The role of endothelial cell on cancer cell metastasis

Chemotaxis是癌症细胞感染和Metastasis过程中的关键步骤。我们研究了非肌肉肌红蛋白光链激酶（nm-MLCK）在癌症细胞化学轴中的作用，朝着使用A549细胞的方向发展的方向发展，一个来自肺腺癌瘤的细胞模型。我们的分析用Western blot描述纳米-MLCK的表征;测试Boyden室中的HGF化学轴正在向用8 μm pore聚碳酸酯膜检测8 μm pore聚碳酸酯膜;用抗肌蛋白II抗免疫细胞检测肌红蛋白II膜;和测试肌红蛋白光链（MLC）用肌红蛋白II免疫受体磷酸化，后续用抗磷酸盐和抗磷酸盐酮抗体进行免疫抑制沉淀。To assess the dependency on intracellular Ca D12+ Ca D1 and MLCK activity， experiments were performed in presence and absence of the Ca D12+ Ca D1 chelator， BAPTA， or the specific MLCK antagonist，ML-7。A549细胞已发现可快速提取214 kDa nm-MLCK，在HGF、磷酸化MLC两种丝氨酸和三氟醚的存在中。HGF也是常见的肌红蛋白II膜形成和化学酶，目前30纳克/毫升HGF的最大值为24小时。所有以前的反应都被BAPTA和ML-7所抑制。因此，它出现在Ca-D12 +-D1-依赖的nm-MLCK活动调节HGF诱导的A549细胞化学轴。

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.