PI3K/Akt信号通路及SP1在VEGF上调胃癌细胞MRP1中的作用

Role of PI3K/Akt and SP1 in VEGF induced up-regulation of MRP1 in gastric cancer cell line BGC-823

目的:从多药耐药相关蛋白1(multidrug resistanceassociated protein 1,MRP1)基因转录调控入手,初步探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)上调MRP1表达的机制.方法:(1)以人胃癌BGC-823细胞为模型,一组常规培养24 h,另一组VEGF作用24 h,最后一组PI3K/Akt抑制剂LY294002预处理1 h后再加VEGF作用24 h,Western blot法分别检测各实验组MRP1、Akt、p-Akt及SP1蛋白表达水平,EMSA方法检测各实验组转录因子SP1与D N A的结合活性;(2)构建分别含M R P1基因启动子序列及SP1结合位点突变的MRP1基因启动子序列的重组荧光素酶报告基因载体(PGL3-Basic-MRP1w、PGL3-Basic-MRP1m),荧光素酶活性分析突变后的MRP1基因启动子在BGC-823细胞中活性的改变及VEGF对其转录活性的影响.结果:(1)VEGF 32 ng/m L作用24 h组与未处理组相比,MRP1、p-Akt、SP1在蛋白水平均上调,且SP1的DNA结合活性明显增强;LY294002 50μmol/m L预处理1 h再联合VEGF32 ng/m L作用24 h后,与VEGF 32 ng/m L单独作用组相比,MRP1、p-Akt、SP1在蛋白水平均下调,SP1的DNA结合活性明显减弱;(2)在BGC-823细胞中,PGL3-Basic-MRP1m具有启动子活性(110.000±2.603),其转录活性为空载体PGL3-Basic的1.8倍(t=-8.936,P<0.01),但与SP1结合位点突变之前的MRP1启动子(PGL3-Basic-MRP1w)活性(144.000±6.888)相比,其转录活性下降23.6%(t=4.617,P<0.05);V E G F作用12 h后,其活性增强,且呈剂量依赖关系(r=0.911,P<0.01),VEGF作用浓度为32 ng/m L时达最大值(191.000±14.799),与无V E G F作用组(112.000±11.358)相比,活性增高0.7倍(t=-7.335,P<0.01);VEGF作用24 h后,也能以剂量依赖的方式上调SP1结合位点突变后MRP1启动子区的转录活性(r=0.945,P<0.01),与无VEGF作用组(133.000±6.083)相比,最大活性(426.000±7.000)增高2.2倍(t=-56.032,P<0.01).结论:VEGF对MRP1启动子活性的上调作用与激活PI3K/Akt信号通路及增强转录因子SP1的表达及活性相关;MRP1基因启动子区SP1结合位点突变后其启动子活性减弱,VEGF对其活性的上调作用不如突变之前明显.

AIM： To explore the mechanism by which vascular endothelial growth factor （VEGF） up-regulates multidrug resistance-associated protein 1 （MRP1） in gastric cancer cell line BGC-823. METHODS： BGC-823 cells cultured in the absence or presence of VEGF for 24 h were pretreated with phosphatidylinositol 3 kinase, PI-3K/protein kinase B, and PKB （PI3K/Akt） inhibitor LY294002 for 1 h before stimulation with VEGF. Western blot assay was applied to assess the expression of MRP1, Akt, p-Akt and specificity protein 1 （SP1） proteins in the three groups of cells described above, and electrophoretic mobility shift assay （EMSA） was adopted to detect the DNA binding activity of transcriptional factor SP1. The sequences of MRP1 promoter and MRP1 promoter with SP1 binding site mutants were synthesized and cloned into the luciferase reporter gene vector PGL3-Basic to result in recombinant plasmids PGL3-Basic-MRP1w and PGL3-Basic-MRP1m, respectively. The recombinant plasmid was transiently co-transfected into BGC-823 cells using lipofectamine 2000 reagent, and the alteration of the mutant MRP1 promoter activity and the effect of VEGF on MRP1 promoter activity were then investigated. RESULTS： Compared with the control group, the MRP1, p-Akt and SP1 proteins were all up-regulated, and the DNA binding activity of SP1 was significantly enhanced in BGC-823 cells treated with 32 ng/mL VEGF for 24 h. Contrarily, the protein levels of MRP1, p-Akt and SP1 were down-regulated, and the DNA binding activity of SP1 was remarkably decreased in the LY294002 pretreated group when compared with the VEGF 32 ng/mL group. The analysis of the luciferase reporter gene activity indicated that the recombinant plasmid PGL3-Basic-MRP1m possessed its promoter activity （110.000 ± 2.603） in BGC-823 cells, and compared with the control vector PGL3-Basic, its transcriptional activity was increased by 1.8-fold （t = -8.936, P 〈 0.01）. On the contrary, the transcriptional activity of PGL3-Basic-MRP1m was reduced by 23.6% compared with PGL3-Basic-MRP1w （t = 4.617, P 〈 0.05）, and this was dose-dependently enhanced at the 12-h time point after the transfected cells were treated with VEGF （r = 0.911, P 〈 0.01）. When the concentration of VEGF was increased to 32 ng/mL to continuously stimulate the cells for 12 h, the transcriptional activity of PGL3-Basic-MRP1m （191.000 ± 14.799） was 0.7-fold increased than the control group （112.000 ± 11.358, t = -7.335, P 〈 0.01）. Similarly, 24 h after the transfected cells were treated with VEGF, the mutant MRP1 promoter activity was also up-regulated in a dose-dependent manner （r = 0.945, P 〈 0.01）. Compared with untreated cells （133.000 ± 6.083）, the greatest activity （426.000 ± 7.000） of about 2.2-fold was observed （t = -56.032, P 〈 0.01）. CONCLUSION： The PI3K/Akt signaling pathway and transcriptional factor SP1 are two critical factors involved in VEGF-mediated augmentation of the activity of the MRP1 promoter; the transcriptional activity of the MRP1 promoter is decreased by SP1 binding site mutation, and the enhancing effect of VEGF on the promoter activity is weakened when compared with the wild type MRP1 promoter.