基于Hippo/YAP信号通路探究粉防己碱抗乳腺癌耐药机制  

Mechanism of tetrandrine against drug resistance in breast cancer based on Hippo/YAP signaling pathway

作　　者：辛国松 王毛毛[1] 侯妍秀 杨燚 于淼 季宇彬 李文兰[1,2] 李海茹 XIN Guo-song;WANG Mao-mao;HOU Yan-xiu;YANG Yi;YU Miao;JI Yu-bin;LI Wen-lan;LI Hai-ru(Engineering Research Center for Medicine,Harbin University of Commerce,Harbin 150076,China;National Ministry of Education Anti-tumor Natural Drug Engineering Research Center,Harbin 150076,China;Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China)

机构地区：[1]哈尔滨商业大学药物工程技术研究中心,黑龙江哈尔滨150076 [2]国家教育部抗肿瘤天然药物工程研究中心,黑龙江哈尔滨150076 [3]哈尔滨医科大学附属第二医院,黑龙江哈尔滨150086

出　　处：《中草药》2023年第18期5960-5967,共8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);哈尔滨商业大学产业化项目支持计划(XL0086)。

摘　　要：目的研究粉防己碱通过调控Hippo/Yes-相关蛋白同源癌蛋白(homologous oncoproteins Yes-associated protein,YAP)信号通路抗乳腺癌多重耐药的分子机制。方法采用CCK-8法检测粉防己碱对乳腺癌MCF-7/ADR细胞增殖的影响;采用倒置显微镜、荧光显微镜观察细胞形态;采用流式细胞仪检测细胞凋亡率;采用细胞集落实验检测细胞克隆形成能力;采用Transwell实验检测细胞侵袭能力;采用Western blotting检测细胞耐药外排蛋白[P-糖蛋白(P-glycoprotein,P-gp)、乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)、多药耐药相关蛋白(multidrug resistance associated protein,MRP)],Hippo通路关键节点蛋白[YAP1、大肿瘤抑制激酶1(large tumor suppressor kinase 1,LATS1)、哺乳动物不育系20样激酶1(mammalian Sterile 20-like kinase 1,MST1)、转录共活化因子(transcriptional co-activator with PDZ-binding motif,TAZ)]和凋亡关键蛋白[半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)、细胞色素C(cytochrome-C,Cyt-C)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)]表达。结果粉防己碱对MCF-7/ADR细胞具有增殖抑制作用,半数抑制浓度(half inhibitory concentration,IC50)值为14.20μmol/L;粉防己碱能够改变细胞形态,部分细胞出现凋亡迹象;粉防己碱显著诱导MCF-7/ADR细胞发生凋亡(P<0.01),抑制细胞的增殖分化和侵袭能力,显著下调P-gp、BCRP、MRP1、YAP1、TAZ和Bcl-2蛋白表达(P<0.01),显著上调MST1、LATS1、Caspase-3、Cyt-C和Bax蛋白表达(P<0.01)。结论粉防己碱能够通过下调MCF-7/ADR细胞P-gp、BCRP和MRP1蛋白表达,抑制外排蛋白活性,增加细胞内粉防己碱含量蓄积;并通过激活Hippo信号通路,上调MST1蛋白表达,激活LATS1,进而调控下游靶点YAP1和TAZ表达,进一步上调Caspase-3、Cyt-C和Bax蛋白表达,下调Bcl-2蛋白表达,启动细胞凋亡程序,发挥抗肿瘤作用。Objective To study the molecular mechanism of tetrandrine against multidrug resistance in breast cancer by regulating Hippo/homologous oncoproteins Yes-associated protein(YAP)signaling pathway.Methods CCK-8 method was used to detect the effect of tetrandrine on proliferation of MCF-7/ADR cells;Inverted microscopy and fluorescence microscopy were used to observe cell morphology;Flow cytometry was used to detect cell apoptosis rate;Cell colony assay was used to detect cell clone formation ability;Transwell experiment was used to detect cell invasion ability;Western blotting was used to detect expressions of resistance efflux proteins[P-glycoprotein(P-gp),breast cancer resistance protein(BCRP)and multidrug resistance associated protein(MRP)],Hippo pathway key node proteins[YAP1,large tumor suppressor kinase 1(LATS1),mammalian Sterile 20-like kinase 1(MST1),transcriptional co activator with PDZ binding motif(TAZ)],and apoptosis key proteins[cysteine aspartate protease-3(Caspase-3),cytochrome C(Cyt-C),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)].Results Tetrandrine had a proliferative inhibitory effect on MCF-7/ADR cells,with a half inhibitory concentration(IC50)value of 14.20μmol/L;Tetrandrine could change cell morphology and some cells showed signs of apoptosis;Tetrandrine significantly induced apoptosis in MCF-7/ADR cells(P<0.01),inhibited cell proliferation,differentiation and invasion ability,significantly down-regulated the expressions of P-gp,BCRP,MRP1,YAP1,TAZ and Bcl-2 proteins(P<0.01),and significantly up-regulated the expressions of MST1,LATS1,Caspase-3,Cyt-C and Bax proteins(P<0.01).Conclusion Tetrandrine can inhibit efflux protein activity and increase intracellular accumulation of tetrandrine by down-regulating the expressions of P-gp,BCRP and MRP1 proteins in MCF-7/ADR cells.And tetrandrine exerts anti-tumor effects by activating Hippo signaling pathway,upregulation of MST1 protein expression,activation of LATS1,and regulation of downstream target YAP1 and TAZ expressions,further upregu

关 键 词：粉防己碱 乳腺癌 阿霉素 耐药性 Hippo/YAP信号通路 

分 类 号：R285.5[医药卫生—中药学]