阿米洛利对高转移肺癌细胞侵袭能力和uPA 系统的影响*

目的:观察阿米洛利对人高转移肺癌细胞 PGCL3 体外侵袭能力和尿激酶型纤溶酶原激活物（urokinase-type plasminogen activator，uPA ）系统的影响。方法:不同浓度（25 μmol/L、50 μmol/L 和100μmol/L）的阿米洛利作用于PGCL3 细胞6h 后，用Transwell 小室法检测对细胞侵袭能力和运动能力的影响。阿米洛利作用 24 h 后，用发色底物法检测对细胞分泌的 uPA 和纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor- 1，PAI-1）活性的变化。RT-PCR 检测阿米洛利对细胞uPA 、尿激酶型纤溶酶原激活物受体（urokinase-type plasminogen activator receptor ，uPAR）和PAI-1 mRNA 表达的影响。Western blot 检测阿米洛利对细胞uPA 、细胞外调节蛋白激酶2（extracellular regulated protein kinase 2，ERK 2）和ras蛋白表达情况的影响。结果:侵袭实验和运动实验结果均显示，经
阿米洛利处理后，穿膜细胞数明显减少，在100μmol/L 时，对侵袭和运动的抑制率分别为（37 .7±4.1）%和（64 .9±4.9）%，与对照组相比具有显著性差异（P<0.01 ）。同时，细胞分泌的 uPA 和PAI-1 活性降低，当浓度达到 100μmol/L 时，差异具有统计学意义（P<0.05 ）。从25 μmol/L 开始，阿米洛利就可显著抑制PAI-1 mRNA 的表达，当达 100μmol/L 时可明显下调uPA mRNA 的表达，但各浓度对uPAR mRNA的表达均无影响。随着阿米洛利浓度的增加，uPA 蛋白表达量逐渐减少，但对 ras和ERK 2 蛋白表达量无明显影响。结论:阿米洛利能够抑制高转移肺癌细胞PGCL3 的侵袭和运动，其作用机制与抑制uPA 和PAI-1 的活性和表达有关，有成为抗肿瘤转移药物的潜力。 

Effect of Amiloride on Invasive Activity and uPA System of Lung Carcinoma Cell Line

Objective: To investigate the effect of amiloride on in vitro invasive activity and uPA (urokinase-type plasminogen activator)system of human highly metastatic lung carcinoma cell line PGCL3. Methods: At 6 hours after treatment with amiloride at the concentrations of 25μmol/L，50μmol/L and 100μmol/L for PGCL3 cells，Transwell Chamber assay was performed to detect the effect of amiloride on the invasive and migratory capacity of PGCL3 cells.Effect of amiloride on the activity of uPA and PAI-1(plasminogen activator inhibitor-1)secreted by PGCL3 cells were measured by chromogenic substrate assay after PGCL3 cells were incubated with amiloride for 24 hours.RT-PCR was used to analyze the effect of amilorede on mRNA levels of uPA，uPAR(urokinase-type plasminogen activator receptor)and PAI-1.The expression levels of uPA，ERK2(extracellular regulated protein kinases 2)and ras protein were assessed by Western blot. Results: The number of cells through membrane was significantly decreased in invasion and migration test in vitro.The inhibitory rates of invasion and migration after treatment with amiloride of 100μmol/L were 37.7%±4.1%and 64.9%±4.9%.respectively,with a significant difference from those in the control group(P<0.01).At 24 hours after amiloride treatment，the chromogenic substrate assay showed direct inhibition of the activity of uPA and PAI-1 secreted by PGCL3 cells.No effect on the expression of uPAR in mRNA level was observed,but the expression of PAI-1 in mRNA level was significantly inhibited.Amiloride of 100μmol/L dramatically inhibited the expression of uPA mRNA.The expression level of uPA protein was decreased with the increase of the concentration of amiloride,but no effect was observed on the expression of ERK2 and ras in protein level.Conclusion: Amiloride can inhibit the invasion and migration of PGCL3 cells,through inhibiting the expression and activity of uPA and PAI-1.Amiloride is a potential agent to inhibit cancer invasion and metastasis.