Transforming growth factor­ß/miR­143­3p/cystatin B axis is a therapeutic target in human ovarian cancer.

We previously reported that cystatin B (CSTB) is a progression marker of human ovarian cancer (OC); however, the regulatory mechanism of CSTB and its function in OC remain unclear. The present study aimed to explore the mechanism underlying transforming growth factor-ß (TGF­ß) 1­mediated CSTB regulation, and to examine the function of CSTB on OC cell proliferation and apoptosis. Using the online program, miRWalk, a microRNA (miR)­143­3p was detected, which contains a homologous sequence of the potential binding site to the 3'­untranslated region (3'­UTR) of CSTB. A dual­luciferase reporter assay confirmed the interaction between miR­143­3p and CSTB 3'­UTR. Treating OC cells with miR­143­3p mimics or inhibitors resulted in a decrease or an increase of CSTB expression at mRNA and protein levels, respectively. Additionally, CSTB was significantly overexpressed, whereas miR­143­3p was downregulated in human OC tissues compared with normal ovarian tissues. A negative correlation between miR­143­3p and CSTB mRNA expression was observed in ovarian malignant tumors. The levels of primary and mature miR­143­3p expression were upregulated in OC cells after TGF­ß1 treatment; the action of TGF­ß1 was abolished in the presence of an inhibitor of TGF­ß type I receptor. These results indicated an axis between TGF­ß, miR­143­3p and CSTB in OC cells. Furthermore, high levels of CSTB expression were associated with the poor overall survival of patients with OC. Knockdown of CSTB resulted in a decrease in OC cell proliferation and arrested cells in G2/M phase. In addition, suppression of CSTB induced cell apoptosis. In conclusion, CSTB was overexpressed and miR­143­3p was downregulated in ovarian malignant tumors. Mature miR­143­3p directly bound CSTB 3'­UTR, leading to a decrease in CSTB expression in OC cells, which was regulated by TGF­ß1. Our findings suggest the potential therapeutic application of targeting the TGF­ß/miR­143­3p/CSTB axis for treating patients with OC.

Proteomics analysis of melanoma metastases: association between S100A13 expression and chemotherapy resistance.

BACKGROUND: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. METHODS: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples (n=14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. RESULTS: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry (n=48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. CONCLUSIONS: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ.

Cystatin B is a progression marker of human epithelial ovarian tumors mediated by the TGF-ß signaling pathway.

Advanced ovarian cancer is a devastating disease. Gaining biomarkers of early detection during ovarian tumorigenesis may lead to earlier diagnosis and better therapeutic strategies. Cystatin B (CSTB) functions as an inhibitor to suppress intracellular cysteine proteases and has been implicated in several types of cancers. The present study explored the expression of CSTB in human ovarian tumors, to investigate CSTB expression associated with clinicopathological features, and to examine the effect of transforming growth factor-ß (TGF-ß), which plays a key role in ovarian tumorigenesis, on CSTB expression in ovarian cancer cells. The ovarian tissue samples from 33 patients were retrieved. The expression of CSTB in ovarian tissue was examined by immunohistochemistry. We found that CSTB was over-expressed in human ovarian surface epithelial tumors, including serous, mucinous and clear cell tumors. The immunoreactive staining of CSTB was strong in borderline and malignant tumors, weak in benign tumors, and negative in normal tissue counterparts, but was not correlated with the clinicopathological features of patients with ovarian tumors, such as age, histological types, tumor size, lymph node metastasis and clinical stages. The CSTB at mRNA and protein levels in two types of epithelial ovarian cancer cells, OVCAR-3 and SK-OV-3, was decreased after TGF-ß1 treatment detected by quantitative PCR and western blot analysis, respectively. The inhibitory effect of TGF-ß1 on CSTB expression was abolished in the presence of SB-431542, a TGF-ß type I receptor kinase inhibitor. Our data suggest that CSTB is tumor tissue-specific and overexpressed in ovarian borderline and malignant tumors. The increased CSTB expression in ovarian tissue represents tumor progression and is dysregulated by the TGF-ß signaling pathway. CSTB may become a novel diagnostic intracellular biomarker for the early detection of ovarian cancer.

Cathepsins and their endogenous inhibitors cystatins: expression and modulation in multiple sclerosis.

Cathepsins are involved in a variety of physiological processes including antigen processing and presentation and extracellular matrix degradation. In the present study, we evaluated whether expression levels of cathepsins S and B and their inhibitors cystatins B and C are affected by multiple sclerosis (MS) disease state (relapse and remission) and therapies (interferon-ß [IFN-ß] and the glucocorticoid [GC] methylprednisolone), and whether they are associated with the IFN-ß response phenotype. Real-time PCR was employed to compare RNA expression levels in peripheral blood leucocytes (PBLs) and ELISA to determine serum protein levels of MS patients and matched healthy individuals. Cathepsin S RNA was higher in MS patients in the relapse state compared to controls (by 74%, P = 3 × 10(-5), n = 30 versus n = 18) with a similar increase observed in serum (66%, P = 0.002, n = 18 versus n = 20). GC treatment reduced cathepsin S levels in PBL RNA (by 44%, P = 6 × 10(-6), n = 27) and serum proteins (by 27%, P = 1 × 10(-5), n = 26), reduced the serum protein levels of pro-cathepsin B (by 8%, P = 0.0007, n = 23), and in parallel increased the serum levels of their inhibitor cystatin C (by 82%, P = 8 × 10(-6), n = 26). IFN-ß therapy significantly elevated the RNA levels (n = 16) of cathepsin B (by 16%, P = 0.03), cystatin B (44%, P = 0.004) and cystatin C (48%, P = 0.011). In the serum, only cathepsin S levels were reduced by IFN-ß (16%, P = 0.006, n = 25). Interestingly, pre-treatment serum cathepsin S/cystatin C ratio was higher in 'good responders' to IFN-ß therapy compared to patients without a good response (by 94%, P = 0.003). These results suggest that cathepsin S and cystatin C may contribute to disease activity in MS, specifically in a subgroup of patients that are responsive to IFN-ß therapy, and that these proteins should be further evaluated as biomarkers in MS.

Development of a fluorescent microsphere immunoassay for cystatin B (CSTB) in serum of patients with hepatocellular carcinoma.

BACKGROUND: Serum cystatin B (CSTB) concentrations have been reported to be increased in patients with hepatocellular carcinoma compared to concentrations seen in normal subjects. In this study, we developed a "fluorescent microsphere immunoassay" (FMI) capable of specifically detecting CSTB in serum. METHODS: The FMI used a microparticle conjugated polyclonal antibody to CSTB and biotinylated monoclonal antibody as capture protein and probe protein, respectively. The results were obtained using the Bio-Plex(200) system. RESULTS: The dose-response relationship between CSTB and fluorescent intensity showed linearity in the range 0-1000 pg/mL and 7 pg/mL, sensitivity lower than 11.2 pg/mL. This result revealed that the FMI system was more sensitive than enzyme-linked immunoassay (ELISA). Additionally, the FMI system used smaller sample volumes compared to ELISA. CONCLUSIONS: We measured CSTB with both the FMI and an ELISA procedure and compared the two methods. The CSTB concentrations in serum specimens as measured with the FMI assay system were similar to those measured with ELISA. Thus, the new FMI using the Bio-Plex system may be useful for detection of CSTB in human serum.