天然冰片通过触发铁死亡增强他莫昔芬对人乳腺癌T47D细胞的杀伤作用  

Natural borneol enhances killing effect of tamoxifen on human breast can⁃cer T47D cells by triggering ferroptosis

作　　者：李雪[1] 王凯[1] 李超[2] 侯秀颖 何伟丽 朱林燕[2] 杨海峰[4] LI Xue;WANG Kai;LI Chao;HOU Xiuying;HE Weili;ZHU Linyan;YANG Hai-feng(The Second Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510120,China;School of Basic Medicine and Public Health,Jinan University,Guangzhou 510632,China;The First Affiliated Hospital of Jinan University(Guangzhou Overseas Chinese Hospital),Guangzhou 510630,China;The Second Affiliated Hospital of Guang-zhou University of Chinese Medicine(Guangdong Provincial Hospital of Chinese Medicine),Guangzhou 510120,China)

机构地区：[1]广州中医药大学第二临床医学院,广东广州510120 [2]暨南大学基础医学与公共卫生学院,广东广州510632 [3]暨南大学附属第一医院(广州华侨医院),广东广州510630 [4]广州中医药大学第二附属医院(广东省中医院),广东广州510120

出　　处：《中国病理生理杂志》2023年第5期779-787,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81872133);广东省医学科研基金资助项目(No.A2023148)。

摘　　要：目的:探讨天然冰片(NB)增强人乳腺癌T47D细胞对他莫昔芬(TAM)的敏感性及其机制。方法:以6.25~800μmol/L梯度浓度的NB与20μmol/L TAM联合作用于体外培养的T47D细胞,处理时间为24 h,MTT实验检测细胞活力并计算TAM的半数抑制浓度(IC50),集落形成实验测定细胞集落形成率,以评估NB对TAM的增敏效应,并以流式细胞术检测NB联用TAM条件下细胞周期分布和凋亡率的变化。20μmol/L TAM与400μmol/L NB联合作用于T47D细胞24 h,以流式细胞术检测线粒体膜电位(MMP)和细胞活性氧(ROS)水平;比色法检测胞内谷胱甘肽(GSH)浓度;Western blot法检测铁死亡相关蛋白核因子E2相关因子2(Nrf2)、胱氨酸/谷氨酸反向转运体(xCT)和谷胱甘肽过氧化物酶4(GPX4)的表达水平,以评估NB联合TAM是否引起T47D细胞铁死亡。以ROS抑制剂N-乙酰-L-半胱氨酸(NAC)或铁死亡抑制剂ferrostatin-1(Fer-1)进行预处理,MTT实验检测细胞活力并计算TAM的IC50,评估主动干扰铁死亡是否影响NB增强TAM的细胞杀伤作用。结果:(1)联用NB可促进TAM对T47D细胞的杀伤作用:NB(200和400μmol/L)与TAM联用导致TAM对T47D细胞的IC50降低(P<0.01);细胞集落形成率受到抑制(P<0.01);400μmol/L NB联合20μmol/L TAM可诱导T47D产生细胞周期S期阻滞(P<0.05)和增加细胞凋亡(P<0.01),晚期凋亡细胞占凋亡细胞的(78.0±6.8)%,而早期凋亡细胞仅占(22.0±6.8)%。(2)400μmol/L NB联合20μmol/L TAM触发T47D细胞铁死亡,表现为ROS积累增加(P<0.01)、MMP降低(P<0.01)、GSH浓度降低(P<0.01)以及铁死亡相关蛋白Nrf2、xCT和GPX4表达下调(P<0.05)。(3)1μmol/L NAC及1μmol/L Fer-1均可逆转NB联合TAM诱导的T47D细胞增殖抑制(P<0.01)。结论:NB联合TAM通过触发T47D细胞铁死亡的机制,增强TAM对人乳腺癌T47D细胞的杀伤作用。AIM:To investigate the effect of natural borneol(NB)on the sensitivity of human breast cancer T47D cells to tamoxifen(TAM)and its mechanisms.METHODS:The T47D cells were treated with 6.25~800μmol/L NB combined with 20μmol/L TAM for 24 h.The cell viability,50%inhibitory concentration(IC50)of TAM and cell colo⁃ny formation rate were measured to evaluate whether NB enhanced the cytotoxicity of TAM to T47D cells.Cell cycle distri⁃bution and apoptosis of the T47D cells were detected by flow cytometry under the condition of NB combined with TAM.To evaluate whether NB combined with TAM triggered ferroptosis,the T47D cells were treated with 20μmol/L TAM com⁃bined with 400μmol/L NB for 24 h.The mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were measured by flow cytometry,intracellular glutathione(GSH)concentration was determined by colorimetry,and the expression levels of ferroptosis-related proteins nuclear factor E2-related factor 2(Nrf2),cystine/glutamate antiporte(xCT)and glutathione peroxidase 4(GPX4)were detected by Western blot.After pretreatment with ROS inhibitor N-ace⁃tyl-L-cysteine(NAC)or ferroptosis inhibitor ferrostatin-1(Fer-1),the cell viability and IC50 of TAM were detected by MTT assay to evaluate the effect of active interference in ferroptosis on NB-enhanced TAM cytotoxicity to T47D cells.RE⁃SULTS:(1)After treatment with combination of NB and TAM,the cytotoxicity of TAM to T47D cells was increased.The IC50 of TAM was decreased in NB(200μmol/L and 400μmol/L)+TAM groups(P<0.01),colony formation rate was de⁃creased(P<0.01),and S phase arrest was induced in T47D cells(P<0.05).Treatment with 400μmol/L NB combined with 20μmol/L TAM induced S phase arrest and increased apoptosis of T47D cells(P<0.01).The results of flow cytome⁃try showed that late apoptotic cells accounted for(78.0±6.8)%of apoptotic cells,while early apoptotic cells only account⁃ed for(22.0±6.8)%.(2)Treatment with NB+TAM triggered ferroptosis of T47D cells.This treatment increased ROS ac⁃cumula

关 键 词：天然冰片 他莫昔芬 乳腺癌 活性氧 铁死亡 

分 类 号：R979.1[医药卫生—药品] R737.9[医药卫生—药学] R363.2