[Buzhong Yiqi Decoction induces ferroptosis of A549/DDP cells to reverse cisplatin resistance in non-small cell lung cancer via AMPK/ACC1 signaling pathway].

Authors

Jia-Lu Lyu, Jian-Guang Wang, Qiu-Yu Zhao, Wen-Jun Liu, Si-Jia Bai, He Li, Chun Wang

Published in

Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica. Volume 51. Issue 7. Pages 1953-1962.

Abstract

This study aimed to investigate the molecular mechanism by which Buzhong Yiqi Decoction(BZYQD), a representative TCM formula for invigorating the spleen and replenishing Qi, regulates fatty acid metabolism, induces ferroptosis in cisplatin-resistant lung adenocarcinoma A549/DDP cells, and reverses cisplatin resistance in non-small cell lung cancer(NSCLC) via the AMP-activated protein kinase(AMPK)/acetyl-CoA carboxylase 1(ACC1) signaling pathway. The sensitivity of parental A549 cells and cisplatin-resistant A549/DDP cells to different concentrations of cisplatin(0-128 μmol·L~(-1)) was compared, and the optimal cisplatin concentration for subsequent intervention was selected via the CCK-8 assay. The BZYQD-containing serum was prepared, and the effects of cisplatin, low-(L), medium-(M), and high-concentration(H) BZYQD, as well as the combinations of low-, medium-, and high-concentration BZYQD with cisplatin on the viability of A549/DDP cells were evaluated via the CCK-8 assay. RNA-seq was employed to analyze the effect of BZYQD-H+cisplatin treatment on the gene expression profile of A549/DDP cells. A549/DDP cells were randomly allocated into the control group, cisplatin group, and BZYQD-L+cisplatin, BZYQD-M+cisplatin, BZYQD-H+cisplatin groups. Western blot was performed to quantify the protein levels of AMPK, phosphorylated AMPK(p-AMPK), ACC1, fatty acid synthase(FASN), ATP-citrate lyase(ACLY), and carnitine palmitoyltransferase 1A(CPT1A). Immunofluorescence was used to observe lipid droplet synthesis in cells of each group under the intervention of BZYQD+cisplatin and AMPK inhibitor. The CCK-8 assay was adopted to examine the effect of the inhibitor on the viability of A549/DDP cells treated with BZYQD-H+cisplatin, and thus the underlying molecular mechanism was clarified. A ferrous ion assay kit was used to measure intracellular ferrous ion levels. The DCFH-DA fluorescent probe was employed to assess reactive oxygen species(ROS) levels. Western blot was conducted to determine the expression levels of ferroptosis-related proteins, including glutathione peroxidase 4(GPX4), acyl-CoA synthetase 4(ACSL4), and transferrin receptor 1(TFR1). The Liperfluo kit was used to assess intracellular lipid peroxidation levels, and the JC-1 probe was employed to quantify mitochondrial membrane potential. The results showed that cisplatin demonstrated the half-maximal inhibitory concentrations(IC_(50)) of 12.2 μmol·L~(-1) and 41.2 μmol·L~(-1) against A549 cells and A549/DDP cells, respectively, with a resistance index(RI) of 3.38. Compared with the cisplatin group, BZYQD-L+cisplatin, BZYQD-M+cisplatin, and BZYQD-H+cisplatin significantly inhibited the viability of A549/DDP cells. RNA-seq results revealed that the differentially expressed genes in the BZYQD-H+cisplatin group were associated with signaling pathways such as fatty acid synthesis and catabolism, AMPK, and ferroptosis. Compared with the cisplatin group, the BZYQD-L+cisplatin, BZYQD-M+cisplatin, and BZYQD-H+cisplatin groups showed a significant upregulation of p-AMPK, a significant downregulation of fatty acid synthesis-related proteins(FASN, ACC1, and ACLY), and a significant upregulation of CPT1A. Immunofluorescence results indicated that BZYQD+cisplatin treatment inhibited lipid droplet formation by activating the AMPK/ACC1 pathway. Meanwhile, CCK-8 results showed that the AMPK inhibitor significantly reversed the decrease in viability of cells treated by BZYQD-H+cisplatin. Compared with the cisplatin group, the BZYQD-M+cisplatin and BZYQD-H+cisplatin groups exhibited significant increases in intracellular ferrous ion levels, along with significant elevations in intracellular ROS levels and lipid peroxidation levels and a significant reduction in mitochondrial membrane potential. The expression of the ferroptosis-related protein GPX4 was significantly downregulated, suggesting an imbalance in the cellular antioxidant system, while the expression levels of ACSL4 and TFR1 were significantly upregulated. These findings indicate that BZYQD+cisplatin can induce ferroptosis in A549/DDP cells and reverse cisplatin resistance. In summary, the results indicate that BZYQD synergizes with cisplatin to inhibit fatty acid synthesis and induce ferroptosis in A549/DDP cells through the AMPK/ACC1 pathway, thereby reversing cisplatin resistance in NSCLC.

PMID:
42392776
Bibliographic data and abstract were imported from PubMed on 03 Jul 2026.

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