The molecular pathway regulating gastric carcinoma (GC) invasiveness and metastasis remains elusive. Here, we detected significant increase in the phosphorylated epidermal growth factor receptor (pEGFR), MMP7, and MMP13 in the resected GC, compared with the adjacent normal tissue, in patients. Moreover, strong positive correlation was detected between pEGFR and MMP7, and between pEGFR and MMP13 in GC. To examine whether a causal link exists, we used two human GC lines, SNU-5 and AGS, to study the cross talk between EGFR signaling activation, and expression of MMP7 and MMP13. We found that EGF-induced EGFR phosphorylation activated both MMP7 and MMP13, and consequently cancer invasiveness. EGF-induced activation of MMP7 and MMP13 can be both inhibited by use of an inhibitor for EGFR. EGF-induced activation of MMP7 can be also significantly inhibited by use of an inhibitor for Akt, but not an inhibitor for ERK1/2, while EGF-induced activation of MMP13 can be significantly inhibited by use of an inhibitor for ERK1/2, but not by an inhibitor for Akt. These data suggest that EGF-induced activation of MMP7 and MMP13 in GC is through phosphatidylinositol 3-kinase (PI3K) and extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway, respectively. Our study thus highlights EGFR signaling regulated MMP7 and MMP13 activation as molecular basis for metastasis of GC, and further demonstrate that different signaling pathway cascades are involved in the downstream signaling transduction.

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-19 (CK-19) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-19 mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-19 in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

No abstract

Hepatic stellate cell (HSC) activation serves a key role in liver fibrosis, and is associated with chronic liver diseases. Bilirubin, a product of heme degradation, has been demonstrated to have antioxidant properties. The present study investigated the effects of physiological concentrations of bilirubin on rat HSC activation. Rat HSCs were isolated and cultured for several generations to induce activation. The activated HSCs were subsequently treated with 0, 1, 10 or 20 mg/l bilirubin and assayed for parameters of cell activation. As the bilirubin concentration increased, HSCs demonstrated reduced production of reactive oxygen species, reduced protein expression levels of α-smooth muscle actin, a decreased mRNA expression ratio of tissue inhibitor of matrix metalloproteinase-1/matrix metalloproteinase-2, decreased proliferation and increased apoptosis. In conclusion, elevated bilirubin levels, within its physiological concentration range, appeared to inhibit HSC activation. These findings suggested a potential role for bilirubin in the treatment of fibrosis that requires further investigation.