通关藤注射液逆转乳腺癌多柔比星耐药的作用机制  

Mechanism of Tongguanteng Injection on reversing doxorubicin resistance inbreast cancer

作　　者：张小帅 杨柳青 王晓艳[1,2,3,4] 汪彬 孟高全 孟伟亭 李伟霞 陈小菲[1,3] 张明亮 张辉[1,3] 吴娅丽 唐进法 ZHANG Xiaoshuai;YANG Liuqing;WANG Xiaoyan;WANG Bin;MENG Gaoquan;MENG Weiting;LI Weixia;CHEN Xiaofei;ZHANG Mingliang;ZHANG Hui;WU Yali;TANG Jinfa(Department of Pharmacy,The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450003,China;School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan Engineering Research Center for Clinical Application,Evaluation and Transformation of Traditional Chinese Medicine,Henan Key Laboratory for Clinical Pharmacy of Traditional Chinese Medicine,Henan Engineering Research Center of Safety Evaluation and Risk Management of Traditional Chinese Medicine,Zhengzhou 450003,China;Provincial and Ministerial Coconstruction Collaborative Innovation Center for Prevention and Treatment of Respiratory Diseases with Traditional Chinese Medicine,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学第一附属医院药学部,河南郑州450003 [2]河南中医药大学药学院,河南郑州450046 [3]河南省中药临床应用、评价与转化工程研究中心,河南省中药临床药学中医药重点实验室,河南省中药安全评价与风险防控工程研究中心,河南郑州450003 [4]河南中医药大学呼吸疾病中医药防治省部共建协同创新中心,河南郑州450046

出　　处：《中草药》2024年第15期5112-5124,共13页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金青年基金资助项目(82204838);河南省卫生健康委国家中医临床研究基地科研专项(2022JDZX103);河南省医学科技攻关计划省部共建项目(SBGJ202302100);河南省高校科技创新团队(23IRTSTHN026);河南省中医药科学研究专项课题(2024ZY3012);河南省国家中医药传承创新中心科研专项(2023ZXZX1166)。

摘　　要：目的 探究通关藤注射液(Tongguanteng Injection,TGT)对乳腺癌多柔比星(doxorubicin,DOX)耐药的逆转作用及潜在作用机制。方法 分别用DOX、TGT、DOX+TGT干预人乳腺癌DOX耐药MCF-7/DOX细胞株,采用CCK-8法、克隆形成实验检测细胞增殖;Combenefit软件定量分析药物联合作用指数;Transwell实验观察细胞迁移能力;TUNEL染色检测细胞凋亡率;利用罗丹明外排实验检测TGT对MCF-7/DOX细胞外排功能的影响;采用生物信息学手段预测TGT逆转乳腺癌DOX耐药的作用机制,并采用Western blotting实验检测TGT对MCF-7/DOX细胞中耐药相关蛋白表达的调控作用。结果 TGT可逆转MCF-7/DOX细胞的耐药性,且能够协同DOX抑制MCF-7/DOX细胞的增殖、迁移,促进细胞凋亡;此外,TGT增加细胞内Rho-123的蓄积(P<0.001)。TGT成分靶标的基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析结果表明其主要参与调控磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、凋亡等信号通路。GEO数据库筛选得到MCF-7和MCF-7/DOX细胞差异表达基因823个,KEGG富集分析表明乳腺癌DOX耐药主要与PI3K/Akt、Ras相关蛋白1(Ras-proximate-1,Rap1)、凋亡等信号通路相关。TGT联合DOX显著下调MCF-7/DOX细胞中p-PI3K、p-Akt、B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、三磷酸腺苷结合转运蛋白B1(adenosine triphosphate-binding cassette transporter B1,ABCB1)、ABCG2蛋白表达(P<0.05、0.01),且上调Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)表达(P<0.05)。结论 基于生物信息学结合细胞实验验证TGT联合DOX可通过抑制PI3K/Akt介导的凋亡相关信号通路,同时下调ABCB1、ABCG2蛋白表达逆转乳腺癌DOX耐药。为临床TGT与DOX联合用药提供了实验依据,为乳腺癌耐药的治疗提供新的思路。Objective To explore the reversal effect and potential mechanism of Tongguanteng Injection(通关藤注射液,TGT)on doxorubicin(DOX)resistance in breast cancer.Methods MCF-7/DOX cells were treated with DOX,TGT and DOX+TGT,respectively.Cell proliferation was detected by CCK-8 assay and clonal formation assay;Combenefit software was used to analyze the drug combination index quantitatively;Cell migration ability was examined by Transwell assay;Cell apoptosis was detected by TUNEL staining;The effect of TGT on efflux function of MCF-7/DOX cells was detected by rhodamine efflux assay;The mechanism of TGT on reversing DOX resistance in breast cancer was predicted by bioinformatics,and the regulation of TGT on expressions of drug resistance-related proteins in MCF-7/DOX cells was studied by Western blotting.Results TGT could reverse the drug resistance of MCF-7/DOX cells.Meanwhile,TGT combined with DOX could synergistically inhibit the proliferation and migration of MCF-7/DOX cells and promote apoptosis.In addition,TGT increased intracellular accumulation of Rho-123(P<0.001).The results of Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis of TGT component targets showed that they were mainly involved in the regulation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt),mitogen-activated protein kinase(MAPK),apoptosis and other signaling pathways.A total of 823 differentially expressed genes in MCF-7 and MCF-7/DOX cells were screened by GEO database.KEGG enrichment analysis showed that DOX resistance in breast cancer was mainly related to PI3K/Akt,Ras-proximate-1(Rap1),apoptosis and other signaling pathways.TGT combined with DOX significantly down-regulated the protein expressions of p-PI3K,p-Akt,B-cell lymphoma-2(Bcl-2),adenosine triphosphate-binding cassette transporter B1(ABCB1)and ABCG2 in MCF-7/DOX cells(P<0.05,0.01),and up-regulated Bcl-2 associated X protein(Bax)protein expression(P<0.05).Conclusion Based on bioinformatics and cell experiments,it was verified that TGT combined with DOX co

关 键 词：通关藤注射液 多柔比星 乳腺癌 耐药性 PI3K/AKT信号通路 

分 类 号：R285.5[医药卫生—中药学]