【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。更多还原

【Abstract】 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 14.20 μmol/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 antitumor effects by activating Hippo signaling pathway, upregulation of MST1 protein expression, activation of LATS1, and regulation of downstream target YAP1 and TAZ expressions, further upregulation of Caspase-3, Cyt-C, and Bax protein expressions, downregulation of Bcl-2 protein expression, initiation of cell apoptosis program.更多还原