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Study on the Effect of Vascular Endothelial Growth Factor on the Expression of Multidrug Resistance-Associated Protein1 in Cancer Cells

Author: LiJuan
Tutor: XuWenLin
School: Jiangsu University
Course: Internal Medicine
Keywords: tumor vascular endothelial growth factor multidrug resistance-associated protein1 luciferase reporter gene promoter activity PI3K/Akt signaling pathway SP1
CLC: R73-3
Type: Master's thesis
Year: 2010
Downloads: 97
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Abstract

Objective:1.To assess the changes of the expression of multidrug resistance-associated proteinl (MRP1) mRNA and protein levels in human leukemia cell line K562 and human gastric cancer cell line BGC-823 after cells being treated with recombinant human vascular endothelial growth factor(VEGF),and explore the relationship between VEGF and multidrug resistance(MDR) in human malignancies.2.To construct the recombinant luciferase reporter gene vector which contains MRP1 promoter, and then observe the alteration of the promoter transcriptional activity that regulated by VEGF.3. Setting about the transcriptional control of MRP1 gene,the protein levels of Akt, p-Akt, SP1 and the activity of SP1 binding with DNA were detected. Moreover, the mechanism of VEGF up-regulating MRP1 was explored.4.To construct the recombinant luciferase reporter gene vector that containing MRP1 promoter with SP1 binding sites mutants, and then analyze its transcriptional activity and the effect of VEGF on it.Methods:1.24 hours after the cancer cells being treated with different concentrations of VEGF, the expression of MRP1 of all groups, at mRNA and protein levels, were respectively detected by the SYBR Green I real time fluorescent quantitative PCR (qRT-PCR) method and Western blot assay.2. The promoter of MRP 1 was synthesized and cloned into the luciferase reporter gene vector PGL3-Basic.The recombinant plasmid was transiently co-transfected into BGC-823 cells with control vector P-gal by lipofectamine 2000 reagent, and then detect the alteration of the MRP1 promoter activity after the cells being treated with VEGF. Subsequently, co-transfected cells were pretreated with LY294002, the inhibitor of PI3K/Akt signaling pathway, and the effect of VEGF on the MRP1 promoter activity was evaluated afterwards.3. BGC-823 cells were cultured with absence or presence of VEGF for 24 h or pretreated with LY294002 for 1 h before stimulating with VEGF. Western blot assay was applied to assess the expression of MRP1,Akt,p-Akt or SP1 protein in the three groups described above,and electrophoretic mobility shift assay(EMSA) was adopted to detect the DNA binding activity of transcription factor SP1.4. The sequence of MRP 1 promoter with SP1 binding sites mutants was synthesized and cloned into the luciferase reporter gene vector PGL3-Basic.The recombinant plasmid was transiently co-transfected into BGC-823 cells with control vector P-gal by lipofectamine 2000 reagent, and then investigate the alteration of the mutational MRP1 promoter activity after the cells being treated with VEGF.Results:1.As shown by qRT-PCR, the expression of MRP1 mRNA levels in K562 cells and BGC-823 cells were remarkably enhanced by VEGF in a dose-dependent manner. Compared with the control group, the expression of MRP1 mRNA in the VEGF 32 ng/mL group was increased to 2.0-fold and 3.5-fold(P<0.01), respectively,and the changes of MRP1 protein levels were consisted with mRNA.2. The consequence of restriction enzyme digestion and nucleotide sequence analysis confirmed that the recombinant plasmid was successfully constructed; The analysis of the luciferase reporter gene activity demonstrated that the recombinant plasmid displayed marked activity(144±6.888) in BGC-823 cells,about 2.4-fold as powerful as the activity showed in the control vector PGL3-Basic(60±4.910,P<0.01). Importantly, this activity was up-regulated by VEGF in a dose-dependent manner as well (r=0.944,P<0.01), and with the most pronounced concentration,32 ng/mL, the activity(924±19.975) was at least 4.4-fold increased, compared with the cells which were cultured without VEGF(171±6.083,P<0.01); Furthermore, LY294002 significantly supressed the effect of VEGF on the MRP1 promoter activity.Compared with cells cultured in the absence of LY294002(1001±11.533),the activity of the MRP1 promoter was reduced 60.8% when the cells were exposured to 50μmol/mL LY294002 (392±25.541,P<0.01).3. Compared with the control group, the MRP1, p-Akt and SP1 protein were all up-regulated, and the DNA binding activity of SP1 was significantly enhanced in BGC-823 cells which were 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.4. The analysis of the luciferase reporter gene activity indicated that the recombinant plasmid PGL3-Basic-MRP1m possessed its promoter activity (110±2.603) in BGC-823 cells,and compared with the control vector PGL3-Basic, its transcriptional activity was increased to 1.8-fold(P<0.01).On the contrary,the transcriptional activity of PGL3-Basic-MRP1m was reduced 23.6%, which compared with PGL3-Basic-MRP1w(P<0.05), and it was dose-dependently potentized at the 12-hour time point after the transfected cells being treated with VEGF(r=0.911,P<0.01). When the concentration of VEGF was increased to 32 ng/mL and then continuously stimulated the cells for 12 h, the transcriptional activity of PGL3-Basic-MRPlm(191±14.799) was 0.7-fold increased than the control group(112±11.358, P<0.01). Similarily, 24-hour after the transfected cells being treated with VEGF,the mutational MRP1 promoter activity was also up-regulated in a dose-dependent manner(r=0.945,P<0.01). Compared with untreated cells(133±6.083), the greatest activity (426±7.000)was increased about 2.2-fold(P<0.01).Conclusions:1.The expression of MRP1 mRNA and protein levels in malignancy cells were significantly up-regulated by VEGF.2. VEGF could augment the activity of the MRP1 promoter, and PI3K/Akt signaling pathway and transcriptional factor SP1 were acting as two critical factors underlying this mechanism.3.The transcriptional activity of the MRP1 promoter was decreased by SP1 binding sites mutating, and the enhancement of VEGF on the promoter activity was weakened when compared with the wide MRP1 promoter.In conclusion,our results demonstrated that VEGF could effectively promote the development of the multidrug resistance in human malignancies by up-regulating MRP1 mRNA and protein,and it could be a new strategy for clinical tumor chemotherapy.

Study on the Effect of Vascular Endothelial Growth Factor on the Expression of Multidrug Resistance-Associated Protein1 in Cancer Cells

Abstract

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