Zhang Ting, Liu Yong-Jiao, Zhang Lei, Zhou Xin-Yu, Jia Xiu-Hong
Department of Pediatrics， Binzhou Medical University Hospital， Binzhou 256603, Shandong Province， China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2024 Dec;32(6):1696-1703. doi: 10.19746/j.cnki.issn.1009-2137.2024.06.010.
To investigate the reversal effect and mechanism of asiatic acid (AA) on multidrug resistance in human adriamycin (ADR) chronic myeloid leukemia K562/ADR cells.
CCK-8 assay was used to detect the resistance of K562 cells and K562/ADR cells to ADR. CCK-8 assay was used to detect the effect of AA on K562/ADR cell viability and adriamycin sensitization. After K562/ADR cells were treated with non-toxic doses of AA(10, 20 μmol/L), the average fluorescence intensity of ADR was detected by flow cytometry. Real-time quantitative PCR was used to detect the expression levels of and mRNA. Western blot was used to detect the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 proteins. Western blot assay was used to detect the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 proteins in K562/ADR cells treated with 20 μmol/L AA and Wnt/β-catenin pathway agonist WAY-262611 (5 μmol/L).
The CCK-8 assay showed that the drug resistance of K562/ADR cells was 56.57 times that of K562 cells, showing stable drug resistance, and the difference was statistically significant ( < 0.05). AA inhibited the proliferative activity of K562/ADR cells in a concentration-dependent manner（ =0.9666）. Compared with 0 μmol/L AA group, the 10 and 20 μmol/L AA groups could significantly enhance the average fluorescence intensity of intracellular ADR ( < 0.05), and reverse the cell resistance to ADR ( < 0.05). The mRNA and protein expressions of MRP1, P-gp, β-catenin, C-myc and cyclinD1 in cells were down-regulated ( < 0.05). Compared with 20 μmol/L AA group, the expression levels of MRP1, P-gp, β-catenin, C-myc and cyclinD1 protein in 20 μmol/L AA+WAY group were significantly increased ( < 0.05).
AA inhibits K562/ADR cell proliferation in a concentration-dependent manner and reverse their resistance to ADR, the reversal mechanism may be related to the down-regulation of MRP1 and P-gp expression after inhibiting Wnt/β-catenin signaling pathway.
探讨积雪草苷（AA）对人阿霉素（ADR）慢性髓系白血病K562/ADR细胞多药耐药的逆转作用及机制。
采用CCK-8法检测K562细胞和K562/ADR细胞对ADR的耐药性。采用CCK-8法检测AA对K562/ADR细胞活力及阿霉素增敏作用。用无毒剂量的AA（10、20μmol/L）处理K562/ADR细胞后，通过流式细胞术检测ADR的平均荧光强度。采用实时定量PCR检测 和 mRNA的表达水平。采用蛋白质免疫印迹法检测MRP1、P-gp、β-连环蛋白、C-myc和细胞周期蛋白D1蛋白的表达水平。采用蛋白质免疫印迹法检测20μmol/L AA和Wnt/β-连环蛋白通路激动剂WAY-262611（5μmol/L）处理的K562/ADR细胞中MRP1、P-gp、β-连环蛋白、C-myc和细胞周期蛋白D1蛋白的表达水平。
CCK-8法检测显示，K562/ADR细胞的耐药性是K562细胞的56.57倍，呈现稳定耐药性，差异具有统计学意义（<0.05）。AA以浓度依赖方式抑制K562/ADR细胞的增殖活性（=0.9666）。与0μmol/L AA组相比，10和20μmol/L AA组可显著提高细胞内ADR的平均荧光强度（<0.05），并逆转细胞对ADR的耐药性（<0.05）。细胞中MRP1、P-gp、β-连环蛋白、C-myc和细胞周期蛋白D1的mRNA和蛋白表达下调（<0.05）。与20μmol/L AA组相比，20μmol/L AA+WAY组中MRP1、P-gp、β-连环蛋白、C-myc和细胞周期蛋白D1蛋白的表达水平显著升高（<0.05）。
AA以浓度依赖方式抑制K562/ADR细胞增殖并逆转其对ADR的耐药性，其逆转机制可能与抑制Wnt/β-连环蛋白信号通路后MRP1和P-gp表达下调有关。
