摘要目的 探讨p38丝裂原活化蛋白激酶(MAPK)通路介导的早期生长反应基因(EGR)-1活性与乳腺癌细胞表柔比星耐药的关系.方法 SB203580(15 μmol/L)干预后,激光共聚焦显微镜观察;流式细胞术、四甲基偶氮唑蓝(MTT)、凝胶电泳迁移率法(EMSA)、RT-PCR及Western blot分别检测耐药MCF-7/Adr及亲本MCF-7细胞内磷酸化p38MAPK蛋白表达、细胞凋亡及细胞内表柔比星浓度、EGR-1蛋白活性改变、细胞对表柔比星敏感性;EGR-1 mRNA、P糖蛋白、磷酸化p53及p38蛋白表达.结果 p38MAPK通路激活的MCF-7/Adr细胞经SB203580(15 μmol/L)干预24和48 h后,(1)MCF-7/Adr细胞(早+晚期)凋亡率分别由(0.54±0.17)%和(0.81±0.16)%提高为(25.36±1.17)%和(38.21±1.25)%,P<0.05,并呈一定时间依赖性;(2)MCF-7/Adr细胞的平均荧光强度分别为(32.45±2.36)及(41.66±3.12),均高于空白对照组及DMSO组MCF-7/Adr细胞的(14.17±1.45)及(16.28±0.63),P<0.01;MCF-7/Adr细胞对表柔比星药物的耐受性显著降低;(3)增加了MCF-7/Adr细胞的EGR-1活性,IC50分别为(21.53±2.17)和(8.77±1.02),低于空白对照组(40.74±2.56);伴随p38MAPK通路活性抑制和EGR-1 mRNA表达增加,磷酸化p53蛋白表达显著上调,而P糖蛋白显著下调.结论 p38MAPK通路与乳腺癌表柔比星耐药密切相关,可能与p38MAPK通路介导的EGR-1表达相关,EGR-1激活抑制了其下游耐药基因转录,从而使表柔比星耐药得以逆转.

abstractsObjective To investigate the relationship between activities of early growth response gene 1 (EGR-1) of p38 mitogen-activated protein kinase(MAPK) pathway and in the epirubicin resistance of breast carcinoma cells.Methods Protein expression of phosphorylated p38MAPK was detected by confocal spectral microscopy.Using specific inhibitor SB203580, the effect of p38MAPK on cell apoptosis was analyzed by FITC-Annexin-V/PI double staining.The concentration of epirubicin was detected by flow cytometry (FCM).The 50% inhibition concentration (ICS0) of epirubicin on MCF-7/Adr cells was determined by MTT method.Electrophoretic motility shift assay (EMSA) was performed to examine the affinity of EGR-1.EGR-1 mRNA was assessed by RT-PCR.The expression levels of p-glycoprotein, phosphorylated p53 and p38 were detected by Western blot.Results After treatment with SB203580 (15 μmol/L)24 h and 48 h, (1)the early and late apoptosis of MCF-7/Adr cells expressing the phosphorylated p38MAPK protein was (25.36 ± 1.17 ) % and ( 38.21 ± 1.25 ) %, respectively, P < 0.05.And the tendency was in a time-dependent manner.(2)The average fluorescence intensity of MCF-7/Adr cells expressing the phosphorylated p38MAPK protein was (32.45 ± 2.36) and (41.66 ± 3.12), higher than the blank group ( 14.17 ± 1.45 ) and DMSO group ( 16.28 ± 0.63 ), P < 0.01.The epirubicin resistance of MCF-7/Adr cells significantly decreased.( 3 ) SB203580 demonstrated a significantly higher level of EGR-1 activity.The IC50 was (21.53 ± 2.17) and ( 8.77 ± 1.02), lower than the DMSO group (40.74 ± 2.56) .MCF-7/Adr cells treated with SB203580 down-regulated the p38MAPK pathway activity, but up-regulated the EGR-1 mRNA expression.SB203580 significantly increased the cellular phosphorylated p53 protein level, but decreased the p-glycoprotein level in MCF-7/Adr cells.Conclusions There is a close relationship between p38MAPK pathway activity and the epirubicin resistance of breast carcinoma cells.The activation of EGR-1 mediated by p38MAPK pathway plays a critical role in epirubiein resistance.