从银杏叶中提取的银杏萜内酯通过铁死亡介导的 Nrf2/HO-1 轴破坏增强 EGFR 野生型非小细胞肺癌中顺铂的治疗效果。

Ginkgetin derived from Ginkgo biloba leaves enhances the therapeutic effect of cisplatin via ferroptosis-mediated disruption of the Nrf2/HO-1 axis in EGFR wild-type non-small-cell lung cancer.

机构信息

College of Pharmacy, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, 311121, China; Key Laboratory of Elemene Class Anti-Cancer Chinese Medicines, Engineering Laboratory of Development and Application of Traditional Chinese Medicines, Collaborative Innovation Center of Traditional Chinese Medicines of Zhejiang Province, Hangzhou Normal University, Hangzhou, Zhejiang, 31121, China.

出版信息

Phytomedicine. 2021 Jan;80:153370. doi: 10.1016/j.phymed.2020.153370. Epub 2020 Oct 9.

DOI:10.1016/j.phymed.2020.153370

PMID:33113504

Abstract

BACKGROUND

Cisplatin (DDP) is the first-in-class drug for advanced and non-targetable non-small-cell lung cancer (NSCLC). A recent study indicated that DDP could slightly induce non-apoptotic cell death ferroptosis, and the cytotoxicity was promoted by ferroptosis inducer. The agents enhancing the ferroptosis may therefore increase the anticancer effect of DDP. Several lines of evidence supporting the use of phytochemicals in NSCLC therapy. Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, showed anticancer effects on NSCLC by triggering autophagy. Ferroptosis can be triggered by autophagy, which regulates redox homeostasis. Thus, we aimed to elucidate the possible role of ferroptosis involved in the synergistic effect of ginkgetin and DDP in cancer therapy.

METHODS

The promotion of DDP-induced anticancer effects by ginkgetin was examined via a cytotoxicity assay and western blot. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blot, and qPCR. With ferroptosis blocking, the contribution of ferroptosis to ginkgetin + DDP-induced cytotoxicity, the Nrf2/HO-1 axis, and apoptosis were determined via a luciferase assay, immunostaining, chromatin immunoprecipitation (CHIP), and flow cytometry. The role of ferroptosis in ginkgetin + DDP-treated NSCLC cells was illustrated by the application of ferroptosis inhibitors, which was further demonstrated in a xenograft nude mouse model.

RESULTS

Ginkgetin synergized with DDP to increase cytotoxicity in NSCLC cells, which was concomitant with increased labile iron pool and lipid peroxidation. Both these processes were key characteristics of ferroptosis. The induction of ferroptosis mediated by ginkgetin was further confirmed by the decreased expression of SLC7A11 and GPX4, and a decreased GSH/GSSG ratio. Simultaneously, ginkgetin disrupted redox hemostasis in DDP-treated cells, as demonstrated by the enhanced ROS formation and inactivation of the Nrf2/HO-1 axis. Ginkgetin also enhanced DDP-induced mitochondrial membrane potential (MMP) loss and apoptosis in cultured NSCLC cells. Furthermore, blocking ferroptosis reversed the ginkgetin-induced inactivation of Nrf2/HO-1 as well as the elevation of ROS formation, MMP loss, and apoptosis in DDP-treated NSCLC cells.

CONCLUSION

This study is the first to report that ginkgetin derived from Ginkgo biloba leaves promotes DDP-induced anticancer effects, which can be due to the induction of ferroptosis.

摘要

背景

顺铂（DDP）是治疗晚期和不可靶向非小细胞肺癌（NSCLC）的一线药物。最近的一项研究表明，DDP 可以轻微诱导非凋亡性细胞死亡铁死亡，并且细胞毒性可以通过铁死亡诱导剂促进。因此，增强铁死亡的药物可能会增加 DDP 的抗癌效果。有几条证据支持在 NSCLC 治疗中使用植物化学物质。银杏素是银杏叶中的一种生物类黄酮，通过触发自噬对 NSCLC 表现出抗癌作用。铁死亡可以通过调节氧化还原平衡的自噬来触发。因此，我们旨在阐明铁死亡在银杏素和 DDP 联合治疗癌症中的协同作用中的可能作用。

方法

通过细胞毒性测定和 Western blot 检测银杏素对 DDP 诱导的抗癌作用的促进作用。通过脂质过氧化测定、不稳定铁池测定、Western blot 和 qPCR 观察银杏素在 DDP 处理的 NSCLC 中触发的铁死亡。通过荧光素酶测定、免疫染色、染色质免疫沉淀（CHIP）和流式细胞术，确定铁死亡阻断对银杏素+DDP 诱导的细胞毒性、Nrf2/HO-1 轴和细胞凋亡的贡献。通过应用铁死亡抑制剂来阐明铁死亡在银杏素+DDP 处理的 NSCLC 细胞中的作用，并在异种移植裸鼠模型中进一步证明。

结果

银杏素与 DDP 协同作用增加 NSCLC 细胞的细胞毒性，同时增加不稳定铁池和脂质过氧化。这两个过程都是铁死亡的关键特征。银杏素诱导的铁死亡通过 SLC7A11 和 GPX4 的表达降低以及 GSH/GSSG 比值降低得到进一步证实。同时，银杏素破坏了 DDP 处理细胞中的氧化还原平衡，表现为 ROS 形成增加和 Nrf2/HO-1 轴失活。银杏素还增强了 DDP 诱导的培养 NSCLC 细胞中线粒体膜电位（MMP）丧失和细胞凋亡。此外，阻断铁死亡逆转了银杏素诱导的 Nrf2/HO-1 失活以及 DDP 处理的 NSCLC 细胞中 ROS 形成、MMP 丧失和细胞凋亡的增加。