背景:顺铂(DDP)是治疗晚期和不可靶向非小细胞肺癌(NSCLC)的一流药物。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凋亡,铁凋亡诱导剂促进了细胞毒性。因此,增强发酵作用的试剂可以增加DDP的抗癌作用。支持在NSCLC治疗中使用植物化学物质的几个证据。银杏素,一种来源于银杏叶的生物类黄酮,显示通过触发自噬对NSCLC的抗癌作用。自噬可以触发铁凋亡,调节氧化还原稳态。因此,我们旨在阐明在银杏素和DDP在癌症治疗中的协同作用中涉及的铁死亡的可能作用。
方法:通过细胞毒性试验和蛋白质印迹研究了银杏素对DDP诱导的抗癌作用的促进作用。通过脂质过氧化试验观察到在DDP治疗的非小细胞肺癌中由银杏素引发的铁凋亡,不稳定的铁池检测,westernblot,和qPCR。铁性凋亡阻断,铁凋亡对银杏素+DDP诱导的细胞毒性的贡献,Nrf2/HO-1轴,细胞凋亡是通过荧光素酶测定法测定的,免疫染色,染色质免疫沉淀(CHIP),和流式细胞术。通过应用铁凋亡抑制剂说明了铁凋亡在银杏素DDP处理的NSCLC细胞中的作用。这在异种移植裸鼠模型中进一步证明。
结果:银杏素与DDP协同作用以增加NSCLC细胞的细胞毒性,伴随着不稳定的铁库和脂质过氧化的增加。这两个过程都是铁死亡的关键特征。通过SLC7A11和GPX4的表达降低以及GSH/GSSG比率降低,进一步证实了银杏素对铁凋亡的诱导。同时,银杏素破坏了DDP处理细胞的氧化还原止血,如通过增强的ROS形成和Nrf2/HO-1轴的失活所证明的。银杏素还增强了DDP诱导的线粒体膜电位(MMP)损失和培养的NSCLC细胞凋亡。此外,阻断铁凋亡逆转了银杏素诱导的Nrf2/HO-1失活以及ROS形成的升高,MMP损失,和DDP处理的NSCLC细胞中的凋亡。
结论:本研究首次报道了来自银杏叶的银杏素促进DDP诱导的抗癌作用,这可能是由于铁性凋亡的诱导。
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.
CONCLUSIONS: 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.