NF-κB介导白血病多药耐药细胞中葡萄糖神经酰胺合成酶对P-糖蛋白的调节作用

摘要

Objective To investigate whether transcription factor-kappaB (NF-κB) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κB and extracelluar signal-regulated kinase (ERK).Methods K562/A02 cells were treated with GCSsiRNA,pyrrolidine dithiocarbamate (PDTC,a NF-κB specific inhibitor) and U0126 (a MEK1/2 inhibitor),respectively.The expression of GCS and multidrug resistance protein 1 (MDR1)mRNA were analyzed with qRT-PCR.Various proteins of different groups were measured by Western blotting.Results After transfected with GCSsiRNA for 48 h,GCS mRNA were reduced by 62％ (51％-73％) and MDR1 mRNA was reduced by 52％ (43％-61％) in the K562/A02 cells.Compared with the negative control,relative expression of NF-κB p65 in nuclear and PERK1/2 were both down-regulated,and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P＜0.05).In addition,the expression of P-gp was decreased at 24 h with 80 μmol/L PDTC and 48 h with 20 μmol/L PDTC.P-ERK1/2 was inhibited significantly when the cells were treated with 20 μmol/L U0126 for 48 h.The expression of NF-κB p65 in nuclear and P-gp were also down-regulated.Conclusion NF-κB can modulate the effect of GCS on P-gp in K562/A02 cells.P-ERK1/2 can activate NF-κB in above signal transduction pathway.%目的 探讨在白血病耐药细胞K562/A02中核因子κB(nuclear transcription factor-kappaB,NF-κB)是否参与葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)对P-糖蛋白(P-glycoprotein,PgP)的调节作用,并研究在该调节作用信号通路中NF-κB与细胞外信号调节激酶(extracelluar signalregulated kinase,ERK)的相互关系.方法 应用特异性的GCS小干扰RNA(GCS small interfering RNA,GCSsiRNA)抑制GCS基因后,实时荧光定量PCR方法检测靶基因抑制率以及对多药耐药蛋白1(multidrug resistance protein 1,MDRl) mRNA表达水平的影响；同时应用Western印迹检测各种蛋白的表达情况.使用NF-κB的特异性抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocarbamate,PDTC)抑制NF-κB后,应用Western印迹检测P-gp的表达情况.U0126抑制P-ERK(phosphorylated-ERK)后,Western印迹检测细胞核内以及总的NF-κB p65和P-gp的表达.结果 GCSsiRNA转染48 h后,GCSmRNA的表达被抑制,抑制率为62％(51％～73％),同时下调MDR1mRNA的表达,抑制率为52％(43％～61％),与阴性干扰对照组比较,差异均有统计学意义(P＜0.05)；GCSsiRNA转染72 h后,GCSsiRNA组细胞核内NF-κB p65及P-ERK的表达水平均明显下调(P＜0.05),同时对P-gp的表达亦有显著的抑制作用(P＜0.05).NF-κB的抑制剂PDTC处理K562/A02细胞后,对P-gp有明显的抑制作用(P＜o.05).P-ERK抑制剂U0126处理K562/A02细胞,除了能显著抑制P-ERK1/2的表达外(P＜0.01),还可明显抑制细胞核内NF-κB p65的表达和P-gp的表达(P＜0.01).结论 NF-κB介导了白血病多药耐药细胞中GCS对P-gp的调节作用,且在这一调节作用信号通路中位于ERK下游.