Zhu Y, Shi Y J, Zhao Y L, Zhu P
Department of Oncology, Peking University First Hospital,Beijing 100034, China.
Department of Hematology,Peking University First Hospital,Beijing 100034, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2018 Apr 18;50(2):318-325.
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.
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 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.
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.
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.
研究临床广泛应用的化疗药物对乳腺癌细胞主要组织相容性复合体I类相关链A和B（MICA/B）表达的影响，并探讨其相关分子机制。
分别采用实时逆转录聚合酶链反应（RT-PCR）和流式细胞术检测化疗药物处理后的乳腺癌细胞中MICA/B mRNA和表面蛋白的表达。研究共济失调毛细血管扩张症突变基因和Rad3相关激酶（ATM/ATR）抑制剂咖啡因及核因子κB（NF-κB）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对依托泊苷上调的MICA/B mRNA和表面蛋白表达的阻断作用。采用电泳迁移率变动分析（EMSA）研究依托泊苷是否增强NF-κB与MICA/B基因启动子的结合。
三种拓扑异构酶抑制剂依托泊苷、喜树碱和阿霉素上调了乳腺癌细胞MCF-7中MICA和MICB mRNA的表达。与未用药处理的细胞相比，在浓度为5、20和100 μmol/L的依托泊苷处理的癌细胞中，MICA mRNA水平分别增加到（1.68±0.17）、（2.54±0.25）和（3.42±0.15）倍，MICB mRNA水平分别增加到（1.82±0.24）、（1.56±0.05）和（5.84±0.57）倍（P<0.05）。当MCF-7细胞与特定浓度的喜树碱或阿霉素孵育时，MICA和MICB mRNA水平也显著增加（P<0.05）。在另一种乳腺癌细胞SK-BR-3中，拓扑异构酶II抑制剂依托泊苷和喜树碱处理后，MICB mRNA表达也略有增加（P<0.05）。此外，依托泊苷和喜树碱上调了MCF-7细胞中MICA/B表面蛋白的表达（P<0.05），且在活细胞和凋亡细胞中均有上调。我们的研究表明，不同浓度的咖啡因可抑制依托泊苷诱导的MCF-7细胞中MICA/B的表达。当癌细胞用1、5和10 mmol/L的咖啡因处理时，MICA mRNA水平分别从（3.75±0.25）降至（0.89±0.05）、（0.81±0.02）和（0.48±0.04）倍（P<0.001），MICB mRNA水平分别从（6.85±0.35）降至（1.36±0.13）、（0.76±0.06）和（0.56±0.03）倍（P<0.05），而MICA/B蛋白水平分别从（3.42±0.05）降至（1.32±0.03）、（1.21±0.06）和（1.14±0.03）倍（P<0.001），表明ATM/ATR抑制剂可抑制依托泊苷诱导的MICA/B表达。同样，当MCF-7细胞与浓度为10、50和100 μmol/L的PDTC孵育时，NF-κB抑制剂PDTC也显著抑制了依托泊苷诱导的MICA/B mRNA和蛋白表达（P<0.05），表明NF-κB也参与了这一过程。EMSA显示，依托泊苷处理后MCF-7细胞中NF-κB与MICA/B启动子的结合增强。
拓扑异构酶抑制剂增加了乳腺癌细胞中MICA/B mRNA和蛋白的表达，表明化疗药物可能增强免疫细胞对乳腺癌细胞的识别和杀伤能力。ATM/ATR和NF-κB信号通路可能参与其中。
