丙戊酸和2-己基-4-戊炔酸对乳腺癌易感基因2失活的成纤维细胞放射增敏作用及其机制  

Effects of valproic acid and 2-hexyl-4-pentyne acid on radiosensitization of breast cancer susceptibility gene 2 deficient fibroblast cells and its mechanism

作　　者：韩永涛 蔡祖超 凤志慧 HAN Yongtao;CAI Zuchao;FENG Zhihui(Department of Pharmacy,Qilu Hospital,School of Public Health,Cheeloo College of Medicine,Shandong University,Jinan 250000,China;Department of Occupational Health and Occupational Medicine,School of Public Health,Cheeloo College of Medicine,Shandong University,Jinan 250000,China;Stomatology Hospital,School of Medicine,Zhejiang University,Hangzhou 310000,China)

机构地区：[1]山东大学齐鲁医学院齐鲁医院药剂科,山东济南250000 [2]山东大学齐鲁医学院公共卫生学院职业卫生与职业医学系,山东济南250000 [3]浙江大学医学院附属口腔医院,浙江杭州310000

出　　处：《中国药理学与毒理学杂志》2023年第9期695-702,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(82173460);山东省自然科学基金(ZR2020MH330);浙江省基础公益研究计划(Q23H-140011);浙江省博士后科研项目择优资助(ZJ2022076)。

摘　　要：目的研究丙戊酸(VPA)及其衍生物2-己基-4-戊炔酸(HPTA)对乳腺癌易感基因2(BRCA2)失活的成纤维细胞放射敏感性的影响及其机制。方法取生长良好BRCA2双等位基因失活的EUFA423成纤维细胞,实验分组:(1)电离辐射(IR)0(细胞对照),2,4和6 Gy组,VPA+IR 0,2,4和6 Gy组(VPA 500μmol·L^(-1)预处理24 h后进行IR)和HPTA+IR 0,2,4和6 Gy组(HPTA 15μmol·L^(-1)预处理24 h后进行IR);(2)细胞对照、VPA、HPTA、IR对照、VPA+IR和HPTA+IR组,除IR剂量为8 Gy外,其他处理同(1)。细胞克隆形成实验检测细胞增殖能力;中性和碱性彗星实验检测细胞核拖尾尾距,并计算DNA双链断裂(DSB)百分率;细胞免疫荧光实验和Western印迹法分别检测磷酸化组蛋白(γH2AX)、53BP1、重组酶Rad51和BRCA1蛋白焦点阳性细胞百分率及其蛋白表达。结果细胞克隆形成实验结果显示,给予EUFA423细胞IR 2,4和6 Gy处理后,与同剂量IR对照组比较,VPA+IR和HPTA+IR组细胞克隆形成率均显著降低(P<0.05,P<0.01)。中性和碱性彗星实验结果均显示,与同时间IR对照组比较,IR 8 Gy处理后0,30和60 min VPA+IR和HPTA+IR组细胞核拖尾尾距均明显增加,DNA DSB百分率明显增加(P<0.01)。细胞免疫荧光实验和Western印迹法结果显示,IR 8 Gy处理后6 h,VPA+IR和HPTA+IR组γH2AX和53BP1焦点阳性细胞百分率及γH2AX和53BP1蛋白表达水平与IR对照组比较明显增加(P<0.01),BRCA1和Rad51焦点阳性细胞百分率及BRCA1和Rad51蛋白表达水平显著降低(P<0.01)。上述指标VPA+IR和HPTA+IR组之间均无显著差异。结论VPA和HPTA对BRCA2失活的EUFA423细胞具有放射增敏作用,VPA 500μmol·L^(-1)和HPTA15μmol·L^(-1)对细胞放射增敏作用相似。OBJECTIVE To study the effect of valproic acid(VPA)and its derivative 2-hexyl-4-pentyne acid(HPTA)on radiosensitivity of breast cancer susceptibility gene 2(BRCA2)deficient fibroblast cells and the mechanism.METHODS EUFA423 cells with BRCA2 deficiency were divided into the following experimental groups:①Ionizing radiation(IR)0(cell control),2,4 and 6 Gy groups,VPA+IR 0,2,4 and 6 Gy groups(VPA 500μmol·L^(-1)pretreated for 24 h before IR)and HPTA+IR 0,2,4 and 6 Gy groups(HPTA 15μmol·L^(-1)pretreated for 24 h before IR);②cell control,VPA,HPTA,IR control,VPA+IR and HPTA+IR groups,except for the IR dose of 8 Gy,other treatments were the same as in①.Cell proliferation ability was detected by cell clone formation assay.Neutral and alkaline comet assays were used to detect the tailing distance of nuclei and calculate the percentage of DNA double strand breaks(DSB).The percentage of cells containing focus formation and the expression levels of phosphorylated histone(γH2AX),53BP1,recombinase Rad51 and BRCA1 were detected by immunofluorescence assay and Western blotting.RESULTS The results of cell clone formation assay showed that compared with the IR group,the colony-forming efficiency of EUFA423 cells in VPA+IR and HPTA+IR groups decreased significantly(P<0.05,P<0.01)after EUFA423 cells were treated with IR 4 and 6 Gy.The results of neutral and alkaline comet assays showed that compared with the IR group,the tailing distance of nuclei in VPA+IR and HPTA+IR groups increased significantly at 0,30 and 60 min after IR 8 Gy treatment(P<0.01),so did the percentage of DNA DSB(P<0.01).The results of immunofluorescence assay and Western blotting showed that at 6 h after IR 8 Gy treatment,the percentage of cells containing focus formation and the expression levels of γH2AX and 53BP1 proteins in VPA+IR and HPTA+IR groups were significantly increased compared with the IR control group(P<0.01),but the percentage of cells containing focus formation and the expression levels of Rad51 and BRCA1 proteins were significantly dec

关 键 词：丙戊酸 2-己基-4-戊炔酸 放射增敏 DNA损伤 乳腺癌易感基因2 

分 类 号：R979.1[医药卫生—药品] R815.2[医药卫生—药学]