表皮生长因子受体酪氨酸激酶抑制剂AG1478对卵巢癌细胞生物活性及纤溶酶原激活物抑制物1的作用机制研究

Study on the mechanism of epidermal growth factor receptor tyrosine kinase inhibitor AG1478 on the biological activity of ovarian cancer cells and plasminogen activator inhibitor type 1

目的探讨AG1478对卵巢癌细胞生物活性及纤溶酶原激活物抑制物1(PAI-1)的作用机制。方法将CaOV-3细胞分为空白组和干预组,空白组为无药物干预的CaOV-3细胞,干预组分别为干预A组(5μmol的AG1478溶液)、干预B组(10μmol的AG1478溶液)、干预C组(15μmol的AG1478溶液)和干预D组(20μmol的AG1478溶液)。分别采用四甲基偶氮唑蓝(MTT)法、流式细胞仪及Transwell小室检测细胞抑制率、凋亡率及侵袭数目,采用蛋白质印迹(Western blot)法和逆转录聚合酶链反应(RT-PCR)分别检测PAI-1、尿激酶型纤溶酶原激活剂受体(uPAR)蛋白和mRNA水平。结果不同时间点干预B组、干预C组和干预D组CaOV-3细胞抑制率均高于干预A组,干预D组CaOV-3细胞抑制率均高于干预B组,差异均有统计学意义(P﹤0.05)。干预组CaOV-3细胞凋亡率均高于空白组,细胞侵袭数目均少于空白组,干预B组、干预C组、干预D组CaOV-3细胞凋亡率均高于干预A组,细胞侵袭数目均少于干预A组,干预D组CaOV-3细胞凋亡率高于干预B组,细胞侵袭数目少于干预B组,差异均有统计学意义(P﹤0.05)。干预组CaOV-3细胞中PAI-1、uPAR蛋白和mRNA表达量均低于空白组,干预B组、干预C组及干预D组CaOV-3细胞中PAI-1、uPAR蛋白和mRNA表达量均低于干预A组,干预D组CaOV-3细胞中PAI-1、uPAR蛋白和mRNA表达量均低于干预B组,差异均有统计学意义(P﹤0.05)。结论AG1478能够有效降低卵巢癌CaOV-3细胞存活率及侵袭性,加快凋亡,作用机制可能与阻碍PAI-1和uPAR信号通路表达有关。

Objective To investigate the mechanism of AG1478 on the biological activity of ovarian cancer cells and plasminogen activator inhibitor type 1(PAI-1).Method The CaOV-3 cells were divided into blank group and intervention group.The blank group was CaOV-3 cells without drug intervention,and the intervention group was divided into intervention group A(5μmol AG1478 solution),intervention group B(10μmol AG1478 solution),intervention C group(15μmol AG1478 solution),and intervention D group(20μmol AG1478 solution).The cell inhibition rate,apoptosis rate,and the number of invasions were detected by the methyl thiazolyl terazolium(MTT)method,flow cytometer,and Transwell chamber assay.Western blot and reverse transcription-polymerase chain reaction(RT-PCR)were used to detect PAI-1,urokinase type plasminogen activator receptor(uPAR)protein and mRNA levels,respectively.Result The inhibitory rate of CaOV-3 cells in intervention group B,intervention group C,and intervention group D were higher than those in intervention group A at different time points,and the inhibitory rate of CaOV-3 cells in intervention group D was higher than that in intervention group B,and the differences were statistically significant(P<0.05).The apoptosis rate of CaOV-3 cells in the intervention groups were higher than those of the blank group,and the number of invasion cells were less than those of the blank group.The apoptosis rate of CaOV-3 cells in intervention group B,intervention group C,and the intervention group D were all higher than that of intervention group A,while the number of invasion cells were less than that of intervention group A,the rate of apoptosis of CaOV-3 cells in intervention group D was higher than that of intervention group B,the number of invasion cells was less than that of intervention group B,the differences were statistically significant(P<0.05).The expression levels of PAI-1,uPAR protein and mRNA in CaOV-3 cells in the intervention group were lower than those in the blank group,the expressions of PAI-1,uPAR protein and mRNA in CaOV-3 cells of intervention group B,intervention C,and intervention group D were lower than those of intervention group A,the expression of PAI-1,uPAR protein and mRNA in CaOV-3 cells of intervention group D was lower than the intervention group B,and the differences were statistically significant(P<0.05).Conclusion AG1478 could effectively reduce the survival rate of ovarian cancer CaOV-3 cells and inhibit their invasiveness while inducing apoptosis,the underlying mechanism may be related to blocking the expression of PAI-1 and relevant uPAR signaling pathways.