新型补骨脂酚氨基胍衍生物诱导人三阴性乳腺癌细胞凋亡  

A novel bakuchiol aminoguanidine derivative induces apoptosis in human triple-negative breast cancer cells

作　　者：张振海 朱静 王建安 陈洁 庞颖颖 吴成柱 ZHANG Zhenhai;ZHU Jing;WANG Jian’an;CHEN Jie;PANG Yingying;WU Chengzhu(Department of Emergency Surgery,the First Affiliated Hospital of Bengbu Medical University,Bengbu 233004,Anhui Province,China;School of Pharmacy,Bengbu Medical University,Bengbu 233030,Anhui Province,China;Department of Respiratory Disease,the First Affiliated Hospital of Bengbu Medical University,Bengbu 233004,Anhui Province,China;Anhui Provincial Biochemical Pharmaceutical Technology Research Center,Bengbu 233030,Anhui Province,China)

机构地区：[1]蚌埠医科大学第一附属医院急诊外科,安徽蚌埠233004 [2]蚌埠医科大学药学院,安徽蚌埠233030 [3]蚌埠医科大学第一附属医院呼吸科,安徽蚌埠233004 [4]安徽省生化药物技术研究中心,安徽蚌埠233030

出　　处：《浙江大学学报（医学版）》2024年第4期509-518,共10页Journal of Zhejiang University(Medical Sciences)

基　　金：安徽省高校自然科学研究项目(2022AH051523);蚌埠医学院512人才培育计划(By51202202)。

摘　　要：目的:合成新型补骨脂酚氨基胍衍生物,并探讨其对人三阴性乳腺癌MDA-MB-231细胞死亡的影响。方法:以补骨脂酚为起始化合物,通过甲酰化和席夫碱反应获得补骨脂酚衍生物1、2,并经氢谱、碳谱和高分辨质谱鉴定其结构;用噻唑蓝(MTT)法检测补骨脂酚及其衍生物对细胞存活率的影响;利用荧光显微镜、Anexxin V/PI双染结合流式细胞术法检测补骨脂酚及其衍生物对MDA-MB-231细胞凋亡的影响;蛋白质印迹法检测凋亡相关蛋白的表达;JC-1试剂盒检测细胞线粒体膜电位;DCFH-DA法检测细胞内的活性氧水平。结果:根据波谱数据的分析,本研究合成获得1个新结构的补骨脂酚氨基胍衍生物,其化学名为2-{(E)-5-[(S,E)-3,7-dimethyl-3-vinylocta-1,6-dien-1-yl]-2-hydroxybenzylidene}hydrazine-1-carboximidamide(衍生物2)。MTT法结果显示,补骨脂酚及其衍生物对MDA-MB-231细胞均表现出一定的细胞毒性,其中衍生物2对MDA-MB-231细胞显示较强的细胞毒性,其作用24、48、72 h的IC_(50)值分别为(13.11±1.09)、(6.91±1.78)和(2.23±1.32)μmol/L,但对小鼠正常肝细胞AML-12毒性较低,作用72 h的IC_(50)值为(31.23±1.58)μmol/L。荧光显微镜下观察和流式细胞术检测结果显示,衍生物2诱导乳腺癌细胞凋亡,且呈浓度依赖性。进一步研究发现,衍生物2作用后的MDA-MB-231细胞中Bax/Bcl-2比值增加,细胞色素C蛋白表达水平上调,胱天蛋白酶3激活,线粒体膜电位下降、细胞内活性氧水平显著增高。结论:新型补骨脂酚氨基胍衍生物可诱导MDA-MB-231细胞发生凋亡,且对正常肝细胞的毒性较小,可作为抗三阴性乳腺癌的先导化合物。Objective:To synthesize new bakuchiol aminoguanidine derivatives and test their effect on viability and apoptosis of human triple-negative breast cancer(TNBC)cells.Methods:Two bakuchiol derivatives 1 and 2 were obtained by formylation and Shiff base reaction of bakuchol.The structures of derivatives 1 and 2 were identified by 1H-NMR,13C-NMR,and high-resolution electrospray ionization mass spectrometry(HRESI-MS)analysis.Human TNBC MDA-MB-231 cells were treated with bakuchiol and its derivatives and cell viability was measured by MTT assay.Apoptosis was detected by fluorescence microscopy and flow cytometry with Annexin V-FITC/PI staining.The expressions of apoptosis-related proteins were analyzed with Western blotting.The JC-1 and reactive oxygen species(ROS)assay kits were used to determine the effect of new bakuchiol derivatives on mitochondrial function.Results:Based on spectroscopic analysis,a new bakuchiol schiff base derivative was elucidated as 2-{(E)-5-[(S,E)-3,7-dimethyl-3-vinylocta-1,6-dien-1-yl]-2-hydroxylbenzylidene}hydrazine-1-carboximidamide(derivative 2).Bakuchiol and its derivatives 1 and 2 all showed cytotoxic activity against the MDA-MB-231 cells.Derivative 2 exhibited the most potent cytotoxic activity to MDA-MB-231 cell with IC_(50) of(13.11±1.09),(6.91±1.78),and(2.23±1.32)μmol/L after 24,48,and 72 h.It had low toxicity to normal mouse liver(AML-12)cells with IC_(50) of(31.23±1.58)μmol/L at 72 h.Fluorescence microscopy and flow cytometry demonstrated apoptosis in breast cancer cells after treating with derivative 2 in a concentration dependent manner.Western blotting showed that after derivative 2 treatment,the expression of apoptosis-related proteins cytochrome C,cleaving caspase-3 and Bax/Bcl-2 radio in MDA-MB-231 cells increased;in addition,apoptosis was associated with the decreased mitochondrial membrane potential and increased reactive oxygen species accumulation.Conclusion:The novel bakuchiol aminoguanidine derivative(derivative 2)is capable of inducing apoptosis in MDA-MB-231 cell

关 键 词：补骨脂酚 氨基胍 三阴性乳腺癌 凋亡 线粒体功能 

分 类 号：R979.1[医药卫生—药品]