Src激酶和p38激酶在乙酰肝素酶促进人胃癌细胞迁移和侵袭中的作用及相互关系

目的 探讨Src激酶和p38激酶在乙酰肝素酶促进人胃癌细胞迁移和侵袭中的作用及相互关系.方法 实验设正常对照组(细胞未作其他处理)、人乙酰肝素酶重组蛋白处理组(5、10 μg/ml)以及提前3 h加入Src激酶特异性抑制剂pp2(5 μmol/L)或p38激酶特异性抑制剂SB 203580(1 μmol/L)再加入人乙酰肝素酶重组蛋白(10 μg/ml)作用24 h组.利用划痕损伤实验和Transwell小室基质侵袭实验分别检测各处理组对人胃癌MGC-803细胞迁移和侵袭能力的影响;Western blot法检测各处理组对人胃癌MGC-803细胞、高表达乙酰肝素酶的人胃癌SGC-7901细胞和乙酰肝素酶表达被下调的SGC-7901-Heparanasel(-)细胞中Src激酶、p38激酶、磷酸化Src激酶(p-Src)和磷酸化p38激酶(p-p38)蛋白表达的影响.结果 与正常对照组比较,5和10 μg/ml人乙酰肝素酶重组蛋白均能明显增强人胃癌MGC-803细胞迁移和侵袭能力(P<0.05);与未加抑制剂的人乙酰肝素酶重组蛋白比较,抑制剂pp2和SB 203580均能削弱人乙酰肝素酶重组蛋白增强的人胃癌MGC-803细胞的迁移和基质侵袭能力(P<0.05).10 μg/ml人乙酰肝素酶重组蛋白促进人胃癌MGC-803细胞Src激酶和p38激酶的磷酸化,而SGC-7901-Heparanasel(-)细胞中磷酸化的Src激酶和p38激酶表达水平较SGC-7901细胞明显下调(P<0.01);抑制剂pp2和SB 203580对人胃癌SGC-7901细胞中乙酰肝素酶蛋白表达无明显影响;在人胃癌SGC-7901细胞和MGC-803细胞中,Src激酶的抑制剂pp2抑制磷酸化p38蛋白的表达,而p38激酶的抑制剂SB 203580对磷酸化Src蛋白表达无明显影响.结论 乙酰肝素酶可能通过促进Src激酶磷酸化或p38激酶的磷酸化或先促进Src激酶磷酸化再促进p38激酶的磷酸化,从而促进人胃癌细胞迁移和侵袭能力.乙酰肝素酶-Src激酶磷酸化-p38激酶磷酸化可能是人胃癌细胞内存在的与细胞迁移和侵袭有关的信号转导通路.

Role and relationship of Src kinase and p38 kinase in heparanase promoting the migration and invasion of human gastric cancer cells

Objective To study the role and relationship of Src kinase and p38 kinase in heparanase promoting migration and invasion of human gastric cancer cells.Methods Rats were divided into 3 groups.The first group was normal control group (no other treatment to the cells);the second group was treated with human recombinant heparanase protein (5 μg/ml and 10 μg/ml), and the third group was added into Src kinase specific inhibitor pp2 (5 mol/L) or p38 kinase specific inhibitor SB 203580 (1 mol/L) 3 hours in advance, and then was treated by human recombinant heparanase protein (10 μg/ml) for 24 hours.Scratch wound assay and Transwell chamber stromal invasion assay were used to detect the effects of each treatment group on the migration and invasion ability of human gastric cancer MGC-803 cell.Western blot was used to detect the effects of each treatment group on the expression of Src kinase, p38 kinase,phosphorylation of Src kinase (p-Src) and phosphorylation of p38 kinase (p-p38) in human gastric cancer cell line MGC-803 cells, SGC-7901 cells and the SGC-7901-Heparanasel (-) in which the expression of heparanase was down regulated.Results Compared with the normal control group, both 5 and 10 μg/ml recombinant heparanase protein could significantly enhance the migration and invasion ability of human gastric cancer MGC-803 cell (P<0.05);compared with human recombinant heparanase protein group without inhibitors, both Src kinase specific inhibitor pp2 and p38 kinase specific inhibitor SB 203580 could weaken the migration and invasion ability of the MGC-803 cell, which was enhanced by recombinant human heparanase protein (P<0.05).Human recombinant heparanase protein (10 μg/ml) could promote the phosphorylation of Src kinase and p38 kinase in human gastric cancer MGC-803 cells, and the expression level of phosphorylation of Src kinase and p38 kinase in SGC-7901-Heparanasel (-) cells was lower than that in SGC-7901 cells (P<0.01).Src kinase specific inhibitor pp2 and p38 kinase specific inhibitor SB 203580 had no obvious effect on the expression of heparanase protein in human gastric cancer SGC-7901 cells.In human gastric cancer SGC-7901 cells and MGC-803 cells, Src kinase inhibitor pp2 inhibited the expression of p-p38 protein, while p38 kinase inhibitor SB 203580 had no significant effect on the expression of p-Src protein.Conclusion Heparanase, probably through promoting the phosphorylation of Src kinase or p38 kinase, or probably through first promoting the phosphorylation of Src kinase and then the phosphorylation p38 kinase, promotes the migration and invasion ability of human gastric cancer cell.Heparanase-Src kinase phosphorylation-p38 kinase phosphorylation may be the signal transduction pathway related to cell migration and invasion in human gastric cancer cells.