本研究探讨了紫花提取物(SCE)对乳腺癌多药耐药的影响及其机制。以化疗敏感的乳腺癌细胞株MCF-7和阿霉素(ADR)耐药细胞株MCF-7/ADR为实验对象。MTT法检测细胞增殖活性。Pi染色用于检测细胞周期。4\',6-Diamidino-2-苯基吲哚,二盐酸盐(DAPI)染色和流式细胞术检测细胞凋亡。采用Dansylcadaverine(MDC)染色和GFP-LC3B-Mcherry腺病毒转染检测自噬。Bcl-2、Bax蛋白的表达,caspase-9,caspase-3,LC3B,p62和Beclin-1通过蛋白质印迹检测。结果表明,SCE可以显着抑制敏感和耐药乳腺癌细胞系的增殖。耐药因子为0.53,明显低于ADR的59。同时,SCE处理后G_0/G_1期敏感/耐药细胞比例明显增加。此外,DAPI染色显示一系列细胞凋亡现象如核固缩,染色加深,SCE给药后,敏感/抗性细胞系中出现了核碎片。此外,流式细胞术双染色结果显示,SCE给药后敏感/耐药细胞系中凋亡细胞的比例显著增加。此外,Westernblot显示,SCE给药后,两种乳腺癌细胞系中caspase-3,caspase-9和Bcl-2的蛋白表达水平均显着降低,而Bax蛋白的表达水平均显着升高。此外,SCE还可以增加MDC染色后的阳性荧光斑点和GFP-LC3B-mcherry转染后的黄色荧光斑点,并上调自噬相关蛋白LC3B-Ⅱ的表达水平,p62和Beclin-1在乳腺癌细胞中的表达。总之,SCE可能通过阻断乳腺癌多药耐药细胞的细胞周期发挥抗多药耐药的作用,阻断自噬流,并最终干扰耐药细胞的凋亡抗性。
This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4\',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.