Epithelial to Mesenchymal Transition in Human Mesothelial Cells Exposed to Asbestos Fibers: Role of TGF-ß as Mediator of Malignant Mesothelioma Development or Metastasis via EMT Event.

Asbestos exposure increases the risk of asbestosis and malignant mesothelioma (MM). Both fibrosis and cancer have been correlated with the Epithelial to Mesenchymal Transition (EMT)-an event involved in fibrotic development and cancer progression. During EMT, epithelial cells acquire a mesenchymal phenotype by modulating some proteins. Different factors can induce EMT, but Transforming Growth Factor ß (TGF-ß) plays a crucial role in promoting EMT. In this work, we verified if EMT could be associated with MM development. We explored EMT in human mesothelial cells (MeT-5A) exposed to chrysotile asbestos: we demonstrated that asbestos induces EMT in MeT-5A cells by downregulating epithelial markers E-cadherin, ß-catenin, and occludin, and contemporarily, by upregulating mesenchymal markers fibronectin, α-SMA, and vimentin, thus promoting EMT. In these cells, this mechanism is mediated by increased TGF-ß secretion, which in turn downregulates E-cadherin and increases fibronectin. These events are reverted in the presence of TGF-ß antibody, via a Small Mother Against Decapentaplegic (SMAD)-dependent pathway and its downstream effectors, such as Zinc finger protein SNAI1 (SNAIL-1), Twist-related protein (Twist), and Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), which downregulate the E-cadherin gene. Since SNAIL-1, Twist, and ZEB-1 have been shown to be overexpressed in MM, these genes could be considered possible predictive or diagnostic markers of MM development.

MMP23B expression and protein levels in blood and urine are associated with bladder cancer.

Urothelial bladder cancer (UBC) represents a public health problem because of its high incidence/relapse rates. At present, there are no suitable biomarkers for early diagnosis or relapse/progression prognosis. To improve diagnostic accuracy and overcome the disadvantages of current diagnostic strategies, the detection of UBC biomarkers in easily accessible biofluids, such as urine, represents a promising approach compared with painful biopsies. We investigated the levels of MMP23 genes (microarray and qPCR) and protein (western blot and enzyme-linked immunosorbent assay) in a set of samples (blood, plasma and urine) from patients with UBC and controls as biomarkers for this cancer. MMP23B and its pseudogene MMP23A resulted downregulated in blood cells from UBC compared with controls (66 cases, 70 controls; adjusted P-value = 0.02 and 0.03, respectively). In contrast, MMP23B protein levels in plasma (53 UBC, 49 controls) and urine (59 UBC, 57 controls) increased in cases, being statistically significant in urine. MMP23B dosage observed in urine samples was related to both tumor risk classification and grading. As the lack of correlation between mRNA and protein levels could be due to a posttranscriptional regulation mediated by microRNAs (miRNAs), we investigated the expression of urinary miRNAs targeting MMP23B. Five miRNAs resulted differentially expressed between cases and controls. We reported the first evidence of MMP23B secretion in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC as a potential non-invasive biomarker for this cancer. Further analyses are needed to elucidate the mechanism of regulation of MMP23B expression by miRNAs.

Combination of urinary fibrinogen ß-chain and tyrosine-phosphorylated proteins for the detection of bladder cancer.

AIM: To evaluate the performance of urinary fibrinogen ß-chain (FBC) - either alone or associated with urinary tyrosine-phosphorylated proteins (UPY) - as bladder cancer (BCa) diagnostic biomarker. MATERIALS & METHODS: 164 subjects were tested. RESULTS: Significantly different FBC and UPY levels were found between BCa patients and controls, as well as between low-grade and high-grade cancers. The diagnostic accuracy was 0.84 for FBC and 0.87 for UPY. The combination of FBC and UPY improved the accuracy to 0.91. The addition of clinical variables (age, gender, and smoking habit) to FBC and UPY into a model for BCa prediction significantly improved the accuracy to 0.99. The combination of FBC and UPY adjusted for clinical variables associates with the highest sensitivity and good specificity. CONCLUSION: Urinary FBC and UPY could be used as biomarkers for BCa diagnosis.

Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients.

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000 ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.