银杏素通过GPX4/OTUB1轴诱导铁死亡抑制三阴性乳腺癌细胞增殖与迁移

doi:10.12092/j.issn.1009-2501.2026.07.001

中国临床药理学与治疗学 ›› 2026, Vol. 31 ›› Issue (7): 865-874.doi: 10.12092/j.issn.1009-2501.2026.07.001

• 基础研究 • 下一篇

银杏素通过GPX4/OTUB1轴诱导铁死亡抑制三阴性乳腺癌细胞增殖与迁移

杨锋¹(), 史伟¹, 王慧玲²^,*(), 郑自通³^,*()

1. 湖南省人民医院（湖南师范大学第一附属医院）药学部，长沙 410006，湖南
2. 湖南省人民医院（湖南师范大学第一附属医院）乳甲二科，长沙 410006，湖南
3. 南华大学附属第二医院药学部，衡阳 421000，湖南

收稿日期:2025-09-01 修回日期:2025-12-19 出版日期:2026-07-26 发布日期:2026-08-04
通讯作者: 王慧玲,郑自通 E-mail:yangyayue201612@163.com;jonnce.1018@163.com;usczzt@163.com
作者简介:杨锋，男，研究方向：炎症通路与分子肿瘤药理学研究。E-mail：yangyayue201612@163.com
基金资助:
湖南省自然科学基金（2021JJ80015，2026JJ81320）

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

Feng YANG¹(), Wei SHI¹, Huiling WANG²^,*(), Zitong ZHENG³^,*()

1. Department of Pharmacy, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha 410006, Hunan, China
2. Department of Breast and Thyroid Surgery, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha 410006, Hunan, China
3. Department of Pharmacy, The Second Affiliated Hospital of University of South China, Hengyang 421000, Hunan, China

Received:2025-09-01 Revised:2025-12-19 Online:2026-07-26 Published:2026-08-04
Contact: Huiling WANG,Zitong ZHENG E-mail:yangyayue201612@163.com;jonnce.1018@163.com;usczzt@163.com

摘要/Abstract

摘要：

目的: 观察银杏素对三阴性乳腺癌（TNBC）细胞铁死亡的影响，并探讨其通过调控GPX4/OTUB1轴发挥抗肿瘤作用的机制。方法: 将MDA-MB-231和MDA-MB-468细胞分为溶媒对照组（0.1% DMSO）、银杏素组（5、10、20 μmol/L）和联合干预组（20 μmol/L银杏素+10 μmol/L Fer-1或20 μmol/L DFP）。采用CCK-8法检测细胞活力；EdU染色观察细胞增殖；集落形成实验评估细胞增殖能力；Transwell实验检测细胞迁移和侵袭；流式细胞术检测细胞内活性氧（ROS）、脂质过氧化和Fe²⁺水平；透射电镜（TEM）观察线粒体形态；Western blot检测GPX4、OTUB1等蛋白表达；免疫共沉淀分析GPX4与OTUB1的相互作用；免疫荧光观察GPX4和OTUB1的共定位。结果: CCK-8、EdU染色实验、集落形成实验和Transwell实验表明，银杏素处理可引起MDA-MB-231和MDA-MB-468细胞的增殖侵袭迁移能力显著降低，且呈剂量依赖性。铁死亡相关指标检测显示，银杏素处理组细胞内ROS水平、脂质过氧化产物和Fe²⁺含量显著升高（P<0.01），GSH水平显著降低（P<0.01），而铁死亡抑制剂Fer-1和DFP可部分逆转这些效应。铁死亡抑制剂对侵袭迁移的恢复效果也得到了证实。TEM观察到银杏素诱导的典型铁死亡线粒体形态改变。Western blot显示银杏素显著下调GPX4蛋白表达（P<0.01），促进其泛素化降解。免疫共沉淀证实银杏素抑制OTUB1与GPX4的相互作用（P<0.01）。结论: 银杏素可通过抑制OTUB1与GPX4的相互作用，促进GPX4泛素化降解，进而诱导铁死亡，发挥抗TNBC作用。

关键词: 银杏素, 三阴性乳腺癌, 铁死亡, 谷胱甘肽过氧化物酶4, 去泛素化酶OTUB1

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

中图分类号:

杨锋, 史伟, 王慧玲, 郑自通. 银杏素通过GPX4/OTUB1轴诱导铁死亡抑制三阴性乳腺癌细胞增殖与迁移[J]. 中国临床药理学与治疗学, 2026, 31(7): 865-874.

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.

图/表 5

图 1

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.

图 2

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.

图 3

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.

图 4

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.

图 5

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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银杏素通过GPX4/OTUB1轴诱导铁死亡抑制三阴性乳腺癌细胞增殖与迁移

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

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