拓扑异构酶抑制剂通过ATM/ATR和NF-κB途径上调乳腺癌细胞MICA/B的表达  

Topoisomerase inhibitor upregulates MICA/B expression in breast cancer cells through ATM/ATR and NF-κB pathway

作　　者：朱燕[1] 石永进[2] 赵玉亮[1] 朱平[2] ZHU Yan;SHI Yong-jin;ZHAO Yu-liang;ZHU Ping(Department of Oncology,Peking University First Hospital,Beijing 100034,China;Department of Hematology,Peking University First Hospital,Beijing 100034,China)

机构地区：[1]北京大学第一医院肿瘤化疗科,北京100034 [2]北京大学第一医院血液科,北京100034

出　　处：《北京大学学报（医学版）》2018年第2期318-325,共8页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81370612);国家自然科学青年基金(81102211)资助~~

摘　　要：目的:分析常用化疗药物对免疫识别乳腺癌细胞重要的靶标主要组织相容性复合体Ⅰ类相关分子A和B(major histocompatibility complex classⅠ-related chain A and B,MICA/B)的影响,探讨其调节MICA/B表达的分子机制。方法:用荧光定量RT-PCR法检测化疗药物对乳腺癌细胞MICA和MICB mRNA表达的影响,流式细胞术检测化疗药物对MICA/B表面蛋白表达的作用。加入咖啡因抑制毛细血管扩张性共济失调突变和Rad3相关激酶(ataxia telangiectasia mutated and Rad3-related kinase,ATM/ATR),加入吡咯烷二硫氨基甲酸酯(pynolidine dithiocarbamate,PDTC)抑制核因子κB(nuclear factorκB,NF-κB),观察其是否能够抑制化疗药物中拓扑异构酶抑制剂对乳腺癌细胞MICA和MICB mRNA及蛋白的表达。凝胶阻滞实验(electrophoretic mobility shift assay,EMSA)检测药物是否影响乳腺癌细胞NF-κB与MICA/B启动子区的结合。结果:三种拓扑异构酶抑制剂足叶乙甙、拓扑替康和阿霉素可使乳腺癌MCF-7细胞的MICA和MICB mRNA水平升高。足叶乙甙在浓度为5、20、100μmol/L时,与不加药处理组相比,MICA mRNA水平分别升高到(1.68±0.17)、(2.54±0.25)、(3.42±0.15)倍(P<0.05);MICB mRNA水平分别升高到(1.82±0.24)、(1.56±0.05)、(5.84±0.57)倍(P<0.05)。拓扑替康和阿霉素在特定浓度时也使MCF-7的MICA和MICB mRNA水平显著升高(P<0.05)。拓扑异构酶Ⅱ抑制剂足叶乙甙和拓扑替康处理另一乳腺癌细胞系SK-BR-3后,MICB mRNA的表达也轻度升高(P<0.05)。足叶乙甙和拓扑替康还增加MCF-7细胞表面MICA/B蛋白的表达(P<0.05),且存活和凋亡细胞MICA/B蛋白的表达均增高。用不同浓度的咖啡因处理足叶乙甙损伤的乳腺癌细胞,MICA/B mRNA和蛋白表达均显著降低,当咖啡因浓度为1、5、10 mmol/L时,MICA mRNA相对表达水平由不用咖啡因处理组的(3.75±0.25)倍分别降为(0.89±0.05)、(0.81±0.02)、(0.48±0.04)倍(P<0.001),MICB mRNA由(6.85±0.35)倍降为(1.36±0.13)、(0Objective:To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex classⅠ-related chain A and B(MICA/B)expression in breast cancer cells,and to explore the molecular mechanisms involved.Methods:We examined MICA/B mRNA and surface protein expressions in breast cancer cells treated with chemotherapeutic agents by real-time RT-PCR and flow cytometry respectively.The blocking effects of ataxia telangiectasia mutated and Rad3-related kinase(ATM/ATR)inhibitor caffeine and nuclear factorκB(NF-κB)inhibitor pynolidine dithiocarbamate(PDTC)on etoposide-upregulated MICA/B mRNA and surface protein expressions were investigated.Electrophoretic mobility shift assay(EMSA)was taken to investigate whether etoposide enhanced the binding of NF-κB to MICA/B gene promoter.Results:Three topoisomerase inhibitors etoposide,camptothecin and doxorubicine upregulated MICA and MICB mRNA expressions in breast cancer cell MCF-7.Comparing to no-drug-treated cells,MICA mRNA levels increased to(1.68±0.17),(2.54±0.25)and(3.42±0.15)fold,and levels of MICB mRNA increased to(1.82±0.24),(1.56±0.05)and(5.84±0.57)fold respectively in cancer cells treated by etoposide at the concentrations of 5,20 and 100μmol/L(P<0.05).MICA and MICB mRNA levels also increased significantly when MCF-7 cells were incubated with camptothecin or doxorubicine at the specific concentrations(P<0.05).MICB mRNA expression also increased slightly in another breast cancer cell SK-BR-3 treated by topoisomeraseⅡinhibitors etoposide and camptothecin(P<0.05).Furthermore,etoposide and camptothecin upregulated MICA/B surface protein expression in MCF-7 cells(P<0.05),and the upregulation was found in both living and apoptotic cells.Our study showed that etoposide induced-MICA/B expression in MCF-7 was inhibited by caffeine at different concentrations.When cancer cells were treated by caffeine with 1,5 and 10 mmol/L,MICA mRNA levels decreased from(3.75±0.25)to(0.89±0.05),(0.81±0.02)and(0.48±0.04)fold respe

关 键 词：拓扑异构酶抑制剂 MHCⅠ类链相关分子A/B 毛细血管扩张性共济失调突变蛋白 毛细血管扩张性共济失调Rad3相关激酶 NF-ΚB 乳腺肿瘤 

分 类 号：R730.53[医药卫生—肿瘤]