CAJ | 학술논문

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(25μmol/L、50μmol/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24h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,ERK2)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25μmol/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uPAmRNA的表达,但各浓度对uPA RmRNA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ERK2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.
목적:관찰아미락리대인고전이폐암세포PGCL3체외침습능력화뇨격매형섬용매원격활물(urokinase-type plasminogen activator,uPA)계통적영향.방법:불동농도(25μmol/L、50μmol/L화100μmol/L)적아미락리작용우PGCL3세포6h후,용Transwell소실법검측대세포침습능력화운동능력적영향.아미락리작용24h후,용발색저물법검측대세포분비적uPA화섬용매원격활물억제제-1(plasminogen activator inhibitor-1,PAI-1)활성적변화.RT-PCR검측아미락리대세포uPA、뇨격매형섬용매원격활물수체(urokinase-type plasminogen activator receptor,uPAR)화PAI-1 mRNA표체적영향.Western blot검측아미락리대세포uPA、세포외조절단백격매2(extracellular regulated protein kinase 2,ERK2)화ras단백표체정황적영향.결과:침습실험화운동실험결과균현시,경아미락리처리후,천막세포수명현감소,재100μmol/L시,대침습화운동적억제솔분별위(37.7±4.1)%화(64.9±4.9)%,여대조조상비구유현저성차이(P<0.01).동시,세포분비적uPA화PAI-1활성강저,당농도체도100μmol/L시,차이구유통계학의의(P<0.05).종25μmol/L개시.아미락리취가현저억제PAI-1 mRNA적표체,당체100μmol/L시가명현하조uPAmRNA적표체,단각농도대uPA RmRNA적표체균무영향.수착아미락리농도적증가,uPA단백표체량축점감소,단대ras화ERK2단백표체량무명현영향.결론:아미락리능구억제고전이폐암세포PGCL3적침습화운동,기작용궤제여억제uPA화PAI-1적활성화표체유관,유성위항종류전이약물적잠력.
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.