药学学报, 2019, 54(5): 861-866
引用本文:
张如月, 李朵璐, 杨哲, 郭金秀, 周玉冰. 外泌体介导乳腺癌MCF-7细胞对多柔比星耐药性的机制[J]. 药学学报, 2019, 54(5): 861-866.
ZHANG Ru-yue, LI Duo-lu, YANG Zhe, GUO Jin-xiu, ZHOU Yu-bing. Mechanisms of doxorubicin resistance of breast cancer MCF-7 cells mediated by exosomes[J]. Acta Pharmaceutica Sinica, 2019, 54(5): 861-866.

外泌体介导乳腺癌MCF-7细胞对多柔比星耐药性的机制
张如月, 李朵璐, 杨哲, 郭金秀, 周玉冰
郑州大学第一附属医院药学部, 河南省精准临床药学重点实验室, 河南 郑州 450052
摘要:
本研究选用乳腺癌亲本细胞株(MCF-7)、多柔比星(doxorubicin)耐药株(MCF-7/Dox)及多柔比星耐药株上清液共培养的敏感株(MCF-7/Exo)为模型,探讨外泌体(exosomes)在乳腺癌细胞多柔比星耐药传递中的作用及初步分子机制。应用CCK8法和显微镜检测多柔比星对MCF-7、MCF-7/Exo、MCF-7/Dox细胞增殖活性的影响。通过荧光显微镜观察多柔比星对3种细胞凋亡的影响。超速离心法提取3种细胞上清液中的外泌体,用透射电子显微镜、BCA法、DiI标记法检测外泌体的含量,Western blot法检测外泌体特异分子CD63和Flotillin-1的表达水平。应用激光共聚焦显微镜观察MCF-7细胞对MCF-7/Dox细胞来源的外泌体的摄取情况。Western blot检测3种细胞中多药耐药蛋白(ATP-binding cassette subfamily B member 1,ABCB1)的表达水平。细胞增殖活性检测显示,MCF-7/Exo细胞对多柔比星的半数抑制浓度(IC50)为0.83 ±0.09 μmol · L-1,明显高于MCF-7细胞的IC50值0.15 ±0.05μmol·L-1P<0.01),其耐药性提高了5.5倍。细胞凋亡检测显示,多柔比星作用后,MCF-7细胞明显发生凋亡(P<0.001),而与耐药细胞上清共培养的MCF-7/Exo细胞凋亡不明显(P>0.05)。外泌体定量及特异性标记物检测显示,MCF-7/Exo细胞的外泌体明显多于MCF-7细胞(P<0.05)。PKH67示踪标记显示,MCF-7/Dox细胞来源的外泌体可以被MCF-7摄取。Western blot显示,MCF-7/Exo细胞内ABCB1的表达水平明显高于MCF-7。本研究结果表明,多柔比星耐药乳腺癌细胞的外泌体可以向敏感细胞传递耐药性,其机制可能与外泌体介导的ABCB1蛋白转运有关。
关键词:    外泌体      乳腺癌      耐药性      多柔比星      多药耐药蛋白     
Mechanisms of doxorubicin resistance of breast cancer MCF-7 cells mediated by exosomes
ZHANG Ru-yue, LI Duo-lu, YANG Zhe, GUO Jin-xiu, ZHOU Yu-bing
Department of Pharmacy, The First Affiliated Hospital, Zhengzhou University, Henan Key Laboratory for Precision Clinical Pharmacy, Zhengzhou 450052, China
Abstract:
This study aimed to explore the roles of exosomes in doxorubicin-resistance in breast cancer cells. Using breast cancer parental cell line (MCF-7), doxorubicin-resistant cell line (MCF-7/ADR) and sensitive cell line co-cultured with doxorubicin-resistant supernatant (MCF-7/EXO) as models, the effects of doxorubicin on proliferation or apoptosis of MCF-7, MCF-7/EXO and MCF-7/ADR cells were detected by CCK8, and light or fluorescent microscopy. Exosomes in the supernatants of cell culture were extracted by ultracentrifugation, and the quantity of exosomes was determined by transmission electron microscopy, BCA and DiI labeling assay. Expression levels of exosome-specific biomarkers CD63 and Flotillin-1 were detected by Western blot. The uptake of MCF-7/ADR cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy. Western blot was used to detect the expression levels of multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1) in all three cell strains. Cell proliferation assays showed that IC50 of MCF-7/EXO cells to doxorubicin was 0.83 ±0.09 μmol·L-1, which was significantly higher than 0.15 ±0.05 μmol·L-1 (P<0.01) of MCF-7 cells, suggesting 5.5 times of increase in drug resistance. Apoptosis of MCF-7 cells was induced after doxorubicin treatment (P<0.001), but MCF-7/EXO cells were not significantly different (P>0.05). Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells (P<0.05). PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells. Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells. Taken together, these results indicate that exosomes of doxorubicin-resistant breast cancer cells can transmit drug resistance to sensitive cells, and the underlying mechanism may involve ABCB1 protein transport mediated by exosomes.
Key words:    exosome    breast cancer    drug resistance    doxorubicin    multidrug resistance protein   
收稿日期: 2018-11-13
DOI: 10.16438/j.0513-4870.2018-1024
基金项目: 国家自然科学基金资助项目(81402266);河南省科技攻关计划项目(162102410057).
通讯作者: 周玉冰,Tel:86-371-66295269,E-mail:zhouyubing1982@163.com
Email: zhouyubing1982@163.com
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