Erastin2

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

Jian-Shu Loua,b *, Li-Ping Zhaoa,b, Zhi-Hui Huanga,b, Xia-Yin Chena,b, Jing-Ting Xud, William Chi-Shing TAIe, Karl W.K. Tsimd, Yi-Tao Chenc,*, Tian Xie a,b *

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 blotting. Ferroptosis triggered by ginkgetin in DDP-treated NSCLC was observed via a lipid peroxidation assay, a labile iron pool assay, western blotting, 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.

Key words: Ginkgetin, cisplatin, ferroptosis, non-small-cell lung cancer, redox homeostasis

Background

Lung cancer is the leading cause of cancer-related deaths worldwide. The global incidence of lung cancer ranked first in 2018 among all types of cancer (Cao et al., 2019). Lung cancer mortality and incidence have increased rapidly in recent years. In China, the mortality and incidence rates are much higher than those in other regions (Yang et al., 2020), and mortality is expected to increase by ~40% between 2015 and 2030 (Cao et al., 2019). Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for more than 80% of all lung cancers. For NSCLC treatment, immunotherapy usually has a low response rate. Targeted therapy is highly dependent on oncogenic mutations, which account for only a small percentage of the total NSCLC cases. Thus, cisplatin (DDP), a platinum-based chemotherapeutic drug, is still the standard treatment for non-targetable NSCLC, especially for EGFR wild-type NSCLC patients as well as for patients at the advanced stages. Combined drug therapy is a promising strategy to treat NSCLC, especial for those classic chemo-therapeutic drugs. DDP is usually combined with other chemotherapeutic drugs to treat NSCLC. However, patients at the advanced stages of cancer, or with poor overall health, may not be able to tolerate the unexpected side effects induced by the combination of chemo-drugs (Vasconcellos et al., 2020). Therefore, the focus has shifted to a combination of phytochemicals with DDP to increase the therapeutic effect or to eliminate side effects. Extensive studies have demonstrated that phytochemicals can enhance the sensitivity of DDP without causing toxicity (Sun et al., 2019). DDP resistance results from an increase in apoptosis resistance and redox homeostasis resetting. These are the two key processes involved in the therapeutic efficacy of DDP. Thus, phytochemicals that trigger non-apoptotic cell death or disrupt the redox homeostasis resetting could be effective in enhancing the chemosensitivity of DDP as well as in preventing the emergence of resistance.
Ferroptosis is a mode of non-apoptotic cell death, whereby cell death is triggered via iron-dependent lipid peroxidation (Hirschhorn et al., 2019). A recent study demonstrated that ferroptosis is a novel anticancer action for DDP (Guo et al., 2018). Erastin, a classic ferroptosis inducer, induces ferroptosis via system Xc− inhibition and has been shown to synergize with DDP in promoting cytotoxicity in different types of tumors, especially in NSCLC (Guo et al., 2018; Roh et al., 2016; Sato et al., 2018). Drug resistance to DDP occurs via apoptotic evasion, and therefore, ferroptosis is being considered as a new therapeutic method for promoting the efficacy of DDP.
The core mechanism of ferroptosis involves lipid peroxidation and iron accumulation. Ferroptosis is driven by lipid peroxidation, which is typically triggered via the suppression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). SLC7A11 is a cystine-glutamate antiporter. The reduction of SLC7A11 expression leads to cystine depletion, glutathione (GSH) shortage, and a consequent increase in lipid peroxidation (Koppula et al., 2018). GPX4, a key inhibitor of lipid peroxidation, oxidizes GSH to glutathione disulfide (GSSG). Repressing GPX4 compromised the capability in neutralizing lipid peroxidation via GSH (Forcina et al., 2019). Transferrin and solute carrier family 40 member 1 (SLC40A1) are crucial for maintaining the intracellular iron concentration; transferrin imports iron into cells, while SLC40A1 exports iron from cells (Mou et al., 2019). Thus, an increase in transferrin and decrease in SLC40A1 could cause intracellular iron accumulation. Cancer cells are usually adapted to iron and exist under persistent oxidative stress. To protect themselves from ferroptosis-induced cell death, cancer cells can promote their antioxidant systems (Hirschhorn et al., 2019). For instance, nuclear factor erythroid 2–related factor 2 (Nrf2), a master antioxidant regulatory transcription factor, prevents ferroptosis-induced cell death by upregulating antioxidant enzymes (Yu et al., 2017), such as heme oxygenase-1 (HO-1), which contains multiple antioxidant response elements (ARE) in its promoter region (Loboda et al., 2016). In line with this notion, cancer cells with mutations in Nrf2 are able to increase the transcription of antioxidant genes. The induction of ferroptosis through SLC7A11 and GPX4 could be mitigated via Nrf2/HO-1 activation through the elimination of lipid oxidation. Therefore, phytochemicals that suppress the Nrf2/HO-1 antioxidant system could be effective in promoting ferroptosis.
Ginkgetin, a bioflavonoid derived from Ginkgo biloba leaves, has been proposed for the treatment of colorectal (Hu et al., 2019), lung (Lou et al., 2017), and breast cancer (Cao et al., 2017). Using a drug screening platform, ginkgetin was identified to have a better synergistic effect with DDP in inducing cytotoxicity in NSCLC. We previously reported that ginkgetin-induced autophagy and apoptosis inhibitors could not reverse ginkgetin-induced cytotoxicity (Lou et al., 2017). Autophagy induction has been proposed for the degradation of the key factors of ferroptosis in cancer cells, which could trigger ferroptosis (Gao et al., 2016; Park et al., 2019). Thus, it is intriguing to elucidate the possible role of ferroptosis induction in accounting for ginkgetin-induced promotion on anticancer effect of DDP in NSCLC.

Materials and Methods

Cell lines, reagents and antibodies

A549 cell line was obtained from the American Tissue and Cell Collection (ATCC, Manassas, VA). NCIH460 and SPC-A-1 cell lines were kindly gifted by Dr. William Dai in The Hong Kong Polytechnic University. Ginkgetin (>98% purity) was obtained from Chengdu Must Bio-technology Ltd. (Chengdu, China). Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (San Jose, CA). fumarate, sulforaphane, desferoxamine and deferiprone were purchased from MedChemExpress (Monmouth Junction, NJ). Cisplatin (DDP), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis, MO). Calcein-acetoxymethyl ester (CA-AM) was purchased from Shanghai Yisheng Biotechnology Co. (Shanghai, China). BODIPY™ 581/591 C11 was obtained from Thermo Fisher Scientific (Waltham, MA). The culture medium and FBS was obtained from Invitrogen Technologies (Carlsbad, CA). The plasmid pARE-Luc was obtained from Promega Corporation (Madison, WI). The antibodies were obtained from the following sources: Nrf2, HO-1, GPX4, and transferrin were purchased from Abcam (Cambridge, UK); α-tubulin from Sigma-Aldrich (St. Louis, MO); cleaved-PARP, cleavedcaspase 3, cleaved-caspase 7, cleaved-caspase 9, SLC7A11, SLC40A1, horseradish peroxidase (HRP)conjugated goat anti-rabbit antibody, HRP-conjugated goat anti-mouse antibody and Alexa Fluor 555conjugated goat anti-rabbit antibody were from Cell Signalling Technology (Danvers, MA).

Cell viability assay

Cell viability assay was conducted as previously described (Lou et al., 2018). In brief, cells were seeded in 96-well plates, after drug treatment, MTT reagent was added (5 mg/µL, 20 µL/well), incubation for 4 hours. After removing the medium, formazan was dissolved in DMSO (200 µL/well), shacking for 15 min, before the reading by spectrophotometric absorbance at 570 nm.

Apoptosis detection

Cells were treated with ginkgetin, DDP and ginkgetin + DDP. The apoptosis rates of untreated and treated cells were detected by Annexin V-FITC Apoptosis Detection Kit as previously described (Lou et al., 2018). Both floating and adherent cells were collected and wash 3 times by PBS. Cells were stained with Annexin V and PI for 15 min in dark, and detected apoptosis via flow cytometry with the acquisition criteria of 10,000 events for each sample.

Total ROS and MMP measurement

The measurement of total ROS and MMP were conducted as previously described (Lou et al., 2016). For total ROS detection, 15 μM DCFH-DA was added in culture medium without FBS for 0.5 hour. After washing cells twice with PBS, cells were collected and analyzed by flow cytometry (Exc=488 nm, Em=530 ± 30). For MMP measurement, cells were stained with JC-1 (5 μg/ml) for 30 min, then washed twice with PBS before analysis. Both JC-1 monomers (Exc=488 nm, Em=530 ± 30) and aggregates were detected (Exc=561 nm, Em=582 ± 25). Each sample met the acquisition criteria of 10,000 events, and the results were analyzed with Flowjo v7.6 software.

Lipid peroxidation measurement

C11-BODIPY (10 μM) was added to drug treated and untreated cells for 0.5 hour, then cells were collected by trypsin. Oxidation of the polyunsaturated butadienyl portion of C11-BODIPY resulted in a shift of the fluorescence emission peak from 590 nm to 510 nm. Cells were analyzed using flow cytometry (Exc=488 nm, Em=510) after washing twice with PBS, and the results were analyzed with Flowjo v7.6 software.

Measurement of labile iron pool (LIP)

Drug treated and untreated cells were collected and washed 2 times with PBS. Then, cells were loaded with CA-AM (0.25 μM) at the density of 0.5 × 106/ml for 15 min. After washing twice with PBS, cells were incubated with iron chelator deferiprone (100 μM) for 1 hour or untreated. Measurement was conducted using fluorescence microplate reader (Exc=488 nm, Em=525). The amount of LIP was reflected via difference on mean fluorescence of each sample with or without deferiprone.

RNA isolation and real-time PCR

RNAzol RT reagent was used to extract total RNAs, which reversed into cDNAs using HiScript®II One Step qRT-PCR Kit (Vazyme, Shanghai, China) according to the manufacturer’s instruction. In brief, collected cells were lysed with RNAzol RT reagent. RNA was separated by adding RNase-free ddH2O to lysate, after centrifuging, the aqueous layer was collected. RNA was precipitated and washed by ethanol. After removing ethanol, RNA was dried and re-suspended in RNase-free ddH2O. The concentration of RNA was quantified by spectrometry. Next, 1 µg RNA was diluted to 12 µL by RNase-free ddH2O, and 4 µL 4 × gDNA wiper Mix was added, mixed and incubated at 42 oC for 2 min. After adding 4 µL 5 × HiScript III qRT SuperMix, reverse transcription was conducted at 37 oC for 15 min, and stopped at 85 ℃ for 5 sec. The following primers were used: 5’-CCA GGC AGA GAA TGC TGA GTT C-3’ (S) and 5’AAG ACT GGG CTC TCC TTG TTG C-3’ (AS) for HO-1; 5’-TCC TGC TTT GGC TCC ATG AAC G-3’ (S) and 5’-AGA GGA GTG TGC TTG CGG ACA T-3’ (AS) for SLC7A11; 5’-ACA AGA ACG GCT GCG TGG TGA A-3’ (S) and 5’-GCC ACA CAC TTG TGG AGC TAG A-3’ (AS) for GPX4; 5’-GAG ACA AGT CCT GAA TCT GTG CC-3’ (S) and 5’-TTC TTG CAG CAA CTG TGT CAC AG-3’ (AS) for SLC40A1; and 5’-AAC GGA TTT GGC CGT ATT GG-3’ (S) and 5’-CTT CCC GTT CAG CTC TGG G-3’ (AS) for GAPDH. Real-time PCR was performed using SYBR Green Master mix (Vazyme) by Bio-Rad qPCR system (Bio-Rad, Hercules, CA). The data were normalized to amount of GAPDH housekeeping genes.

Western blot analysis

Western blot was conducted as described (Lou et al., 2016). Briefly, cells were lysed, and their protein concentrations were measured using Bradford method. SDS-PAGE was used to separate the protein in each sample. Proteins were transferred from gel to membrane. Then, the membrane was blocked and incubated with indicated primary antibodies. The blots were rinsed before probed with secondary antibodies. The reactive bands were visualized by ECL and calibrated by Chemidoc Imaging System (Bio-Rad).

Immunofluorescence

Cultured A549 cells were seeded on coverslips. Ginkgetin, DDP and ginkgetin + DDP were added for 48 hours. Cells were fixed with 4% formaldehyde after rinsing twice with PBS. Specimens were blocked (1X PBS/5% BSA/0.3% Triton™ X-100) for 1 hour. Then, primary antibody was incubated overnight at 4°C. Then, cells were rinsed 3 times, and probed with Alexa Fluor 555-conjugated goat anti-rabbit secondary antibody for 2 hours. After rinsing, coverslips were mounted to slide by Prolong® Gold Antifade Reagent with DAPI (CST, Danvers, MA). Images were taken by FV3000 Confocal Laser Scanning (Olympus, Japan). To analyse nuclear translocation of HO-1, the co-localization coefficients were calculated using Olympus Fluoview FV31S-DT Software.

Chromatin immunoprecipitation

Chromatin immunoprecipitation was performed using ChIP kit (Abcam, Cambridge, UK). In brief, the proteins were cross-linked to DNA by formaldehyde for 15 min at room temperature. Glycine was added to quench the formaldehyde at final concentration of 125 mM. Cells were washed by ice-cold PBS for 3 times, then resuspended in lysis buffer. The cross-linked lysate was sonicated to shear DNA to an average fragment size of 200-800 bp, then centrifuged and transferred the supernatant for immunoprecipitation. The sonicated chromatin (100 μg) was incubated with anti-Nrf2 antibody, or IgG, or H3 antibody overnight at 4 °C with rotation. DNA purification was carried out according to the manufacturer’s instructions. Then, HO-1 DNA was amplified for 45 cycles of PCR with the following primers: 5′- TCA ATA GGC GAT CAG CAA GGG -3′ (S) and 5′- TGG AAT GCG TGG GAC ACT C -3′ (AS).

Luciferase assay

Luciferase assay was conducted as previously described (Lou et al., 2016). In brief, drug treated and untreated cells were washed and lysed. The supernatant was collected and then analysed using a commercial kit (Thermo Fisher Scientific).

Amino acids detection

Culture A549 cells were treated with ginkgetin, DDP and ginkgetin + DDP for 48 hours. Then, cultured cells at least 107 cells for each group was collected. Two hundred µL precipitant (methanol: acetone: water = 2:2:1) was added to each sample, followed by sonication at 4 oC for 10 min. Samples were stayed at -20 oC for 2 hours. Then, the supernatant was lyophilized, and resolved in 160 µL methanol. The levels of glutamate and cystine were detected by Sciex 5500 LC-MS/MS. Data were analysed by MultiQuant software (SCIEX, Framingham, MA).

GSH/GSSG assay

Cultured A549 cells were treated with ginkgetin, DDP and ginkgetin + DDP for 48 hours. The intracellular level of GSH and GSSG were performed by GSH and GSSG Assay Kit (Beyotime, Shanghai, China), according to the protocol given by manufacturer. Briefly, cells were collected and lysed, then centrifuged at 10,000 × g for 10 min. The supernatant was mixed with GSH assay buffer, GSH reductase, 5,5′-dithio-bis 2-nitrobenzoic acid solution and incubated at 25 °C for 5 min, then NADPH was added. The absorbance at 412 nm was measured by microplate reader. The concentration of total glutathione, or GSSG, was calculated via standard curve, and GSH level was calculated as: GSH = (total glutathione-GSSG) × 2. The ratio of GSH/GSSG was calculated as [GSH]/[GSSG].

Xenograft nude mice model

The establishment of xenograft nude mouse model and ginkgetin preparation were conducted as previously described (Lou et al., 2017). Briefly, when tumours on transplanted nude mice reached around 100 mm3, the mice were randomized divided into eight groups: control, ginkgetin, DDP, ginkgetin + DDP, UAMC 3203, ginkgetin + UAMC 3203, DDP + UAMC 3203, ginkgetin + DDP + UAMC 3203. Both DDP (3 mg/kg) and ginkgetin (30 mg/kg) were administered by intraperitoneal injection, with 2 – 3 times per week and once per day, respectively. UAMC 3203 (10 mg/kg) was administered 5 days/week by intraperitoneally injection. Tumour size and body weight were measured 3 times per week. After dosing 31 days, the nude mice were sacrificed, and tumours were removed and weighed. Animal experiments were performed in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals, approved by Hangzhou Hibio Experimental Animal Ethics Committee (Permit Number: HB201510024-B).

Statistical analysis

Statistical analysis conducted by using one-way analysis of variance. Statistically significant changes were classified as significant (*) where p < 0.05, more significant (**) where p < 0.01, as compared with control or indicated group. Results Ginkgetin promotes DDP-induced cytotoxicity Ginkgetin, a biflavonoid from Ginkgo biloba leaves, exhibits cytotoxicity in cultured A549 cells (Lou et al., 2017). In this study, ginkgetin was combined with DDP at various concentrations and applied to cultured A549 cells. Majority of the combinations (ginkgetin + DDP) significantly increased the cytotoxicity compared to the usage of DDP or ginkgetin alone (Fig. 1A). The combination index (Fig. 1B) and normalized isobologram (Fig. S1B) indicated that ginkgetin at 5 µM, together with different concentrations of DDP, showed a lower CI value, suggesting better synergy. The synergistic effect of ginkgetin at 5 µM with DDP was also demonstrated in NCI-H460 and SPC-A-1 NSCLC cells (Fig. S2A&B). All three NSCLC cell lines were harboring wild-type EGFR, which are not sensitive to target therapy, but are more suitable for DDP treatment. Among these combinations, the mixture of ginkgetin and DDP (both at 5 µM) showed a relatively lower CI value, and the lowest CI value at 0.5005 was observed in the A549 cell line (Fig. 1B&C, S2C). To reveal the pharmacodynamic interaction of ginkgetin and DDP on A549 cells, a response surface fitting was constructed. As expected, the combination of ginkgetin and DDP, each at 5 µM, was located at the highest region of the dose–response surface, further confirming that this combination has better anticancer function (Fig. 1D). This combination ratio was therefore chosen for further mechanistic studies. Ginkgetin induces ferroptosis in DDP-treated cells A previous study demonstrated that ginkgetin induces autophagic cell death (Lou et al., 2017); however, this phenomenon was not further promoted in ginkgetin + DDP-treated cells, as demonstrated by the autophagy marker LC3 and the autophagy flux (Fig S3A&B). In addition, blocking apoptosis, necroptosis, and autophagy could not reverse the anticancer effect induced by ginkgetin + DDP (Fig S3C). In recent years, ferroptosis has been considered to be a consequence of autophagy (Gao et al., 2016; Park et al., 2019). Thus, we hypothesized that ferroptosis might be triggered in this combination. The two key characteristics of ferroptosis are lipid peroxidation and intracellular-free iron (Lei et al., 2019). C11‐BODIPY581/591 could be used as a lipid peroxidation probe in mammalian cells (Ortega et al., 2009). Free iron levels could be measured by staining the labile iron pool (LIP) with CA-AM (Gao et al., 2016). Thus, C11‐BODIPY581/591 and CA-AM were employed here to observe lipid peroxidation and LIP, respectively. DDP did not alter the levels of lipid peroxidation and LIP in A549 cells, while ginkgetin significantly increased the levels by ~2.6- and ~2.4-fold, respectively (Fig. 2A&B). The combination of ginkgetin + DDP increased the levels of lipid peroxidation and LIP by ~3.3 and ~7.1-fold, respectively (Fig. 2A&B). The enhancement of LIP was more robust than that of lipid peroxidation in ginkgetin + DDP-treated cells. Ginkgetin-induced promotion of lipid peroxidation and LIP were also observed in DDP-treated NCI-H460 and SPC-A-1 cells (Fig. S4A&B). SLC7A11 and GPX4 are the main targets for ferroptosis induction. Thus, we revealed the expressions of SLC7A11 and GPX4 at the transcriptional and post-transcriptional levels after treatment with the combined drugs. There were no significant changes in SLC7A11 and GPX4 mRNAs after the combined drug treatment in cultured A549 cells (Fig. 2C), NCI-H460, and SPC-A-1 cells (Fig. S4C). In contrast with the mRNA levels, the protein levels of SLC7A11 and GPX4 were markedly decreased following the application of ginkgetin + DDP in A549 (Fig. 2D), NCI-H460, and SPC-A-1 cells (Fig. S4D). These phenomena suggested the role of ginkgetin in increasing protein degradation of SLC7A11 and GPX4 in DDP-treated NSCLC cells. To further demonstrate the ferroptosis induction, we determined another key factor involved in iron accumulation during ferroptosis, namely SLC40A1 and transferrin. SLC40A1 is the sole iron exporter in mammalian cells as well as a downstream target of Nrf2 (Ka et al., 2018), while transferrin imports iron into cells (Stockwell et al., 2017). In the cultures, DDP sharply increased the mRNA and protein levels of SLC40A1 (Fig. 2E&D). Ginkgetin alone did not change the mRNA level of SLC40A1; however, it reversed the DDP-induced elevation of mRNA (Fig. 2E) as well as protein level (Fig. 2D). In the case of transferrin, DDP slightly increased the protein amount in A549 cells (Fig. 2D), while there was no obvious change in NCI-H460 and SPC-A-1 cells (Fig. S4D). Ginkgetin combined with DDP sharply increased transferrin expression in all three NSCLC cell lines (Fig. 2D, S4D). The decreased SLC40A1 and increased transferrin levels may account for the LIP elevation in combination treatment. The inhibition of SLC7A11 triggers ferroptosis via cystine/glutamate transport. The reduction of SLC7A11 is expected to decrease glutamate release and cystine uptake. In accordance with this notion, we measured cystine and glutamate levels in drug treated A549 cells. The intracellular cystine was significantly decreased, accompanied by notably increased glutamate level, after treatment with ginkgetin (Fig. 2F). No significant change in glutamate level was observed in DDP-treated cells, while the cystine level was notably increased, which might contribute to the increased antioxidant activity. The combined treatment sharply reversed DDP-induced elevation of cystine, and significantly increased glutamate level, as compared to the control (Fig. 2F). These results indicated that the anti-porter function of SLC7A11 was partially reversed after application of ginkgetin in DDP-treated A549 cells. GSH is synthesized from cystine and eliminates lipid ROS via the action of GPX4. As the decline of cystine and GPX4 were observed following the combined drug treatment, the intracellular GSH level was determined. As expected, the amount of GSH was significantly decreased by ginkgetin. However, the amount of GSH increased after application of DDP, which might be due to redox resetting via the antioxidant system. The combined drug treatment sharply reversed the DDP-induced increase in GSH (Fig. 2G). GSH is highly reactive with lipid ROS, and their reaction generates glutathione disulfide (GSSG). A reduced ratio of GSH/GSSG is considered to be a marker of oxidative stress. Ginkgetin + DDP application sharply decreased the ratio of GSH/GSSG (Fig. 2H), indicating an elevation of oxidative stress. All these data indicated that ferroptosis was being triggered by the drug combination. Ginkgetin downregulates the Nrf2/HO-1 axis in DDP-treated NSCLC cells Ferroptosis can be downregulated by the well-known antioxidant system Nrf2/HO-1 via neutralization of oxidative stress, which is responsible for the compromised anticancer function of DDP (Dodson et al., 2019; Panieri et al., 2019). Our previous study demonstrated that ginkgetin could reduce Nrf2 activation; thus, we hypothesized that it could downregulate elevated activity on the Nrf2/HO-1 axis induced by DDP. Here, neither ginkgetin nor DDP could change the expression of Nrf2, which however was sharply reduced following treatment with ginkgetin + DDP in A549, NCI-H460, and SPC-A-1 cells (Fig. 3A, S5A&B). DDP slightly increased the expression of HO-1 in A549 cells (Fig. 3A), and there was no significant change in NCI-H460 and SPC-A-1 cells (Fig. S5A&B), while ginkgetin robustly decreased the amount of HO-1 in all three NSCLC cell lines (Fig. 3A, S5A&B). Activated Nrf2 binds to ARE and upregulates HO-1 transcription. To detect the effect of ginkgetin + DDP on ARE-mediated transcriptional activity, a luciferase reporter, pARE-Luc, was applied. This construct contained four repeats of antioxidant response element (ARE) and a luciferase reporter gene luc2P. In pARE-Luc-expressing A549 cells, DDP led to a ~3-fold increase in ARE-mediated transcription; whereas ginkgetin did not show activation of ARE-mediated transcription (Fig. 3B). Application of ginkgetin in DDP-treated A549 cells largely reversed the DDP-induced activation of ARE-mediated transcription, i.e. it counteracted the induction caused by DDP (Fig. 3B). The CHIP assay was applied to identify the binding of Nrf2 to the HO-1 promoter. DDP sharply increased the binding of Nrf2 to the HO-1 promoter by over 40-fold (Fig. 3C). Ginkgetin alone showed no significant change in binding. As expected, ginkgetin sharply reduced the DDP-induced elevation of the binding of Nrf2 to the HO-1 promoter (Fig. 3C). Binding of Nrf2 to the HO-1 promoter leads to the transcription of HO-1. In cultured A549 cells, DDP increased the mRNA expression of HO-1 (Fig. 3D). The application of ginkgetin significantly reversed the DDP-induced upregulation of HO-1 mRNA expression (Fig. 3D). This mRNA regulation was consistent with the change in protein level; ginkgetin reversed the DDP-induced HO-1 protein expression (Fig. 3A). These results indicated that DDP promotes the antioxidant system Nrf2/HO-1 to cope with ferroptosis-induced oxidative stress, and this can be reversed by ginkgetin. The antioxidant activity induced by Nrf2 is further enhanced by nuclear translocation of HO-1 (Vanella et al., 2016). Thus, the change in HO-1 nuclear translocation in ginkgetin-, DDP-, and ginkgetin + DDPtreated A549 cells was revealed by immunostaining. The fluorescence intensity of HO-1 was observed both in the cytosol and nucleus. In the control group, the fluorescence was mainly located in the cytosol, and a faint signal was observed in the nucleus, as demonstrated by co-localization of the DAPI signal. Application of DDP notably increased the HO-1 fluorescence intensity in the nucleus; however, ginkgetin significantly decreased the nuclear expression of HO-1 and sharply reversed DDP-induced HO-1 nuclear translocation (Fig. 3E&F). This result further confirmed that ginkgetin could reverse DDP-induced activation on the Nrf2/HO-1 axis, which contributes to the mitigation of the antioxidant effect in ginkgetin + DDP-treated NSCLC cells. Ferroptosis inhibition reversed ginkgetin-induced promotion of cytotoxicity of DDP To further observe the role of ferroptosis in ginkgetin + DDP-induced anticancer function. Ferroptosis inhibitors, UAMC 3203 and DFO, were applied. Here, both UAMC 3203 and DFO markedly reversed ginkgetin + DDP-induced cytotoxicity in cultured A549, NCI-H460, and SPC-A-1 NSCLC cells (Fig. 4A, S6A&B). However, the Nrf2 activators DMF and SFN could not reverse ginkgetin + DDP induced cytotoxicity in all three NSCLC cell lines (Fig. 4B, S6A&B). The upregulation of DMF and SFN on Nrf2 was demonstrated by western blotting, which showed that the amount of Nrf2 was significantly increased by DMF and SFN in ginkgetin + DDP-treated A549 cells (Fig. S6C). These phenomena indicated that Nrf2 is not the nodal for ginkgetin + DDP-induced cytotoxicity. UAMC 3203 is a novel ferroptosis inhibitor. The reverse effect of UAMC 3203 in ginkgetin + DDPinduced cytotoxicity was more obvious than that of DFO (Fig. 4A, S6A&B), which might be due to its better ferroptosis suppression activity. Thus, we used UAMC 3203 for further observation. To confirm the ferroptosis suppression, the key markers of lipid peroxidation, LIP, SLC7A11, and GPX4, were determined in A549 cultures. The application of UAMC 3203 moderately reversed the ginkgetin-induced elevation of lipid peroxidation (Fig. 4C&D) and LIP (Fig. 4F), and this effect was much more obvious in the scenario of ginkgetin + DDP (Fig. 4C, D&F). In parallel, UAMC 3203 reversed ginkgetin + DDPmediated decline of SLC7A11, while the reverse effect on GPX4 was identified in cultures treated with ginkgetin or ginkgetin + DDP (Fig. 4E). These results indicated that ferroptosis, induced by ginkgetin + DDP, was blocked by UAMC 3203. Ferroptosis induction could directly or indirectly downregulate GPX4, leading to lipid peroxidation. To confirm the role of ferroptosis in ginkgetin + DDP-induced cytotoxicity, we overexpressed GPX4 in A549 cells, and the upregulated expression of GPX4 was confirmed by western blotting in ginkgetin + DDP-treated cells (Fig. 4G). As expected, the cytotoxicity was notably decreased following GPX4 overexpression (Fig. 4H), concomitant with the downregulation of lipid peroxidation (Fig. 4I) and LIP (Fig. 4J). This result further confirmed that ferroptosis contributes to ginkgetin + DDP-induced cytotoxicity. Ferroptosis suppression mitigated the attenuation of Nrf2/HO-1 activation and ROS promotion induced by ginkgetin in DDP-treated NSCLC cells Redox homeostasis is governed by the balance between the antioxidant system and ROS formation. The downregulation of the antioxidant system Nrf2/HO-1 induced by ginkgetin in DDP-treated NSCLC led us to attempt to determine whether ROS levels were further increased to disrupt redox homeostasis. In cultured NSCLC cells, DDP induced ROS formation, which was sharply promoted by ginkgetin in A549, NCI-H460, and SPC-A-1 cells (Fig. 5A&B, S7A). However, blocking ROS formation via Nacetylcysteine failed to reverse the ginkgetin + DDP-induced cytotoxicity (Fig. S7B). Ferroptosis inhibitors, not Nrf2 activators, could largely reverse ginkgetin + DDP-induced cytotoxicity. In addition, the unchanged mRNA levels of SCL7A11 and GPX4 partially indicated that these two ferroptosis genes were not transcriptionally regulated by Nrf2. Thus, we hypothesized that Nrf2/HO-1 antioxidant inactivation and ROS enhancement could be a consequence of ferroptosis. Here, application of UAMC 3203 sharply rescued the ginkgetin + DDP-induced decline in Nrf2 levels (Fig. 5C) as well as ARE-mediated transcription activity (Fig. 5D). In addition, application of UAMC 3203 reversed the ginkgetin induced suppression on mRNA and protein expression of HO-1 in DDP-treated A549 cells (Fig. 5G&C). Consistent with this, the ginkgetin + DDP-induced ROS increase was sharply reversed by the application of UAMC 3203 in A549 (Fig. 5H), NCI-H460, and SPC-A-1 cells (Fig. S7C). These results indicated that ferroptosis suppression mitigated the attenuation of Nrf2/HO-1 activation and the promotion of ROS formation induced by ginkgetin in DDP-treated NSCLC cells. Ginkgetin-promoted DDP-induced apoptosis was alleviated via ferroptosis suppression ROS elevation induced by ginkgetin in DDP-treated cells might result in increased cell sensitivity to ROS. One consequential effect of ROS elevation is the loss of mitochondrial membrane potential (MMP). Here, we demonstrated that ginkgetin notably increased MMP loss in DDP-treated A549 cells (Fig. 5E&F). MMP loss could lead to activation of caspase-9, consequently activating caspase-3 and -7, leading to apoptosis. Apoptosis is the key mechanism for DDP-induced anticancer effects. Thus, we examined whether apoptosis was increased after ferroptosis induction. Here, we found that 5 µM DDP slightly increased the apoptosis rate by ~15%, while ginkgetin at 5 µM induced apoptosis at ~30% (Fig. 6A&B). The combination of DDP and ginkgetin sharply increased the apoptosis rate to over 50%, which was confirmed by increased levels of apoptotic markers, specifically cleaved-caspase 3, cleaved-caspase 7, and cleaved-caspase 9, as revealed by western blotting (Fig. 6C&D). Intriguingly, blocking apoptosis via Q-VD-Oph could significantly reverse DDP-induced apoptosis, but could not reverse ginkgetin-induced promotion of DDP-induced cytotoxicity (Fig. 6E). In contrast, ferroptosis suppression significantly reversed apoptosis in ginkgetin + DDP-treated cells (Fig. 6F). Consistent with this, ferroptosis suppression attenuated ginkgetin + DDP-induced MMP loss (Fig. 5I), as characterized by a sharp increase in the mean FITC fluorescence and a decline in PerCP-Cy5-5 fluorescence (Fig. 5J), which indicated reduced MMP loss. These results indicated that ferroptosis might contribute to ginkgetin-induced promotion of DDP-triggered apoptosis. Ginkgetin enhanced the anticancer effect of DDP is compromised by ferroptosis suppression in a xenograft nude mouse model To further confirm that ginkgetin promoted the anticancer function of DDP, we applied an A549 xenograft nude mouse model. Following treatment for 31 days, the mean body weight of the DDP-treated group significantly declined, while the ginkgetin group showed no significant change, as compared with control mice (Fig. 7A). Combined administration of ginkgetin + DDP significantly increased the mean body weight since day 25, as compared with the DDP group (Fig. 7A), which might indicate that ginkgetin treatment could relieve DDP-induced toxicity. The mean tumor volumes in the DDP, ginkgetin, and ginkgetin + DDP groups decreased, and the greatest reduction was observed in the combined administration group (Fig. 7B). When combined with UAMC 3203 administration, the mean tumor volume was not significantly changed in the control group as well as in the DDP group. The ginkgetin group showed a moderate increase in mean tumor volume in the presence of UAMC 3203. However, a notable increase was identified in the ginkgetin + DDP group after UAMC 3203 treatment (Fig. 7B). Consistent with the change in tumor volume, the mean tumor weight was smallest in the ginkgetin + DDP group (Fig. 7C&D). UAMC treatment significantly reversed the tumor shrinkage in the ginkgetin group; however, the reversal effect was more robust in the ginkgetin + DDP group (Fig. 7C&D). These results are consistent with an in vitro study supporting the notion that ginkgetin-induced promotion of the anticancer effect of DDP in NSCLC could be mediated by ferroptosis. Discussion Combining DDP with phytochemicals is a promising strategy to enhance its anticancer effect in NSCLC. In this study, we revealed the synergistic effect of ginkgetin with DDP on cytotoxicity in EGFR wild-type NSCLC. The synergy of the combined drugs was further confirmed in an animal study. Ginkgetin exhibited much lower toxicity than DDP, as demonstrated by the much lower cytotoxicity in normal lung cells (Fig. S8) as well as the absence of any significant body weight change in the animal study. Apoptosis resistance and redox resetting are the key factors in accounting for the failure of DDP therapy. Therefore, using this strategy to trigger non-apoptotic cell death and disrupt the redox balance could be a promising method to enhance the anticancer function of DDP. Here, ferroptosis, a non-apoptotic cell death, was robustly triggered by ginkgetin application in DDP-treated EGFR wild-type NSCLC cells, concomitant with the inactivation of the Nrf2/HO-1 axis and promotion of total ROS formation. In addition, DDP-induced MMP loss and apoptosis were robustly amplified under ginkgetin application. Furthermore, the suppression of ferroptosis could diminish the synergy of ginkgetin + DDP as well as reverse the inactivation of Nrf2/HO-1, ROS enhancement, MMP loss, and apoptosis. This is the first study to demonstrate that ferroptosis could account for the increased therapeutic effect of DDP induced by ginkgetin both in vitro and in vivo. Ferroptosis is dependent on iron and is characterized by lipid peroxidation, which could ultimately cause oxidative cell death (Dixon et al., 2012). Recently, ferroptosis was considered to be a novel anti-tumor action of DDP, which could trigger ferroptosis at a relatively high level in A549 (Guo et al., 2018). In addition, autophagy could trigger ferroptosis, and some researchers have suggested that ferroptosis is a part of the autophagy process (Hirschhorn et al., 2019). In our study, we used a relatively low concentration of DDP, which alone could not trigger ferroptosis. Considering that ginkgetin could robustly trigger autophagy, we hypothesized that ginkgetin combined with DDP could potentially lead to ferroptosis induction. Ferroptosis is induced by direct or indirect suppression of GPX4 leading to a decrease in GSH-mediated antioxidant activity and contributing to the elevation of lipid peroxidation. GPX4 is a selenoenzyme that catalyzes the oxidation of GSH to GSSG to inhibit lipid peroxidation. As GPX4 is the most effective selenoenzyme in reducing esterified lipid hydroperoxide, its reduction notably increases lipid peroxidation (Maiorino et al., 2018). SLC7A11 is a cystine/glutamate antiporter. Suppression of SLC7A11 could induce autophagy via the activation of lysosomal-associated membrane protein 2a, which in turn degrades GPX4 (Wu et al., 2019). In addition, SLC7A11 inactivation leads to reduced intracellular concentrations of cystine and increases the glutamate level. The unchangeable mRNA level and dramatically decreased level of SLC7A11 and GPX indicated the increased protein degradation of SLC7A11 and GPX4 induced by ginkgetin + DDP in non-small cell lung cancer cells. There are two major pathways involved in protein degradation, the ubiquitin-proteasome pathway and lysosomal proteolysis. For ubiquitin-proteasome pathway, proteins are marked with ubiquitin, subsequently form multiubiquitin chain, which was recognized and degraded by proteasome. For lysosomal proteolysis pathway, proteins must be taken up to lysosome to degrade by lysosomal proteolysis. Autophagy is one of the crucial ways for this process. Proteins are firstly enclosed in vesicles, then fused with lysosome, and degrade by lysosomal enzymes. We have applied the proteasome inhibitors MG-132 and lysosome inhibitor CQ, and only CQ could rescue the decline of GPX4 (Fig. S9). In addition, our previously study has already demonstrated that ginkgetin could trigger autophagy (Lou et al., 2017), which could by blocked by autophagic inhibitors. Thus, the degradation of GPX4 might be due to autophagy mediated degradation. However, further study needs to be conducted to illustrate the exactly way for GPX4 degradation, as well as SLC7A11. Cystine is an essential substrate for GSH synthesis, and the reduction of cystine leads to a decline in GSH. This could disrupt the equilibrium of the antioxidant system, and thereafter, enhance lipid peroxidation (Yu et al., 2016; Fujii et al., 2019). Consistent with this theory, the expression of SLC7A11 and GPX4, the GSH level, and the GSH/GSSG ratio all sharply decreased, which may contribute to a robust increase in lipid peroxidation during the combined drug treatment. Furthermore, SLC40A1 and transferrin contribute to the export and import of iron, respectively. Decreased SLC40A1 and increased transferrin levels were observed here, which contributed to an increase in the LIP. Thus, the phenomena illustrated above indicated that ferroptosis was triggered by ginkgetin + DDP application. The crucial role of ferroptosis in the anticancer effect of the ginkgetin + DDP combination was further demonstrated by ferroptosis inhibition. Blocking ferroptosis significantly reversed the ginkgetin + DDPinduced anticancer effects both in vitro and in vivo. Intriguingly, the key factors of ferroptosis, SLC7A11, GPX4, and SLC40A1, are all transcriptionally regulated by Nrf2. However, only the mRNA expression of SLC40A1 was significantly changed. In addition, ferroptosis blocking largely reversed the ginkgetin + DDP-induced decrease in Nrf2. The inhibition of Nrf2 could not significantly rescue the ginkgetin + DDP-induced cytotoxicity. These phenomena may have been caused by the promoted degradation of SLC7A11 and GPX4, which evoked ferroptosis, consequently downregulating the Nrf2-mediated suppression of SLC40A1 and ultimately increasing the transcriptional and post-transcriptional levels of SLC40A1. Our previous study demonstrated that ginkgetin induces autophagy via p62. The decline of p62 could downregulate Nrf2, and we found a decrease in the p62 levels under the drug combination (Fig. S9); however, the detailed mechanism requires further study. Nrf2 is a master regulator of antioxidants and detoxification, which contributes to ferroptosis resistance by binding to the ARE of its downstream genes (Iton et al., 2010). Among these, HO-1 has the most abundant ARE sites in its promoter region (Loboda et al., 2016). Suppression of HO-1 could promote ferroptosis via its multiple functions: (i) alleviation of ROS formation via suppression of its anti-oxidant activity (Furfaro et al., 2016); (ii) stabilization of nuclear Nrf2 via its nuclear translocation in an enzymatically inactive form (Biswas et al., 2014); and (iii) decreasing the capture of redox-active cells to increase LIP (Mai et al., 2017). Thus, inactivation of the Nrf2/HO-1 axis could contribute to the promotion of ferroptosis. Consistent with this notion, the application of ginkgetin in DDP-treated cells decreased Nrf2 expression, attenuated the DDP-induced increase in the binding of Nrf2 to the HO-1 promoted region as well as the expression and nuclear translocation of HO-1. These results indicate that ginkgetin attenuated DDP-induced Nrf2/HO-1 activation, which could in turn increase LIP and ROS formation, thereby further promoting ferroptosis and the consequent redox homeostasis disruption in ginkgetin + DDP-treated NSCLC cells. LIP consists of free ferrous iron (Fe2+) in the cytosol. The increased LIP level discovered here could contribute to the elevated Fe2+ and promote ROS formation (Mou et al., 2019). Excess ROS generated by iron may promote redox homeostasis, resulting in the development of a tumor. ROS plays a central role in DDP-induced anticancer effects. Cancer cells can evade DDP-induced ROS formation via redox homeostasis resetting. In this regard, persistent ROS elevation contributes to an adaptive response of cancer cells, leading them to survive under high ROS levels. Here, we found that DDP sharply increased ARE-mediated activity, which can increase the binding of Nrf2 to the HO-1 promoter. Supporting this notion, ARE-mediated activity was much more robust in DDP-resistant cells (Fig. S11). In addition, GSH/GSSG, a system that also contributes to antioxidant activity, was upregulated following DDP application. This evidence indicates that DDP could trigger a resetting of redox homeostasis to cope with increased ROS levels in A549 cells. Here, the DDP-induced redox homeostasis resetting could be disrupted by ginkgetin, as demonstrated by the sharply promoted ROS formation, Nrf2/HO-1 inactivation, and GSH/GSSG downregulation in the DDP-treated NSCLC cells. When the ROS level exceeds the threshold, MMP loss can be triggered by the increase in the permeability of mitochondria (Zhou et al., 2016). This further causes cytochrome c release and caspase-9 activation (Kadenbach et al., 2004), which subsequently accelerate the activation of caspase-3 and 7, finally triggering apoptosis (McIlwain et al., 2015). Consistent with this notion, increases in MMP loss, activated caspase-9, caspase-7, caspase-3, and the apoptosis rate were found in ginkgetin + DDP-treated NSCLC cells. This phenomenon indicated that apoptosis was also promoted. However, blocking Nrf2, ROS formation, and apoptosis did not compromise the ginkgetin-induced promotion of DDP-induced cytotoxicity. While pharmaceutical and genetic suppression of ferroptosis compromised ginkgetin-induced promotion of cytotoxicity. In addition, the ginkgetin-induced promotion of ROS formation, MMP loss, and apoptosis in the DDP-treated NSCLC cells were reversed by ferroptosis inhibition. These phenomena indicated the crucial role of ferroptosis in ginkgetin + DDPinduced anticancer effects. Ferroptosis induction may occur at an early stage in ginkgetin + DDP-induced cytotoxicity. The application of ginkgetin to DDP-treated NSCLC cells first triggered the ferroptosis cascade, that is, SLC7A11 and GPX4 suppression, leading to mitigation of the antioxidant activity of GSH/GSSG and the Nrf2/HO-1 axis, which contributes to redox homeostasis disruption, and finally enhances DDP-induced cell death. Conclusions In summary, ferroptosis contributes to the increased cytotoxicity induced by ginkgetin in DDP-treated EGFR wild-type NSCLC. Several studies have reported the benefits of combining ferroptosis and apoptosis inducers in cancer treatment. This phenomenon was confirmed by our study, which showed that ginkgetin-induced ferroptosis and DDP-induced apoptosis were both amplified by the combination of drugs. 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