较好的临床疗效,值得临床推广。     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)