Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Real-time impedance analysis of host cell response to meningococcal infection

Measuring cell proliferation and cell death during bacterial infection involves performing end-point assays that represent the response at a single time point. A new technology from Roche Applied Science and ACEA Biosciences allows continuous monitoring of cells in real-time using specialized cell culture microplates containing micro-electrodes. The xCELLigence system enables continuous measurement and quantification of cell adhesion, proliferation, spreading, cell death and detachment, thus creating a picture of cell function during bacterial infection. Furthermore, lag and log phases can be determined to estimate optimal times to infect cells.In this study we used this system to provide valuable insights into cell function in response to several virulence factors of the meningitis causing pathogen Neisseria meningitidis, including the lipopolysaccharide (LPS), the polysaccharide capsule and the outer membrane protein Opc. We observed that prolonged time of infection with pathogenic Neisseria strains led to morphological changes including cell rounding and loss of cell-cell contact, thus resulting in changed electrical impedance as monitored in real-time. Furthermore, cell function in response to 14 strains of apathogenic Neisseria spp. (N. lactamica and N. mucosa) was analyzed. In contrast, infection with apathogenic N. lactamica isolates did not change electrical impedance monitored for 48 h. Together our data show that this system can be used as a rapid monitoring tool for cellular function in response to bacterial infection and combines high data acquisition rates with ease of handling.     

Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion,proliferation, migration and invasion of colon cancer cells

Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.     

Apoptosis of cholangiocytes modulated by thioredoxin of carcinogenic liver fluke

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini, frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis.     

Dynamic assessment of cell viability,proliferation and migration using real time cell analyzer system (RTCA)

Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.