Objective: To investigate the reversal effect and mechanism of asiatic acid (AA) on multidrug resistance in human adriamycin (ADR) chronic myeloid leukemia K562/ADR cells.
Methods: 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 MRP1, P-gp, β-catenin, C-myc and cyclinD1 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).
Results: 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 (P < 0.05). AA inhibited the proliferative activity of K562/ADR cells in a concentration-dependent manner（r =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 (P < 0.05), and reverse the cell resistance to ADR (P < 0.05). The mRNA and protein expressions of MRP1, P-gp, β-catenin, C-myc and cyclinD1 in cells were down-regulated (P < 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 (P < 0.05).
Conclusion: 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.
题目: 积雪草酸通过Wnt/β-catenin通路逆转白血病K562/ADR细胞的多药耐药性研究.
目的: 探讨积雪草酸(asiatic acid，AA) 对人耐阿霉素（adriamycin，ADR）慢性髓系白血病K562/ADR细胞多药耐药的逆转作用及其机制。.
方法: 采用CCK-8法检测 K562细胞和 K562/ADR细胞对ADR的耐药性；CCK-8法检测AA对K562/ADR 细胞活力的影响及对ADR敏感性的增强作用；无毒剂量的AA(10、20 μmol/L)处理K562/ADR 细胞后，流式细胞术检测细胞内ADR的平均荧光强度；实时荧光定量聚合酶链反应（RT-qPCR）检测多药耐药相关蛋白1(multidrug resistance protein-1，MRP1)、P-糖蛋白(P-glycoprotein，P-gp)、β-连环蛋白（β-catenin）、原癌基因（C-myc）、细胞周期蛋白D1（cyclinD1）的mRNA表达水平；蛋白免疫印迹法（Western blot）法检测MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白的表达水平。20 μmol/L AA联合Wnt/β-catenin通路激动剂WAY-262611（5 μmol/L）处理K562/ADR 细胞后，Western blot 法检测细胞内MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白表达水平的变化。.
结果: CCK-8 检测结果显示，K562/ADR细胞的耐药性是K562细胞的 56.57 倍，具有稳定耐药性，差异具有统计学意义（P < 0.05）。AA可呈浓度依赖性抑制K562/ADR细胞的增殖活力(r =0.9666)。与0 μmol/L AA组相比，10、20 μmol/L AA组可明显增强细胞内ADR的平均荧光强度（P < 0.05），逆转细胞对ADR的耐药性（P < 0.05），明显下调细胞中MRP1、P-gp、β-catenin、C-myc和cyclinD1 mRNA 和蛋白的表达（P 均< 0.05）。与单用20 μmol/L AA组相比，20 μmol/L AA + WAY组细胞中MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白表达水平均明显升高（P 均< 0.05）。.
结论: AA呈浓度依赖性抑制K562/ADR细胞增殖，逆转该细胞对ADR的耐药性，其逆转机制可能与抑制Wnt/β-catenin信号通路后下调耐药相关蛋白MRP1和P-gp的表达有关。.
Keywords: asiatic acid; chronic myeloid leukemia; multidrug resistance; Wnt/β-catenin signaling pathway.