Cui Xi-He, Zeng Rui, Zang Yuan-Long, Yang Qing, Zhu Xiao-Xin, Wang Ya-Jie
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2023 May;48(9):2360-2367. doi: 10.19540/j.cnki.cjcmm.20230103.701.
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.
本研究探讨瑞香狼毒提取物(SCE)对乳腺癌多药耐药性的影响及其潜在机制。将化疗敏感的乳腺癌细胞系MCF-7和阿霉素(ADR)耐药细胞系MCF-7/ADR作为实验对象。采用MTT法检测细胞增殖活性。采用碘化丙啶染色检测细胞周期。采用4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色和流式细胞术检测细胞凋亡。采用丹磺酰尸胺(MDC)染色和GFP-LC3B-Mcherry腺病毒转染检测自噬。通过蛋白质免疫印迹法检测Bcl-2、Bax、半胱天冬酶-9、半胱天冬酶-3、LC3B、p62和Beclin-1的蛋白表达。结果显示,SCE能显著抑制敏感和耐药乳腺癌细胞系的增殖。耐药因子为0.53,显著低于ADR的59。同时,SCE处理后,G_0/G_1期敏感/耐药细胞的比例显著增加。此外,DAPI染色显示,SCE给药后,敏感/耐药细胞系出现了一系列凋亡现象,如核固缩、染色加深和核碎裂。而且,流式细胞术双染结果显示,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,蛋白质免疫印迹法显示,SCE给药后,两种乳腺癌细胞系中半胱天冬酶-3、半胱天冬酶-9和Bcl-2的蛋白表达水平显著降低,Bax蛋白的表达水平显著升高。此外,SCE还能增加MDC染色后的阳性荧光斑点以及GFP-LC3B-mcherry转染后的黄色荧光斑点,并上调乳腺癌细胞中自噬相关蛋白LC3B-Ⅱ、p62和Beclin-1的表达水平。综上所述,SCE可能通过阻断乳腺癌多药耐药细胞的细胞周期、阻断自噬流,最终干扰耐药细胞的凋亡抗性,发挥抗多药耐药的作用。