The intracellular domain of UNC5B facilities proliferation and metastasis of bladder cancer cells

The intracellular domain of UNC5B contains both death domain and caspase-3 cleavage site, and is regarded as a functional domain that mediates apoptosis. However, in our previous studies, we found that the death domain of UNC5B in bladder cancer cells could not be activated to promote apoptosis. In this study, different UNC5B truncates (residue 399-945, residue 412-945) were created to explore whether the caspase-3 cleavage site (site 412), as another potential functional domain of its intracellular portion, could be activated to induce apoptosis in bladder cancer cells. Using mass spectrometry, we acquired a comprehensive and detailed identification of differentially expressed proteins by overexpressing UNC5B and its truncates. Protein-protein-interaction (PPI) network analysis was also applied to investigate the aggregation of related proteins and predict the functional changes. EDU assay, apoptosis, xenograft tumour implantation, migration, invasion and tumour metastasis were performed to comprehensively identify the effects of UNC5B truncates on bladder cancer cells. We demonstrate that the intracellular domain of UNC5B promotes cell proliferation in vitro and tumour formation in vivo, by binding to a large number of ribosomal proteins. The overexpression of intracellular domain also facilitates cells to migrate, invade and metastasize by interacting with fibronectin, beta-catenin and vimentin. In addition, we reveal that overexpressing the intracellular domain of UNC5B cannot bind or activate cleaved caspase-3 to trigger apoptosis in bladder cancer cells.     

LncRNA OIP5-AS1 predicts poor prognosis and regulates cell proliferation and apoptosis in bladder cancer

Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) is a long intergenic noncoding RNA, which has been suggested to be dysregulated in human cancers and served as tumor suppressor or promoter depending on tumor types. However, the role of OIP5-AS1 in bladder cancer was still unknown. In our study, OIP5-AS1 was overexpressed in bladder cancer, and associated with clinical progression and short overall survival. The loss-of-function studies suggested downregulation of OIP5-AS1 expression decreased cell viability, induced cell-cycle arrest and promoted cell apoptosis in bladder cancer. There was a positive association between OIP5-AS1 expression and OIP5 expression in bladder cancer tissues. Moreover, downregulation of OIP5-AS1 expression reduced messenger RNA and protein levels of OIP5 in bladder cancer cell lines. In conclusion, OIP5-AS1 is a useful biomarker for predicting clinical progression and poor prognosis and promotes cell proliferation through modulating OIP5 expression.     

Cell blocks allow reliable evaluation of expression of basal (CK5/6) and luminal (CK8/18) cytokeratins and smooth muscle actin (SMA) in breast carcinoma

W. D. Delgallo, J. R. P. Rodrigues, S. P. Bueno, R. M. Viero and C. T. Soares
Cell blocks allow reliable evaluation of expression of basal (CK5/6) and luminal (CK8/18) cytokeratins and smooth muscle actin (SMA) in breast carcinoma Objective: Gene expression studies have revealed several molecular subtypes of breast carcinoma with distinct clinical and biological behaviours. DNA microarray studies correlated with immunohistochemical profiling of breast carcinomas using cytokeratin (CK) markers, Her2/neu, oestrogen receptor (ER), and basal myoepithelial cell markers have identified five breast tumour subtypes: (i) luminal A (ER+; Her2/neu?), (ii) luminal B (ER+; Her2/neu+), (iii) Her2 overexpression (ER?; Her2/neu+), (iv) basal‐like (ER?; Her2/neu?, CK5/6 and 14+), and (v) negative for all markers. Luminal carcinomas express cytokeratins in a luminal pattern (CK8/18), and the basal‐like type expresses CK5/6 and CK14 or basal epithelial cell markers. CK5/6, CK8/18, and smooth muscle actin (SMA) expression were assessed in cell blocks and compared with expression in surgical specimens. Methods: Sixty‐two cases of breast carcinoma diagnosed by fine needle aspiration cytology with cell blocks and available surgical specimens were included. Cell blocks containing at least 10 high‐power fields each with at least 10 tumour cells and surgical specimens were immunostained for CK5/6, CK8/18 and SMA. Results: Percentage sensitivity, specificity, positive predictive value, negative predictive value and accuracy were, respectively, 77, 100, 100, 92 and 94 for CK5/6; 98, 66, 96, 80 and 95 for CK8/18; and 92, 96, 85, 98 and 95 for SMA. Conclusion: The identification of CK5/6, CK8/18 and SMA by immunohistochemistry in cell blocks can be a reliable method that yields results close to those obtained in surgical specimens, and can contribute to the classification of breast carcinomas with luminal and basal expression patterns, providing helpful information in the choice of treatment and in the evaluation of prognostic and predictive factors.     

NOTCH1 regulates the proliferation and migration of bladder cancer cells by cooperating with long non-coding RNA HCG18 and microRNA-34c-5p

In recent years, the NOTCH signaling pathway has been gradually studied in human malignancies. Inactivation of the NOTCH signaling pathway was uncovered to be correlated with the carcinogenesis of bladder cancer (BCa). Nevertheless, the specific molecular mechanism of NOTCH1 (one of the core factors of the NOTCH signaling pathway) is not well elucidated in BCa. This study focused on the mechanism by which NOTCH1 affects the biological behaviors of BCa cells. According to the experimental results of quantitative real-time polymerase chain reaction, NOTCH1 was dysregulated in BCa tissues and cell lines. The prognostic value of NOTCH1 for the patients with BCa was determined using the Kaplan-Meier method. Mechanism investigations revealed that NOTCH1 is a target of miR-34c-5p in BCa. Furthermore, microarray analysis was used to find the dysregulated long noncoding RNAs (lncRNA), which can bind with miR-34c-5p. Mechanism experiments further demonstrated the rationality of the HCG18-miR-34c-5p-NOTCH1 pathway. Functional assays were then applied to validate the inhibitory influences of NOTCH1 on the proliferation and migration of BCa cells. Furthermore, the inhibitory effects of NOTCH1 could be affected by miR-34c-5p or lncRNA HCG18. All findings in this study revealed that NOTCH1 suppresses the BCa progression by cooperating with lncRNA HCG18 and miR-34c-5p.     

Inhibition of stearoyl CoA desaturase‐1 activity suppresses tumour progression and improves prognosis in human bladder cancer

Urinary bladder neoplasm is one of the most common cancers worldwide. Cancer stem cells (CSCs) have been proven to be an important cause of cancer progression and poor prognosis. In the present study, we established bladder CSCs and identified the crucial differentially expressed genes (DEGs) between these cells and parental bladder cancer cells. Analyses of bioinformatics data and clinical samples from local hospitals showed that stearoyl CoA desaturase‐1 (SCD) was the key factor among the DEGs. A significant correlation between SCD gene expression and poor prognosis among patients with bladder cancer was observed in our data. Loss‐of‐function experiments further revealed that the SCD inhibitor A939572 and SCD gene interference reduced cell proliferation and invasion. The above data suggest that SCD may serve as a novel marker for the prediction of tumour progression and poor prognosis in patients with bladder cancer.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples

OBJECTIVES: Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine. METHODS: Flow cytometry was utilized to detect protoporphyrin IX in an artificial model consisting of normal urinary bladder transitional epithelial cells (NBECs) from healthy volunteers' urine and an established human urinary bladder carcinoma cell line, TCCSUP, after incubation with hexaminolevulinate (HAL). In addition, urine samples from 19 patients with histopathologically confirmed superficial bladder cancer were examined. RESULTS: Incubation of NBECs or TCCSUP cells with HAL for 1 hour resulted in production of protoporphyrin IX only in the TCCSUP cells. Incubation of a mixture of NBECs and TCCSUP cells with HAL gave rise to a separated subpopulation of cells with protoporphyrin IX fluorescence. After cell sorting by flow cytometry the protoporphyrin IX-containing subpopulation of cells was confirmed as TCCSUP cells on cytological examination. It was possible to detect 5% TCCSUP cells in the mixture of NBECs/TCCSUP cells. To test the feasibility of the method in clinica diagnosis, urine samples from patients with bladder cancer were also measured with comparable, although preliminary and limited, results to those of cytological examination. CONCLUSIONS: The preliminary results show that the technique may be feasible for the detection of bladder cancer cells in urine with possible advantages of simplicity, reliability and objectivity.     

ARPC4 promotes bladder cancer cell invasion and is associated with lymph node metastasis

The significance of actin-related protein 2/3 complex subunit 4 (ARPC4) expression in bladder cancer, and its potential role in the invasion and migration of bladder cancer cells, has yet to be determined. This study was to identify the correlation between ARPC4 and lymph node metastasis, and to determine the role of ARPC4 in the invasive migration of T24 bladder cancer cells. One hundred and ninety-eight bladder cancer tissues and 40 normal bladder and lymph node tissues were examined. Tissue microarrays were constructed and subjected to immunohistochemical stating for ARPC4. Multiple logistic analysis was used to determine risk factors associated with bladder cancer metastasis. ARPC4 expression in T24 bladder cancer cells was suppressed using small interfering RNA and changes in protein levels were determined by Western blot analysis. The proliferation of bladder cancer cells after knocking down of ARPC4 was determined by cell counting kit-8. The effects of ARPC4 knockdown on T24 cell invasion and migration was determined using transwell and wound healing assays. Immunofluorescence analysis was performed to examine changes in pseudopodia formation and actin cytoskeleton structure. The expression of ARPC4 was elevated in bladder cancer tissues than normal tissues (84.3% vs 27.5%, P < 0.001). The multivariate logistic analysis demonstrated that the level of ARPC4, as a risk factor, was correlated with lymphatic metastasis (P < 0.05). ARPC4 knockdown attenuated proliferation, migration, invasion, and pseudopodia formation in T24 cells. ARPC4 expression, as a risk factor, is associated with lymphatic metastasis and is upregulated in bladder cancer tissues in comparison with normal tissues. Inhibition of ARPC4 expression significantly attenuates the proliferation, migration, and invasion of bladder cancer cell, possibly due to defects in pseudopodia formation.     

MiR-99a-5p inhibits bladder cancer cell proliferation by directly targeting mammalian target of rapamycin and predicts patient survival

Bladder cancer is a common malignancy and miR-99a-5p has been reported to be downregulated in bladder cancer, but its function and the underlying mechanism in bladder cancer development remains largely unclear. Here, we report that miR-99a-5p expression was decreased in bladder cancer compared with the adjacent normal tissues. Receiver operating characteristic curve revealed that miR-99a-5p expression signature had area under curve value of 0.7989 in differing bladder cancer from the adjacent normal tissues. Bladder cancer patients with low expression of miR-99a-5p had a poor survival rate. Gain-of-function and loss-of-function approaches demonstrated that miR-99a-5p inhibited bladder cell proliferation and cell cycle. Furthermore, we identified that mammalian target of rapamycin (mTOR) was a direct target of miR-99a-5p and mTOR restore could rescue the proliferative ability of bladder cancer cells. Moreover, miR-99a-5p/mTOR axis regulated S6K1 phosphorylation. These suggested that miR-99a-5p/mTOR axis might be a therapeutic target for bladder cancer.     

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3⁺CD8⁺ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.     

应用多重RT—PCR技术检测食管癌患者外周血多种肿瘤基因标志物的研究

目的:建立多重RT-PCR同步检测技术,联合检测黑色素瘤抗原MAGEA3基因、人类斯钙素hsTC1基因、上皮细胞角质蛋白CK20、CK19基因及癌胚抗原CEA基因mRNA在食管癌患者外周血中的表达。方法:在分析MAGEA3、hSTC1、CK20、CK19、CEA和β-actin六种基因mRNA全长序列资料的基础上,自行设计并筛选出6对引物及6条寡核苷酸探针,利用多重RT-PCR技术并结合基因芯片对样本进行检测。结果:全部样品均可检出β-actin,55例食管癌患者外周静脉血中MAGEA3、hSTC1、CK20、CK19和CEA的阳性表达率分别为25.45%、47.27%、49.09%、34.55%和52.73%;多重RT-PCR技术同时检测MAGEA3、hSTC1、CK20、CK19和CEA基因,如果至少表达其中一种即可作为阳性表达者,其阳性表达率可达72.73%。而10例食管癌组织中均为阳性,28例正常健康捐献者的外周血中有2例CEA表达阳性,27例非肿瘤病人的外周血中有3例CK20阳性,3例CK19阳性,3例CEA阳性。测序结果证实RT-PCR产物确为MAGEA3、hSTC1、CK20、CK19和CEA基因。结论:联合检测MAGEA3、hSTC1、CK20、CK19和CEA可以增加外周血循环癌细胞检测的敏感性,本研究建立的多重RT-PCR技术可以做为一种快速、灵敏的检测手段。