摘要
目的研究防己生物碱类活性成分逆转乳腺癌阿霉素耐药潜在的作用机制。方法首先通过TCMSP数据库对防己生物碱类活性成分进行筛选,采用SwissTargetPrediction数据库在线预测防己生物碱类活性成分潜在靶点,通过GeneCards数据库收集疾病靶点,取交集构建“成分-靶点”和蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,然后进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析,最后对预测的防己生物碱类活性成分与核心靶点进行分子对接验证。进一步通过体外实验验证防己生物碱类活性成分抗肿瘤活性,采用CCK-8实验检测防己生物碱类活性成分对乳腺癌MCF-7、MCF-7/ADR细胞的增殖抑制作用,采用倒置光学显微镜、荧光显微镜检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞形态的影响,采用流式细胞仪检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞凋亡的影响,采用Western blotting检测防己生物碱类活性成分对MCF-7、MCF-7/ADR细胞核心靶点蛋白表达的影响。结果N-甲基毛果芸香碱、光千金藤碱、粉防己碱和甜菜碱为逆转乳腺癌阿霉素耐药的防己生物碱类活性成分,可能作用于原癌基因SRC、表皮生长因子受体(epidermal growth factor receptor,EGFR)、热休克蛋白90α家族A类成员1(heat shock protein 90 alpha family class A member 1,HSP90AA1)、蛋白激酶B1(protein kinase b1,AKT1)、有丝分裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、磷脂酰肌醇激酶-3催化亚单位α基因(phosphatidylinositol 3-kinase catalytic alpha polypeptide gene,PIK3CA)、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)等靶点,调控钙信号通路、环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)-cGMP依赖性蛋白激酶(cGMP dependent protein kinase,PKG)信号通路、Apenlin信号通路、维甲酸诱导基因-I(retinoic acid-inducible gene-I,RIG-I)样受体信号通路,从而逆转乳腺癌阿霉素耐药。体外实验结果显示,N-甲基毛果芸香碱、光千金藤碱、粉防己碱和甜菜碱对MCF-7、MCF-7/ADR细胞均具有增殖抑制作用,能够改变细胞形态,诱导细胞发生凋亡,并且对关键通路节点蛋白HSP90AA1、MAPK1、PIK3CA、Caspase-3蛋白表达具有调节作用。结论防己逆转乳腺癌阿霉素耐药主要活性成分为N-甲基毛果芸香碱、光千金藤碱、粉防己碱和甜菜碱,可以通过调控关键靶点蛋白HSP90AA1、MAPK1、PIK3CA、Caspase-3表达,诱导肿瘤细胞发生凋亡,最终逆转乳腺癌阿霉素耐药。防己能够通过多成分、多途径、多靶点逆转乳腺癌阿霉素耐药。
Objective To study the potential mechanism of active components of Fangji(Stephaniae Tetrandrae Radix)alkaloids on reversing adriamycin resistance in breast cancer.Methods Firstly,active components of Stephaniae Tetrandrae Radix alkaloids were screened by TCMSP database,potential targets were predicted online by SwissTargetPrediction database,and disease targets were collected by GeneCards database.Targets were taken by intersection,“component-target”and protein-protein interaction(PPI)networks were constructed,and then gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were carried out.Finally,predicted active components of Stephaniae Tetrandrae Radix alkaloids were verified by molecular docking with core targets.Further,anti-tumor activity of active components of Stephaniae Tetrandrae Radix alkaloids was verified by in vitro experiments,the inhibitory effect of active components of Stephaniae Tetrandrae Radix alkaloids on proliferation of breast cancer MCF-7 and MCF-7/ADR cells was detected by CCK-8 experiment;Effect of active components of Stephaniae Tetrandrae Radix alkaloids on morphology of MCF-7 and MCF-7/ADR cells was detected by inverted optical microscope and fluorescence microscope;Flow cytometry was used to detect the effect of active components of Stephaniae Tetrandrae Radix alkaloids on apoptosis of MCF-7 and MCF-7/ADR cells;Western blotting was used to detect the effect of active components of Stephaniae Tetrandrae Radix alkaloids on core targets protein expressions in MCF-7 and MCF-7/ADR cells.Results N-methylflindersine,stepharine,tetrandrine and betaine were the active components of Stephaniae Tetrandrae Radix alkaloids in reversing adriamycin resistance in breast cancer,which may act on proto-oncogene SRC,epidermal growth factor receptor(EGFR),heat shock protein 90αfamily member 1(HSP90AA1),protein kinase B1(AKT1),mitogen-activated protein kinase 1(MAPK1),phosphoinositide 3-kinase catalyticαpolypeptide gene(PIK3CA),cysteine-aspartate protease-3(Caspase-3)and other targets,regulate calcium signaling pathway,cyclic guanosine monophosphate(cGMP)-cGMP dependent protein kinase(PKG)signaling pathway,Apenlin signaling pathway and retinoic acid-induced gene-I(RIG-I)-like receptor signaling pathway,thus reversing the adriamycin resistance of breast cancer.The in vitro experimental results showed that N-methylflindersine,stepharine,tetrandrine and betaine all had inhibitory effects on the proliferation of MCF-7 and MCF-7/ADR cells,which could change the cell morphology,induce cell apoptosis,and regulate the expressions of key pathway node proteins HSP90AA1,MAPK1,PIK3CA and Caspase-3.Conclusion The main active components of Stephaniae Tetrandrae Radix alkaloids in reversing the adriamycin resistance of breast cancer are N-methylflindersine,stepharine,tetrandrine and betaine,which can induce the apoptosis of tumor cells by regulating the expression of key target proteins HSP90AA1,MAPK1,PIK3CA and Caspase-3,and finally reverse the adriamycin resistance of breast cancer.Stephaniae Tetrandrae Radix can reverse adriamycin resistance of breast cancer through multi-component,multi-channel and multi-target.
作者
杨燚
王毛毛
于淼
季宇彬
李文兰
辛国松
李海茹
YANG Yi;WANG Mao-mao;YU Miao;JI Yu-bin;LI Wen-lan;XIN Guo-song;LI Hai-ru(Engineering Research Center for Medicine,Harbin University of Commerce,Harbin 150076,China;Anti-tumor Natural Drug Engineering Research Center,Ministry of Education,Harbin 150076,China;Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China)
出处
《中草药》
CAS
CSCD
北大核心
2023年第9期2841-2851,共11页
Chinese Traditional and Herbal Drugs
基金
中国博士后面上项目(2021MD703828)
黑龙江省自然科学基金优秀青年项目(YQ2022H002)
黑龙江省博士后资助项目(LBHZ20172)
2022年哈尔滨商业大学产业化项目支持计划(XL0086)
哈尔滨商业大学研究生科研创新项目(YJSCX2022-753HSD)。
关键词
防己
生物碱类活性成分
乳腺癌
多药耐药
网络药理学
N-甲基毛果芸香碱
粉防己碱
光千金藤碱
甜菜碱
Stephaniae Tetrandrae Radix
alkaloid active components
breast cancer
multidrug resistance
network pharmacology
N-methylflindersine
tetrandrine
stepharine
betaine
作者简介
杨燚(1999—),女,硕士研究生,研究方向为抗肿瘤中药药理学。;通信作者:辛国松(1984—),男,副研究员,博士(博士后),硕士生导师,研究方向为抗肿瘤中药药理学。E-mail:13766801150@163.com;通信作者:李海茹(1988—),女,副教授,博士(博士后),硕士生导师,研究方向为中药药理学。E-mail:lihairu0419@126.com。
