缺氧对5-氟尿嘧啶干预的胃癌细胞中HIF-1α/MDR1/VEGF表达的影响

Effect of hypoxia on HIF-1α/MDR1/VEGF expression in gastric cancer cells treated with 5-fluorouracil

目的:5-氟尿嘧啶(5-fluorouracil,5-FU)是胃癌的经典化学治疗药物,但是越来越严重的耐药性限制了其临床应用。本研究旨在探索缺氧微环境是否影响胃癌对5-FU的耐药性,讨论缺氧环境下与耐药性直接相关的基因和蛋白质的变化情况。方法:分别在缺氧(缺氧组)及常氧(常氧组)环境下用5-FU干预胃癌细胞。应用流式细胞术Annexin V/PI双染法检测凋亡,实时反转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RTPCR)和蛋白质印迹法检测缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α),与5-FU耐药相关的多药耐药(multidrug resistance,MDR1)基因、P-糖蛋白(P-glycoprotein,P-gp)以及血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达水平,分析缺氧对5-FU治疗胃癌效果的影响。结果:与常氧组相比,缺氧组的胃癌细胞接受5-FU干预后凋亡明显减少,凋亡启动蛋白caspase 8的表达下降。与常氧组相比,缺氧组HIF-1αmRNA表达水平显著升高(P<0.05),MDR1、P-gp、VEGF的蛋白质表达水平均显著升高(均P<0.05)。MDR1、P-gp、VEGF的表达水平升高与HIF-1α的表达趋势相同。结论:缺氧是导致胃癌细胞对5-FU耐药性的直接因素。为了克服胃癌对5-FU的耐药性,改善胃癌局部缺氧微环境可能是一个新思路。

Objective:Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer.But the problem of drug resistance severely limits their clinical application.This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil(5-FU)and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition.Methods:Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment,and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group.The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining.The real-time reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting were used to detect the expression level of hypoxia inducible factor-1α(HIF-1α),multidrug resistance(MDR1)gene,P-glycoprotein(P-gp),and vascular endothelial growth factor(VEGF)which were related to 5-FU drug-resistance.We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU.Results:Compared with the Normoxic+5-FU group,the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced(P<0.05),and the expression of apoptosis promoter protein caspase 8 was also decreased.Compared with the the Normoxic+5-FU group,HIF-1αmRNA expression in the Hypoxia+5-FU group was significantly increased(P<0.05),and the mRNA and protein expression levels of MDR1,Pgp and VEGF were also significantly increased(all P<0.05).The increased expression of MDR1,P-gp and VEGF had the same trend with the expression of HIF-1α.Conclusion:Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy.Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.