整合素αvβ6通过细胞外调节蛋白激酶介导胃癌细胞对5-氟尿嘧啶的耐受作用

Tolerance of gastric cancer cells to 5-fluorouracil mediated by integrin αvβ6 through extracellular signal-regulated protein kinase

目的 探讨整合素αvβ6通过细胞外调节蛋白激酶介导胃癌细胞对5-氟尿嘧啶的耐受作用.方法 胃癌AGS细胞培养至对数生长期后分4组处理.（1）对照组：向细胞悬液加入含5-氟尿嘧啶的培养基培养24 h.（2）10D5组：向细胞悬液内加入0.1 g/L鼠抗人αvβ6单克隆抗体10D5,培养6 h后换液加入含5-氟尿嘧啶的培养基培养24 h.（3）IgG2a组：加入10D5对照剂IgG2a后用5-氟尿嘧啶处理,余同10D5组.（4）PD98059组：向细胞悬液内加入含5-氟尿嘧啶和20 μmol/L细胞外调节蛋白激酶抑制剂PD98059的培养基培养24 h.MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western blot检测Bcl-2和caspase-3蛋白的表达情况.采用单因素方差分析和LSD检验.结果 对照组、10D5组、IgG2a组和PD98059组的抑制率分别为28.1%±2.7%、84.5%±1.6%、31.4%±5.2%和86.7%±5.2%,组间比较差异有统计学意义（F=342.5,P〈0.05）;凋亡率分别为6.6%±1.4%、30.6%±2.4%、8.1%±1.3%和36.0%±4.0%,组间比较差异有统计学意义（F=105.4,P〈0.05）.caspase-3和Bcl-2的表达水平组间比较,差异有统计学意义（F=292.1,181.6,P〈0.05）.结论 整合素αvβ6可通过细胞外调节蛋白激酶介导胃癌细胞对5-氟尿嘧啶产生耐受.

Objective To explore the role of integrin αvβ6 in mediating the tolerance of gastric cancer AGS cells to 5-fluorouracil, and to determine whether direct β6-extracellular signal-regulated protein kinase（ERK） binding is involved. Methods Gastric cancer AGS cells in the logarithmic phase were incubated with either 5-fluorouracil for 24 hours （ control group）, with 0.1 g/L of mouse anti-αvβ6 monoclonal antibody 10D5 for 6 hours and then with 5-fluorouracil for24 hours （ 10D5 group）, with IgG2a and 5-fluorouracil （ IgG2a group）, or with 5-fluorouracil and 20 μnol/L of ERK inhibitor PD98059 for 24 hours （ PD98059 group）. Cell proliferation,apoptosis and the expression of Bcl-2 and caspase-3 protein were detected by MTT assay, flow cytometry and western blotting, respectively. All data were analysed by one-way analysis of variance and LSD test. Results The cell inhibition rates of the control group, 10D5 group, IgG2a group and PD98059 group were 28.1% ±2.7%,84.5% ± 1.6%, 31.4% ±5.2%, 86.7% ±5.2%, respectively, with a significant difference （ F = 342. 5, P 〈0.05）. The apoptosis rates of the control group, 10D5 group, IgG2a group, and PD98059 group were 6.6% ±1.4%, 30.6% ± 2.4%, 8.1% ± 1.3%, 36.0% ±4.0%, respectively, with a significant difference among the four groups （F = 105.4, P 〈0.05 ）. There was a significant difference in the expression of caspase-3 and Bcl-2 among the four groups （F=292.1, 181.6, P〈0.05）. Conclusion Integrin αvβ6 can mediate the tolerance of gastric cancer AGS cells to 5-fluorouracil through direct β6-ERK binding.