纤连蛋白促进胃癌细胞系AGS中桩蛋白的酪氨酸磷酸化及细胞侵袭。

Fibronectin promotes tyrosine phosphorylation of paxillin and cell invasiveness in the gastric cancer cell line AGS.

作者信息

Li Dan, Ding Jian, Wang Xiaozhong, Wang Chengdang, Wu Ting

机构信息

Digestive Department, the First Affiliated Hospital of Fujian Medical University, Fujian, China.

出版信息

Tumori. 2009 Nov-Dec;95(6):769-79. doi: 10.1177/030089160909500621.

DOI:10.1177/030089160909500621

PMID:20210243

Abstract

AIMS AND BACKGROUND

Paxillin is a central protein within the focal adhesion and serves as a critical transducer of signals from fibronectin. Although abnormal expression of fibronectin and paxillin is often observed during the development of human malignancies, the relationship between paxillin and cell invasion in gastric cancer is still unclear. The current study was designed to investigate the potential role and mechanisms of fibronectin in tyrosine phosphorylation of paxillin and in the invasiveness of gastric cancer cells.

METHODS

Expression of paxillin in human gastric cancer samples was examined by immunohistochemical staining. A gastric cancer cell line, AGS, was stimulated by fibronectin with gradient concentrations, and expression of paxillin and phosphorylation of paxillin tyrosine 118 (tyr118) was detected by immunoprecipitation and Western blotting. The invasiveness of AGS cells was measured by the modified Boyden chamber assay. Small interfering RNA (siRNA) targeting paxillin was used to establish the role of paxillin (tyr118) in the process of cell invasion enhanced by fibronectin. siRNA targeting focal adhesion kinase (FAK) was used to verify the effect of FAK tyrosine 397 (tyr397) on phosphorylation of paxillin (tyr118).

RESULTS

Positivity for paxillin staining in human gastric cancer was associated with tumor stage. AGS cell showed dose dependence on fibronectin for invasiveness and phosphorylation of paxillin (tyr118). Invasiveness and phosphorylation of paxillin (tyr118) in AGS cells reached their peak when the concentration of fibronectin reached 100 nmol/L. siRNA targeting paxillin decreased the phosphorylation of paxillin (tyr118) and the invasiveness of AGS cells significantly as compared with controls. Blockage of FAK (tyr397) can inhibit phosphorylation of paxillin (tyr118) stimulated by fibronectin.

CONCLUSIONS

Fibronectin promotes paxillin (tyr118) phosphorylation and invasiveness of AGS cells. Paxillin silencing by RNA interference inhibits the cell invasiveness stimulated by fibronectin. Paxillin is a key factor in the fibronectin-stimulated invasiveness of AGS cells.

摘要

目的与背景

桩蛋白是粘着斑中的一种核心蛋白，是纤连蛋白信号的关键转导分子。虽然在人类恶性肿瘤发生过程中常观察到纤连蛋白和桩蛋白的异常表达，但桩蛋白与胃癌细胞侵袭之间的关系仍不清楚。本研究旨在探讨纤连蛋白在桩蛋白酪氨酸磷酸化及胃癌细胞侵袭中的潜在作用和机制。

方法

采用免疫组织化学染色法检测人胃癌组织中桩蛋白的表达。用不同浓度的纤连蛋白刺激胃癌细胞系AGS，通过免疫沉淀和蛋白质印迹法检测桩蛋白的表达及桩蛋白酪氨酸118（tyr118）的磷酸化水平。采用改良的Boyden小室法检测AGS细胞的侵袭能力。用靶向桩蛋白的小干扰RNA（siRNA）确定桩蛋白（tyr118）在纤连蛋白增强细胞侵袭过程中的作用。用靶向粘着斑激酶（FAK）的siRNA验证FAK酪氨酸397（tyr397）对桩蛋白（tyr118）磷酸化的影响。

结果

人胃癌组织中桩蛋白染色阳性与肿瘤分期有关。AGS细胞对纤连蛋白的侵袭和桩蛋白（tyr118）磷酸化呈剂量依赖性。当纤连蛋白浓度达到100 nmol/L时，AGS细胞的侵袭和桩蛋白（tyr118）磷酸化达到峰值。与对照组相比，靶向桩蛋白的siRNA显著降低了桩蛋白（tyr118）的磷酸化水平和AGS细胞的侵袭能力。阻断FAK（tyr397）可抑制纤连蛋白刺激的桩蛋白（tyr118）磷酸化。