[Reversal Roles and Its Mechanism of Asiatic Acid on Multidrug Resistance in K562/ADR Cells Through the Wnt/β-catenin Pathway].

Q4 Medicine

中国实验血液学杂志 Pub Date : 2024-12-01 DOI:10.19746/j.cnki.issn.1009-2137.2024.06.010

Ting Zhang, Yong-Jiao Liu, Lei Zhang, Xin-Yu Zhou, Xiu-Hong Jia

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Abstract

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.

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[通过Wnt/β-catenin通路asia - Acid在K562/ADR细胞多药耐药中的逆转作用及其机制]。

目的：探讨积雪酸（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细胞活力及阿霉素致敏作用的影响。用无毒剂量AA（10、20 μmol/L）处理K562/ADR细胞后，流式细胞术检测ADR的平均荧光强度。采用实时荧光定量PCR检测MRP1、P-gp、β-catenin、C-myc和cyclinD1 mRNA的表达水平。Western blot检测MRP1、P-gp、β-catenin、C-myc、cyclinD1蛋白的表达水平。Western blot检测20 μmol/L AA和Wnt/β-catenin通路激动剂WAY-262611 （5 μmol/L）处理后的K562/ADR细胞中MRP1、P-gp、β-catenin、C-myc和cyclinD1蛋白的表达水平。结果：CCK-8法检测显示，K562/ADR细胞的耐药率是K562细胞的56.57倍，呈稳定耐药状态，差异有统计学意义（P < 0.05）。AA抑制K562/ADR细胞增殖活性呈浓度依赖性（r =0.9666）。与0 μmol/L AA组相比，10和20 μmol/L AA组可显著增强细胞内ADR的平均荧光强度（P < 0.05），逆转细胞对ADR的耐药性（P < 0.05）。细胞中MRP1、P-gp、β-catenin、C-myc、cyclinD1 mRNA及蛋白表达下调（P < 0.05）。与20 μmol/L AA组相比，20 μmol/L AA+WAY组MRP1、P-gp、β-catenin、C-myc和cyclinD1蛋白表达量显著升高（P < 0.05）。结论：AA以浓度依赖性的方式抑制K562/ADR细胞增殖，逆转其对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MRP1和P-gp的表达有关。

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