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

doi:10.3969/j.issn.1671-167X.2018.02.019

摘要/Abstract

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 class Ⅰ-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 factor κB，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、100 μmol/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)、(0.76±0.06)、(0.56±0.03)倍（P<0.05），MICA/B蛋白表达由(3.42±0.05)倍降为(1.32±0.03)、(1.21±0.06)、(1.14±0.03)倍（P<0.001），表明足叶乙甙对MICA/B的上调作用能够被ATM/ATR抑制剂所抑制。同样，加入不同浓度的NF-κB的强抑制剂PDTC（10、50、100 μmol/L），MICA/B mRNA和蛋白的表达均明显受到抑制（P<0.05），提示NF-κB也参与这一过程。EMSA实验显示,MCF-7细胞用足叶乙甙处理后，NF-κB与MICA/B基因启动子区的结合活性增强。结论：拓扑异构酶抑制剂诱导并上调乳腺癌细胞MICA和MICB mRNA及表面蛋白的表达，表明化疗药物有可能增强免疫系统对乳腺癌的识别和杀伤，ATM/ATR和NF-κB途径参与了这一过程。

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 -&kappa, B, 乳腺肿瘤

Abstract: Objective： To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex class Ⅰ-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 factor κB (NF-κB) inhibitor pyno-lidine 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 100 μmol/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 topoisomerase Ⅱ inhibitors 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 respectively (P<0.001), and MICB mRNA levels decreased from (6.85±0.35) to (1.36±0.13), (0.76±0.06) and (0.56±0.03) fold (P<0.05), while MICA/B protein levels decreased from (3.42±0.05) to (1.32±0.03), (1.21±0.06) and (1.14±0.03) fold (P<0.001), indicating that etoposide-induced MICA/B expression was inhibited by ATM/ATR inhibitor. Similarly, NF-κB inhibitor PDTC also inhibited MICA/B mRNA and protein expressions induced by etoposide significantly when MCF-7 cells were incubated with PDTC at the concentrations of 10, 50 and 100 μmol/L (P<0.05), indicating that NF-κB was also involved in this process. EMSA showed that the binding of NF-κB to MICA/B promoter enhanced in MCF-7 cells after etoposide treatment. Conclusion: Topoisomerase inhibitor increased MICA/B mRNA and protein expressions in breast cancer cells, indicating that chemotherapeutic agents might increase the recognizing and killing ability of immunocytes to breast cancer cells. ATM/ATR and NF-κB pathways might be involved in it.

Key words: Topoisomerase inhibitors, MHC class I-related chain A/B, Ataxia telangiectasia mutated proteins, Ataxia telangiectasia and Rad3-related kinase, NF-kappa B, Breast neoplasms

中图分类号:

朱燕，石永进，赵玉亮，朱平. 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[J]. 北京大学学报(医学版), 2018, 50(2): 318-325.

ZHU Yan，SHI Yong-jin，ZHAO Yu-liang, ZHU Ping. Topoisomerase inhibitor upregulates MICA/B expression in breast cancer cells through ATM/ATR and NF-κB pathway[J]. Journal of Peking University(Health Sciences), 2018, 50(2): 318-325.

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