Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-ß1 synthesis in indirect co-cultures with normal colon fibroblasts.

PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.

Regulation of proteolytic activity induced by inflammatory stimuli in lung epithelial cells.

A large number of chronic lung diseases such as asthma bronchiale are associated with alveolar and/or bronchial inflammation accompanied by a damage of the alveolocapillary barrier. In this process proteolytic mechanisms may play a crucial role. The aim of the present study was to assess the role of TNF-alpha on the proteolytic activity of pulmonary epithelial cells and to find possible intracellular signaling pathways which may mediate the effect of TNF-alpha. For our studies we have used the A549 human lung epithelial cell line. Plasminogen activator and metalloproteinase activity was measured using zymography. TNF-alpha induced a time and concentration dependent activation of the urokinase type plasminogen activator (u-PA) and tissue type plasminogen activator (t-PA) activity in A549 cells. This effect could be blocked completely by dexamethasone and was reduced significantly by the Rho-kinase inhibitor Y27632. Similarly, an increased activity in the culture medium of the 72 kDa MMP-2 in response to TNF-alpha could be observed as well. This could be reduced by dexamethasone and Y27632. Our results show that TNF-alpha is at least partly responsible for an increased proteolytic activity and beside corticosteroids Rho-kinase may constitute a potential target for future therapeutical approaches.

Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-B1 synthesis in indirect co-cultures with normal colon fibroblasts

Purpose Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-β1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). Methods We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-β1. Results The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-β1, while the NEP downregulation decreased the synthesis of TGF-β1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-β1 production. Conclusions The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-β1 synthesis during paracrine interactions with NCFs (AU)