牛蒡子苷元对白血病K562/A02细胞耐药性的逆转作用及其机制

Zou Lin, Fang Ye, He Wei

General Department, General Hospital of Central Theater Command of the Chinese People's Liberation Army, Wuhan 430070, Hubei Province, China.

General Department, General Hospital of Central Theater Command of the Chinese People's Liberation Army, Wuhan 430070, Hubei Province, China.E-mail:

Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2024 Apr;32(2):409-415. doi: 10.19746/j.cnki.issn.1009-2137.2024.02.013.

OBJECTIVE

To study the effect of arctigenin(ARG) on adriamycin(ADM) resistance of leukemia cell line K562/A02 and the underlying mechanism.

METHODS

Human leukemia cell line K562 and ADM-resistant cell line K562/A02 were cultured and treated with 2.5-50 µmol/L ADM. Cell proliferation was measured using CCK-8 method, and half maximal inhibitory concentration (IC) was calculated. K562/A02 cells were treated with different concentrations of ARG (1, 2, 4, 8, 16 mmol/L) to detect the effect of ARG on K562/A02 cells, and a suitable concentration (2 mmol/L) was selected for subsequent experiments. K562/A02 cells were treated with 2 mmol/L ARG and 5 µmol/L ADM, and cell apoptosis was detected by flow cytometry, the expression of P-gp, MRP, cleaved caspase-3, Bax, Bcl-2 proteins and the TLR4/NF-κB signaling pathway-related proteins were measured by Western blot. TLR4 overexpression plasmid was transfected into K562/A02 cells which were co-treated with ARG and ADM, then drug sensitivity and cell apoptosis were measured.

RESULTS

The IC value of ADM on K562/A02 cells was 36.57 µmol/L, which was significantly higher than that on K562 cells (1.30 µmol/L). ARG with a concentration of ≤2 mmol/L did not have a significant effect on K562/A02 cells. 2 mmol/L ARG significantly reduced the IC of ADM on K562/A02 cells. In 5 µmol/L ADM-treated K562/A02 cells, compared with the control group, the apoptosis rate of K562/A02 cells in the ARG group was significantly increased, the expressions of cleaved caspase-3, Bax proteins were significantly upregulated, the expressions of P-gp, MRP, Bcl-2, TLR4, MyD88, and p-NF-κB proteins were significantly downregulated, and the differences were statistically significant ( < 0.05). After transfection with TLR4 overexpression plasmid, the sensitivity of ARG-treated K562/A02 cells to ADM was reduced ( < 0.05), the cell apoptosis was decreased, and the expressions of P-gp, MRP, Bcl-2 and TLR4/NF-κB signaling pathway-related proteins were significantly elevated, while the expressions of cleaved caspase-3 and Bax proteins were significantly decreased (all < 0.05).

CONCLUSION

ARG may reverse the resistance of human leukemia cell line K562/A02 to ADM by inhibiting TLR4/NF-κB signaling pathway.

目的

研究牛蒡子苷元（ARG）对白血病细胞株K562/A02阿霉素（ADM）耐药性的影响及其潜在机制。

方法

培养人白血病细胞株K562和阿霉素耐药细胞株K562/A02，并用2.5 - 50 μmol/L阿霉素处理。采用CCK - 8法检测细胞增殖情况，并计算半数抑制浓度（IC）。用不同浓度的ARG（1、2、4、8、16 mmol/L）处理K562/A02细胞，检测ARG对K562/A02细胞的作用，选择合适浓度（2 mmol/L）用于后续实验。用2 mmol/L ARG和5 μmol/L阿霉素处理K562/A02细胞，通过流式细胞术检测细胞凋亡情况，采用蛋白质免疫印迹法检测P - gp、MRP、裂解的caspase - 3、Bax、Bcl - 2蛋白以及TLR4/NF - κB信号通路相关蛋白的表达。将TLR4过表达质粒转染至同时用ARG和阿霉素处理的K562/A02细胞中，然后检测药物敏感性和细胞凋亡情况。

结果

阿霉素对K562/A02细胞的IC值为36.57 μmol/L，显著高于对K562细胞的IC值（1.30 μmol/L）。浓度≤2 mmol/L的ARG对K562/A02细胞无显著影响。2 mmol/L ARG显著降低了阿霉素对K562/A02细胞的IC值。在5 μmol/L阿霉素处理的K562/A02细胞中，与对照组相比，ARG组K562/A02细胞的凋亡率显著升高，裂解的caspase - 3、Bax蛋白表达显著上调，P - gp、MRP、Bcl - 2、TLR4、MyD88和p - NF - κB蛋白表达显著下调，差异具有统计学意义（P < 0.05）。转染TLR4过表达质粒后，经ARG处理的K562/A02细胞对阿霉素的敏感性降低（P < 0.05），细胞凋亡减少，P - gp、MRP、Bcl - 2和TLR4/NF - κB信号通路相关蛋白表达显著升高，而裂解的caspase - 3和Bax蛋白表达显著降低（均P < 0.05）。