The role of endothelial cell on cancer cell metastasis

Chemotaxis is a key step in the process of cancer cell invasion and metastasis. We studied the rolenonmuscle myosin light chain kinase (nm-MLCK) in cancer cell chemotaxis toward hepatocyte growthfactor (HGF) using A549 cellsa model cell line derived from lung adenocarcinoma. Our analysis entailed characterizing表达式of nm-MLCK using Western blot；examining chemotaxis toward HGF in Boyden chambers equipped with 8 μm pore polycarbonate membranes；​myosin II filament formation by immunolabeling cells with anti-myosin II antiserum；myosin light chain (MLC) phosphorylation by myosin II immunoprecipitationfollowed by immunoblotting with anti-phosphoserine and anti-phosphotheonine antibodies. To assessthe dependency on intracellular Ca D12+ D12 and MLCK activityexperiments were performed in presence and absence of the Ca D12+ D1 chelator， BAPTA，或特定的MLCK antagonist， ML-7. A549 cells were found to express a 214kDa nm-MLCK，which in the presence of HGF，phosphorylated MLC on both serine and threonine residues. HGF also elicited myosin II filamentformation and chemotaxis which was maximal at 24h in the presence of 30ng /ml HGF. All of theaforementioned reactions were inhibited by both BAPTA and ML-7. Thus，it appears that Ca D12+ D1-dependent nm-MLCK activity regulates HGF-induced A549 cell chemotaxis。

Chemotaxis is a key step in the process of cancer cell invasion and metastasis. We studied the role of nonmuscle myosin light chain kinase (nm-MLCK) in cancer cell chemotaxis toward hepatocyte growth factor (HGF) using A549 cells, a model cell line derived from lung adenocarcinoma. Our analysis entailed characterizing expression of nm-MLCK using Western blot ; examining chemotaxis toward HGF in Boyden chambers equipped with 8 μm pore polycarbonate membranes ; examining myosin II filament formation by immunolabeling cells with anti-myosin II antiserum ; and examining myosin light chain (MLC) phosphorylation by myosin II immunoprecipitation, followed by immunoblotting with anti-phosphoserine and anti-phosphotheonine antibodies. To assess the dependency on intracellular CaィイD12+ィエD1 and MLCK activity, experiments were performed in presence and absence of the CaィイD12+ィエD1 chelator, BAPTA, or the specific MLCK antagonist, ML-7. A549 cells were found to express a 214kDa nm-MLCK, which in the presence of HGF, phosphorylated MLC on both serine and threonine residues. HGF also elicited myosin II filament formation and chemotaxis which was maximal at 24 h in the presence of 30 ng/ml HGF. All of the aforementioned reactions were inhibited by both BAPTA and ML-7. Thus, it appears that CaィイD12+ィエD1-dependent nm-MLCK activity regulates HGF-induced A549 cell chemotaxis.