The function of the extracellular matrix (ECM) in the tumor microenvironment is not limited to forming a barrier against tumor invasion. As demonstrated in pathological specimens, cholangiocarcinoma samples exhibit an enrichment of the ECM surrounding the tumor cells. In this study, we examined involvement of the ECM in the regulation of the invasiveness of cholangiocarcinoma cells. The RMCCA1 cholangiocarcinoma cell line was cultured in culture plates either with or without a coating of reconstituted ECM basement membrane preparation (BD Matrigel matrix). In vitro invasion assays were then performed. In addition, the protein expression profile of the cell line was examined using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. The proteins expressed and their functional associations with cancer progression were determined. Culturing the RMCCA1 cell line in the BD Matrigel matrix induced cell invasion. Numerous proteins were induced by culturing the RMCCA1 cells in the matrix gel. The expression of L-plastin, an actin-binding protein, was significantly upregulated. The knockdown of L-plastin expression by siRNA silencing significantly suppressed the cellular response to matrix gel-stimulated cancer cell invasion. The ECM promotes the invasiveness of cholangiocarcinoma cells by upregulating L-plastin. These findings suggest the potential exploitation of this mechanism as a means of inhibiting the invasiveness of cholangiocarcinoma cells.
The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.
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