Zhou Xinhua, Wang Anqi, Wang Liang, Yin Jianhua, Wang Li, Di Lijun, Hoi Maggie Pui-Man, Shan Luchen, Wu Xu, Wang Yuqiang
State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.
PU-UM Innovative Institute of Chinese Medical Sciences, Zhuhai, China.
Front Pharmacol. 2019 Jun 26;10:722. doi: 10.3389/fphar.2019.00722. eCollection 2019.
We previously demonstrated that a Danshensu-Tetramethylpyrazine conjugate DT-010 enhanced anticancer effect of doxorubicin (Dox) in Dox-sensitive human breast cancer cells, and protected against Dox-induced cardiotoxicity. This work was designed to see whether DT-010 overcomes Dox resistance in resistant human breast cancer cells. The effects of DT-010, Dox or their combination on cell viability of Dox-resistant human breast cancer MCF-7/ADR cells were conducted using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was examined by flow cytometry after Annexin V-FITC/PI co-staining. Dox accumulation in MCF-7/ADR cells was detected by flow cytometry and fluorescence microscopy. A fluorometric multidrug resistance (MDR) assay kit was used to evaluate the effect of DT-010 on MDR transporter activity. P-glycoprotein (P-gp) expression and activity were analyzed by Western blot and rhodamine 123 (Rh123) efflux assay, respectively. The effects of DT-010 on glycolysis and mitochondrial stress were detected using an Extracellular Flux Analyzer. A Succinate Dehydrogenase Activity Assay kit was used to measure mitochondrial complex II activity. At non-cytotoxic concentrations, DT-010 in combination with Dox led to a significant growth inhibition of MCF-7/ADR cells, suggesting a synergy between DT-010 and Dox to reverse Dox resistance. DT-010 restored Dox-mediated apoptosis and p53 induction in MCF-7/ADR cells. DT-010 increased Dox accumulation in MCF-7/ADR cells inhibiting P-gp activity, but without changing P-gp expression. Further studies showed that DT-010 significantly inhibited glycolysis and mitochondrial function of MCF-7/ADR cells. Mitochondrial complex II activity was inhibited by DT-010 or DT-010/Dox combination, but not by Dox. The DT-010-mediated suppression of metabolic process may render cells more vulnerable to Dox treatment and thus result in enhanced efficacy. The results indicate that DT-010 overcomes Dox resistance in human breast cancer cells through a dual action simultaneously inhibiting P-gp-mediated drug efflux and influencing metabolic process.
我们之前证明，丹参素 - 川芎嗪共轭物DT - 010增强了阿霉素（Dox）对阿霉素敏感的人乳腺癌细胞的抗癌作用，并预防了阿霉素诱导的心脏毒性。这项工作旨在研究DT - 010是否能克服耐药人乳腺癌细胞中的阿霉素耐药性。使用3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法检测DT - 010、阿霉素或它们的组合对阿霉素耐药的人乳腺癌MCF - 7/ADR细胞活力的影响。用膜联蛋白V - FITC/PI共染色后通过流式细胞术检测细胞凋亡。通过流式细胞术和荧光显微镜检测阿霉素在MCF - 7/ADR细胞中的蓄积。使用荧光多药耐药（MDR）检测试剂盒评估DT - 010对MDR转运蛋白活性的影响。分别通过蛋白质免疫印迹法和罗丹明123（Rh123）外排试验分析P - 糖蛋白（P - gp）的表达和活性。使用细胞外通量分析仪检测DT - 010对糖酵解和线粒体应激的影响。使用琥珀酸脱氢酶活性检测试剂盒测量线粒体复合物II的活性。在非细胞毒性浓度下，DT - 010与阿霉素联合使用导致MCF - 7/ADR细胞显著生长抑制，表明DT - 010与阿霉素之间存在协同作用以逆转阿霉素耐药性。DT - 010恢复了阿霉素介导的MCF - 7/ADR细胞凋亡和p53诱导。DT - 010增加了阿霉素在MCF - 7/ADR细胞中的蓄积，抑制了P - gp活性，但未改变P - gp表达。进一步研究表明，DT - 010显著抑制了MCF - 7/ADR细胞的糖酵解和线粒体功能。DT - 010或DT - 010/阿霉素组合抑制了线粒体复合物II的活性，但阿霉素未起作用。DT - 010介导的代谢过程抑制可能使细胞对阿霉素治疗更敏感，从而提高疗效。结果表明，DT - 010通过双重作用克服了人乳腺癌细胞中的阿霉素耐药性，同时抑制P - gp介导的药物外排并影响代谢过程。
