基于GPER介导途径探讨隐丹参酮诱导人乳腺癌SKBR-3细胞凋亡的分子机制  

Molecular mechanism of apoptosis of breast cancer SKBR-3 cells induced by cryptanshinone via G protein coupled estrogen receptor( GPER) mediated pathway

作　　者：石丹宁 崔丽霞 赵丕文 孙丽萍 陈梦 牛建昭 SHI Dan-ning;CUI Li-xia;ZHAO Pi-wen;SUN Li-ping;CHEN Meng;NIU Jian-zhao(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China;School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学生命科学学院,北京100029 [2]北京中医药大学中医学院,北京100029

出　　处：《中国中药杂志》2019年第22期4905-4911,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81673764);教育部&国家外国专家局“北京中医药大学中西医结合学科创新引智基地”项目(B07007);北京中医药大学研究生自主课题(2018-JYB-XS100);北京中医药大学中央高校基本科研业务费专项(2017-JYB-JS-001)

摘　　要：基于G蛋白偶联雌激素受体(GPER)及其介导的PI3K/AKT途径探讨隐丹参酮(cryptotanshinone,CPT)诱导人乳腺癌细胞凋亡的效应及其分子机制。选用核ER阴性、GPER阳性人乳腺癌SKBR-3细胞,采用Annexin V-FITC/PI双染法检测CPT对细胞凋亡率的影响,Western blot法检测CPT对凋亡效应蛋白caspase-3表达的影响;Western blot及免疫荧光技术检测CPT对GPER介导的PI3K/AKT通路上关键蛋白表达的调控效应;同时,为进一步明确GPER介导的PI3K/AKT信号通路在CPT诱导SKBR-3细胞凋亡中的作用,选用GPER特异性激动剂G1、特异性拮抗剂G15及PI3K特异性抑制剂LY294002进行干预。结果显示,5,10μmol·L^-1CPT作用48 h后,SKBR-3细胞的凋亡率上升为46.1%,69.0%(P<0.01),caspase-3表达则随着CPT浓度增加而升高(P<0.01)。PI3K,AKT,p-AKT蛋白表达均可被CPT抑制(P<0.05或P<0.01),G1与5μmol·L^-1CPT联合作用48 h后可见AKT与p-AKT的表达量进一步降低(P<0.05),而G15与5μmol·L^-1CPT联合作用后AKT与p-AKT表达的下降被明显抑制(P<0.05);1μmol·L^-1LY294002干预下,可见AKT,p-AKT的表达受到了更为明显的抑制(P<0.01)。AKT与p-AKT的荧光表达强度同样能够受到CPT抑制,G1,G15,LY294002的干预与Western blot结果一致(P<0.05或P<0.01)。此外,CPT对SKBR-3细胞中GPER表达具有一定的抑制效应(P<0.01),加入G1可相对抑制CPT对GPER表达的下调作用;加入G15可使CPT对GPER表达的抑制作用进一步增强。综上,隐丹参酮可诱导人乳腺癌SKBR-3细胞凋亡,其分子途径可能与隐丹参酮对GPER表达及其介导的PI3K/AKT信号通路的调控作用有关。The study aimed to illuminate the role of G protein coupled estrogen receptor( GPER) and its mediated PI3 K/AKT signaling pathway in cryptotanshinone( CPT) induced apoptosis of breast cancer SKBR-3 cells,which is GPER positive and ER negative.The apoptosis rate of SKBR-3 cells was tested by Annexin V-FITC/PI staining and apoptosis effector caspase-3 was determined by Western blot. The key proteins in PI3 K/AKT signaling pathway mediated by GPER were detected by Western blot and immunofluorescence technique. Meanwhile,the agonist G1 and antagonist G15 of GPER and antagonist LY294002 of PI3 K were employed in the test to further clarify the effect of GPER and PI3 K/AKT pathway. The results indicated that the apoptosis rate was increased from 4. 7% to46. 1% and 69. 0% after treatment with 0,5,10 μmol·L-1 CPT for 48 h( P<0. 01). The expression of PI3 K,AKT and p-AKT were inhibited( P<0. 05 or P<0. 01),while caspase-3 level increased obviously after treatment with CPT( P<0. 01). Importantly,inhibitory effect of PI3 K/AKT signaling pathway by CPT was further enhanced by G1 and attenuated by G15. LY294002 also induced a further inhibition of expression of AKT and p-AKT. The mean fluorescence intensity of AKT and p-AKT could be decreased by CPT. Furthermore,CPT could downregulate GPER expression in SKBR-3 cells( P<0. 01),which could be inhibited by G1 and enhanced by G15.In conclusion,CPT could induce the apoptosis of ER negative and GPER positive breast cancer SKBR-3 cells and the molecular mechanism is related to its regulatory effect of GPER and its mediated PI3 K/AKT signaling pathway.

关 键 词：隐丹参酮 GPER 乳腺癌 SKBR-3 PI3K/AKT 细胞凋亡 植物雌激素 

分 类 号：R73[医药卫生—肿瘤]