Ginkgetin suppresses proliferation and migration of triple-negative breast cancer cells by inducing ferroptosis via the GPX4/OTUB1 axis

doi:10.12092/j.issn.1009-2501.2026.07.001

Abstract

Abstract:

AIM: To investigate the effects of Ginkgetin on ferroptosis in triple-negative breast cancer (TNBC) cells and explore its anti-tumor mechanism via regulating the GPX4/OTUB1 axis. METHODS: MDA-MB-231 and MDA-MB-468 cells were divided into vehicle control (0.1% DMSO), Ginkgetin treatment groups (5, 10, 20 μmol/L), and combination intervention groups (20 μmol/L Ginkgetin + 10 μmol/L Fer-1 or 20 μmol/L DFP). Cell viability was detected by CCK-8 assay; cell proliferation was assessed via EdU staining; colony formation assay was used to evaluate proliferative capacity; cell migration and invasion were measured by Transwell assay; intracellular reactive oxygen species (ROS), lipid peroxidation, and Fe²⁺ levels were quantified by flow cytometry; mitochondrial morphology was observed using transmission electron microscopy (TEM); protein expression (GPX4, OTUB1) was analyzed by Western blot; GPX4-OTUB1 interaction was studied by co-immunoprecipitation (Co-IP); and co-localization of GPX4 and OTUB1 was observed by immunofluorescence. RESULTS: CCK-8, EdU staining, colony formation assay, and Transwell assays demonstrated that Ginkgetin treatment significantly reduced the proliferation, invasion, and migration of MDA-MB-231 and MDA-MB-468 cells in a dose-dependent manner. Ferroptosis-related assays revealed that Ginkgetin-treated cells exhibited markedly increased intracellular ROS levels, lipid peroxidation products, and Fe²⁺ content (P<0.01), accompanied by significantly reduced glutathione (GSH) levels (P<0.01). These effects were partially reversed by ferroptosis inhibitors (Fer-1, DFP). Additionally, Fer-1 and DFP restored cell migration and invasion capabilities. TEM confirmed mitochondrial ultrastructural changes characteristic of ferroptosis. Western blot showed that Ginkgetin significantly downregulated GPX4 expression (P<0.01) and promoted its ubiquitination-dependent degradation. Co-IP confirmed that Ginkgetin inhibited the interaction between OTUB1 and GPX4 (P<0.01). CONCLUSION: Ginkgetin exerts anti-TNBC effects by inhibiting the OTUB1-GPX4 interaction, promoting GPX4 ubiquitination and degradation, and thereby inducing ferroptosis.

Key words: Ginkgetin, triple-negative breast cancer, ferroptosis, glutathione peroxidase 4, deubiquitinase OTUB1

CLC Number:

Feng YANG, Wei SHI, Huiling WANG, Zitong ZHENG. Ginkgetin suppresses proliferation and migration of triple-negative breast cancer cells by inducing ferroptosis via the GPX4/OTUB1 axis[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(7): 865-874.

Figures/Tables 5

Fig.1 Inhibitory effect of ginkgetin on the proliferation of TNBC cell lines ($\bar x $±s, n=4) A: representative morphological changes of MDA-MB-231 and MDA-MB-468 cells after treatment with different concentrations of ginkgetin, scale bar=50 μm. B: cell viability of MDA-MB-231 and MDA-MB-468 cells treated with different concentrations of ginkgetin measured by CCK-8. C: EdU staining was used to observe the effect of ginkgetin on cell proliferation, scale bar=100 μm. D: statistical analysis of the effect of ginkgetin on EdU staining. *P<0.05, **P<0.01, ***P<0.001 vs. 0 μmol/L group.

Fig.2 Ginkgetin inhibits colony formation, migration, and invasion in TNBC cells ($\bar x $±s, n=4) A and B: colony formation assays and quantitative analysis of colony numbers in MDA-MB-231 and MDA-MB-468 cells. C-F: transwell migration and invasion assays with quantitative analysis of migration and invasion numbers. Scale bar=100 μm. *P<0.05, **P<0.01, ***P<0.001 vs. 0 μmol/L group.

Fig.3 Ginkgetin suppresses TNBC cells through the ferroptosis pathway ($\bar x $±s, n=4) A: lipid peroxidation level. B: intracellular GSH level. C: intracellular ROS level. D: intracellular Fe2+ content. E: transmission electron microscopy (TEM) images showing mitochondrial morphology (scale bar = 1 μm). F-I: transwell migration and invasion assays with statistical analysis, scale bar=100 μm. ***P < 0.001 vs. Vehicle group; ###P < 0.001 vs. Gin group.

Fig.4 Ginkgetin promotes ubiquitination and degradation of GPX4 in TNBC cells ($\bar x $±s, n=4) A: Western blot analysis of GPX4 protein levels in MDA-MB-231 and MDA-MB-468 cells after treatment with ginkgetin and ferroptosis inhibitors. B and C: Western blot analysis of GPX4 protein levels in MDA-MB-231 and MDA-MB-468 cells treated with 20 μmol/L ginkgetin and co-treated with the proteasome inhibitor MG-132 (10 μmol/L) (B) or the autophagy inhibitor chloroquine (CQ, 20 μmol/L) (C). ***P < 0.001 vs. Vehicle group; ###P < 0.001 vs. Gin group.

Fig.5 Ginkgetin inhibits the interaction between OTUB1 and GPX4 in TNBC cells ($\bar x $±s, n=4) A: immunofluorescence analysis of the co-localization of OTUB1 (red) and GPX4 (green) in MDA-MB-231 and MDA-MB-468 cells. Nuclei are shown by DAPI staining (blue). Scale bar=10 μm. B: Co-immunoprecipitation (Co-IP) assay detects the interaction between GPX4 and OTUB1 in different treatment groups. ***P < 0.001 vs. Vehicle group.

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