血管紧张素抑制剂缬沙坦对膀胱癌细胞表达的影响

Influence of Angiotensin Inhibiter Valsartan on the Expression of Bladder Cancer Cells

目的探讨缬沙坦对不同侵袭能力的膀胱癌细胞株血管紧张素Ⅱ(AngⅡ)1型受体(AT1R)抗原、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)表达的影响。方法对三株不同侵袭能力的细胞株EJM3、EJ、BIU-87进行培养,使用10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9mol/L缬沙坦的1640培养液作用于细胞株,采用细胞生长状态测定法检测细胞增殖状况,确定最终药物浓度。根据细胞株不同分为EJ-M3组、EJ组、BIU-87组,每组细胞分为实验亚组和对照亚组,实验亚组加入含有10-3mol/L缬沙坦的RPMI-1640培养液,对照亚组仅加入无血清RPMI-1640培养液,培养48 h后抽提相应蛋白和mRNA。采用Western blotting法测定AT1R、MMP-2和MMP-9蛋白表达水平,采用RT-PCR法检测AT1R、MMP-2和MMP-9基因的相对表达。采用细胞划痕实验、Transwell体外细胞侵袭实验检测3种膀胱癌细胞株的迁移能力和侵袭能力的改变。结果依据MTT实验测定出的生长曲线选择浓度为10-3mol/L的缬沙坦作为最终药物浓度。Western blotting电泳结果显示,AT1R、MMP-2、MMP-9在3个细胞株中均有表达,但均较对照亚组表达明显降低。3组细胞株对照亚组AT1R、MMP-2、MMP-9 mRNA相对表达均高于实验亚组(P<0.05)。3组细胞株实验亚组细胞迁移距离短于对照亚组(t=7.24、6.14、4.30,P<0.01)。3组细胞株实验亚组侵入细胞数量较对照亚组少(t=6.24、4.33、2.81,P<0.01)。结论缬沙坦可有效抑制膀胱癌细胞AT1R表达,进而抑制MMP-2和MMP-9表达。AT1R抑制剂有可能成为一种新的抑制膀胱癌转移和延长膀胱癌患者生存期的药物。

Objective To study the effect of Valsartan on angiotensin Ⅱ inhibitors（ AngⅡ） type 1 receptor（ AT1R）antigen,matrix metalloproteinase-2（ MMP-2）, matrix metalloproteinase-9（ MMP-9） in bladder cancer（ BC） cell lines of different invasive abilities. Methods Three cell strains（ EJ-M3,EJ,BIU-87） of different invasive abilities were cultured. This study used Valsartan（ 10-2,10-3,10-4,10-5,10-6,10-7,10-8,10-9mol / L） to effect on cell lines in1640 culture solutions,used cell growth status assay to detect cell proliferation to determine the final drug concentration. The cell strains were divided into groups EJ-M3,EJ,BIU-87,and each group subdivided into subgroups study,control. Valsartan RPMI-1640 culture solution containing 10-3mol / L was added into study subgroup,serum-free RPMI-1640 culture into control subgroup and corresponding protein and mRNA were extracted after 48 h of culture. The expressions of AT1 R,MMP-2,MMP-9 proteins were determined by Western blotting,the relative expressions of AT1 R,MMP-2,MMP-9 genes by RT-PCR method. The changes of the abilities of transfer,invasion of 3 BC cell lines were detected by cell wound scratch test,Transwell in vivo cell invasion assay. Results The growth curve measured by MTT assay chose the valsartan of 10-3mol / L asfinal drug concentration. By Western blotting electrophoresis AT1 R,MMP-2,MMP-9 were expressed in 3 cell lines,but lower than in control subgroups. The relative expressions of MMP-2,MMP-9 mRNA were higher in control subgroups than in study subgroups（ P〈0. 05）. The transfer distances were shorter in study subgroups than in control subgroups（ t = 7. 24,6. 14,4. 30,P〈0. 01）. The invasive cell number was fewer in study subgroups than in control subgroups（ t = 6. 24,4. 33,2. 81,P〈0. 01）. Conclusion Valsartan can inhibit the expression of AT1 R effectively, thus to inhibit the expressions of MMP-2,MMP-9. AT1 R inhibitor may become a new drug inhibiting BC metastasis and prolonging patients＇ life span.