GPX4 overexpressed non-small cell lung cancer cells are sensitive to RSL3-induced ferroptosis

Ferroptosis can be induced by inhibiting antioxidant enzymes GPX4 or system Xc−, increased intracellular iron concentrations, and lipid peroxidation. Recently, it has been suggested that ferroptosis can be an effective way to induce cancer cell death, although the specific relevance and mechanism of ferroptosis have not been fully elucidated. Here, we investigated the anticancer effects of ferroptosis inducers erastin and RSL3 on non-small cell lung cancer (NSCLC) cells. RSL3 induced cell death more effectively in NSCLC cells than erastin, with limited cytotoxicity in BEAS-2B normal bronchial epithelial cells. The sensitivity of NSCLC cells to RSL3 induced death was dependent on GPX4 expression levels; the effect of RSL3 was reversed by ferrostatin-1 (a ferroptosis inhibitor) but not by Z-VAD-FMK, chloroquine, bafilomycin A1, or necrostatin-1. RSL3 induced ferroptosis by promoting lipid peroxidation, elevating intracellular LIP concentration and ROS level, and blocking GSH-to-GSSH conversion through the inhibition of GPX4 and induction of Nrf2/HO1. Furthermore, RSL3 induced autophagosomes but disrupted the formation of autolysosomes with lysosomal membrane destabilization. GPX4 knockdown had a similar effect on ferroptosis phenotypes as RSL3. Taken together, RSL3-induced ferroptosis depends on the regulation of GPX4-Nrf2/HO1 in NSCLC cells. These results may be useful in predicting the ferroptosis response in NSCLC as well as drug resistant cancer cells.


Ferroptosis was induced in RSL3 sensitive NSCLC cells by inhibition of GPX4, and induction of KEAP1/Nrf2-HO1.
To verify whether RSL3-induced cell death was due to ferroptosis, the effect of RSL3 co-treatment with ferrostatin-1, a pharmacological ferroptosis inhibitor; Z-VAD-FMK, a pan-caspase inhibitor; chloroquine; bafilomycin A1, an autophagy inhibitor; and necrostatin-1, RIP kinase inhibitor, on cell death and proliferation of NSCLC cells was examined using MTT, flow cytometry, and cell counting. The results showed that RSL3-induced cell death was not rescued by Z-VAD-FMK, chloroquine, bafilomycin A1, or necrostatin-1 co-treatment ( Fig. 2A), but it was rescued by ferrostatin-1 ( Fig. 2A-C). Since GPX4 expression is associated with RSL3-induced cell death, the effect of RSL3 treatment on GPX4 expression was examined using qRT-PCR and western blotting. RSL3 suppressed GPX4 expression, which was restored by cotreatment with ferrostatin-1 (Fig. 2D). In addition, Nrf2/HO1 examination showing increase of Nrf2 and HO1 levels by RSL3, which were restored by ferrostatain-1 (Fig. 2D). However, GPX4 expression was not suppressed by RSL3 in RSL3-nonsensitive NSCLC cells (Fig. 2E). Furthermore, RSL3 treatment did not cause cleavage of apoptosis-related proteins such as caspase-3, cleaved caspase-9, and PARP; however, cleavage was evident with the apoptosis inducer etoposide (Fig. 2F). Taken together, these results show that the type of cell death induced by RSL3 treatment in NSCLC cells is ferroptosis, and that RSL3 also causes the suppression of GPX4 and induction of the Nrf2/HO1 pathway.
RSL3 disrupted formation of autolysosome with lysosomal membrane destabilization. The association between autophagy and ferroptosis is known, and autophagic flux was examined using the mRFP-GFP-LC3 tandem plasmid, with yellow puncta indicating autophagosomes and red puncta indicating autolysosomes. The results showed that the number of autophagosomes increased, but that of autolysosomes decreased, indicating the disruption of autolysosome formation (Fig. 3A,B). Western blotting of the autophagy markers LC3 and p63 showed the accumulation of both LC3-II and p62 after treatment with RSL3, indicating that autophagy was induced; however, p62 was not degraded (Fig. 3C), indicating that autophagic flux was disrupted. To assess the effects of RSL3 on lysosomes, immunofluorescence staining with cathepsin B (CTSB), LAMP1, and acridine orange, and western blotting with cathepsin B were performed. Cathepsin B diffused throughout the cytoplasm and LAMP1 showed enlarged lysosomes (Fig. 3C,D). Acridine orange staining showed an increased massive red signal, indicating that the acidic compartment was localized in the cytoplasmic perinuclear organelles, suggesting a lysosomotropic effect of RSL3 (Fig. 3E). These results indicated destabilization of the lysosomal membrane by RSL3.
RSL3 increased ferroptosis-associated LIP concentration, lipid peroxidation, ROS level, and GSH/GSSG ratio. Increased intracellular LIP concentration, commonly observed during ferroptosis induction, causes lipid peroxidation by generating cellular reactive oxygen species (ROS). Therefore, the effects of RSL3 alone and RSL3 co-treatment with ferrostatin-1 on intracellular LIP levels and lipid peroxidation activity were examined by measuring the fluorescence intensity of FerroOrange and BODIPY 581/591 C11 staining in H1299 and H23 cells. As expected, RSL3 treatment increased LIP levels and co-treatment with ferrostatin-1 blocked www.nature.com/scientificreports/ this effect (Fig. 4A). In addition, RSL3 caused lipid peroxidation, as shown by C11-BODIPY stained cells that oxidized to form C11-BODIPY. This effect was reversed by ferrostatin-1 co-treatment (Fig. 4B). Increased cellular ROS levels after treatment with RSL3 were further confirmed by the DCFDA assay (Fig. 4C). Furthermore, the GSH/GSSH ratio was increased by RSL3 treatment and was reversed by ferrostatin-1 co-treatment (Fig. 4D). Together, these results confirmed that RSL3 increased LIP levels, lipid peroxidation, and the GSH/GSSG ratio, which are characteristics of ferroptosis.
GPX4 knockdown showed effects similar to RSL3. Since RSL3 suppressed GPX4, the effects of GPX4 knockdown on cell proliferation, LIP levels, the GSH/GSSH ratio, and ROS levels in H1299 and H23 cells were examined. GPX4 knockdown reduced cell proliferation and significantly increased LIP levels, the GSH/GSSG ratio, and ROS levels when compared to the control group ( Fig. 5A-D). The Nrf2/HO1 ratio increased, consistent with the RSL3 treatment (Fig. 5E). These results suggested that GPX4 is a key molecule associated with the Nrf2/HO1 pathway for ferroptosis in RSL3-sensitive NSCLC.

RSL3 inhibited NSCLC tumor growth in a zebrafish model. Suppression of tumorigenicity by RSL3
treatment was examined in xenograft zebrafish models using CM-Dil-labeled H1299 or H23 cells. The cells (red) were grafted onto Tg(flk1:EGFP) zebrafish embryos and treated with E3 medium containing either DMSO or RSL3 for 5 days. RSL3 treatment reduced the tumor area in both the H1299 and H23 cell xenografts (Fig. 6A,B). This indicates that RSL3 has anticancer effects, which is consistent with the in vitro results.

Discussion
Lung cancer is a leading cause of cancer-related deaths worldwide. The most common type of lung cancer is NSCLC, accounting for approximately 80% of all lung cancers. Treatment of NSCLC involves a multidisciplinary approach, which consists of surgery, radiotherapy, and systemic treatment with various types of drugs, including www.nature.com/scientificreports/ www.nature.com/scientificreports/ chemotherapeutics, targeted therapies, and immune checkpoint inhibitors. Recent developments in targeted therapy and immunotherapy have changed treatment process for NSCLC 27 . However, the mutation required for targeted therapy did not exist in any patient. In addition, because drug resistance can occur to various agents, there is a need to identify and develop novel agents for the treatment of NSCLC. Cancer therapeutics have long been associated with cell death because many chemotherapeutic agents have been designed to promote the death of malignant cells. Ferroptosis is a unique, non-apoptotic type of cell death that has recently been discovered; thus, ferroptosis-inducing agents have been investigated for use in NSCLC www.nature.com/scientificreports/ treatment 29,30 . Recently, the poor prognostic value of upregulation of ferroptosis associated genes SLC7A11 and GPX4 has also been reported in lung cancer 31,32 . However, studies have examined the effects of erastin and RSL3 on NSCLC cells are very limited. Erastin has been shown to inhibit SLC7A11, which functions in the Nrf2/Keap1 pathway in N5CP and A549 cells 33,34 , and sensitizes NSCLC cells to the chemotherapeutic drug celastrol 35 . RSL3 inhibits GPX4 activity in H1650, HCC827, and PC9 cells 36 . ALOX15, a member of the lipoxygenase family, downregulates Calu-1 cells and increases sensitivity to both erastin and RSL3 14 . Moreover, these cytotoxic effects on NSCLC cells have not been evaluated in normal cells, which is very important. In this study, we evaluated whether erastin and RSL3 induce cell death in NSCLC cells compared to that in normal bronchial epithelial cells, BEAS-2B. Although both erastin and RSL3-induced cell death in all the NSCLC cells tested, only RSL3 showed minimal cytotoxic effects on BEAS-2B cells. Comparison of IC 50 showed that RSL3 was a more effective compound for inducing cell death than erastin (Table 1). In addition, RSL3 showed varying levels of sensitivity in different NSCLC cells, which showed a significant association with the endogenous expression of GPX4, a target of RSL3. We verified that RSL3 induces ferroptosis via GPX4 inhibition and Nrf2/HO1 induction. HO1, a target of Nrf2, is a cytoprotective enzyme that degrades hemoglobin into CO, iron, and biliverdin 37 , and its function in ferroptosis is contradictory, suggesting that dual, protective, or detrimental effects depend on the degree of cellular iron levels and ROS, indicating enhanced ferroptosis via the Nrf2/HO1 axis in our study 23,38 . This effect was recovered only by ferrostatin-1, a ferroptosis inhibitor, and not by the other programmed cell death inhibitors examined. Further studies confirmed that GPX4 knockdown induces Nrf2/HO1 expression, increases lipid peroxidation and cellular LIP levels, and inhibits GSH-to-GSSH conversion. These results indicate that the www.nature.com/scientificreports/ inhibition of GPX4 expression along with Nrf2/HO1 activation is the main factor in RSL3-induced ferroptosis in NSCLC cells, depending on the endogenous levels of GPX4. This may be because GPX4 is necessary or has a more functional role in the GPX4 overexpressing cancer cell, making the cells sensitive to RSL3. Furthermore, an in vivo zebrafish xenograft model confirmed the suppression of tumor growth by RSL3 in NSCLC cells.
Emerging studies have revealed the crosstalk between autophagy and ferroptosis. However, the role of autophagy in ferroptosis is controversial, as autophagy activation can either induce or inhibit ferroptosis, although several studies have shown that ferroptosis induction is associated with autophagy activation [39][40][41] . Our examination of autophagic flux showed an increase in autophagosomes, but not autolysosomes, along with the accumulation of both LC3-II and p62 by RSL3, indicating the inhibition of autophagic flux. Although it was not able to clearly elucidate the mechanism underlying the decrease in autolysosomes relative to autophagosomes in this moment, it can be hypothesized that the fusion of autophagosomes with lysosomes may be disrupted due to lysosome dysregulation. Thus, lysosomal dysregulation was verified by the release of cathepsin B into the cytoplasm with enlarged lysosomes and massively increased acidic compartments, indicating impaired lysosomal membrane stability, which points to the lysomotropic effect of RSL3 42 . These results are consistent with recent reports suggesting an association between lysosomal dysregulation and ferroptosis [43][44][45] . However, the detailed mechanism needs to be further investigated.
Overall, the results of this study suggest that RSL3 is a more effective ferroptotic cell death inducer than erastin in NSCLC cells, and it has very limited cytotoxic effects on normal cells. Because the activity of RSL3 is dependent on GPX4 expression levels, predictions can be made regarding whether ferroptosis will occur in NSCLC cells. Further research is required to investigate the synergistic effect of RSL3 with other targeted or immunotherapy agents, as well as the effect of RSL3 on EGFR-TKI-resistant NSCLC cells. Cell viability and proliferation assays. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded in 96 well culture plates at a density of 1 × 10 3 cells/well and treated with the indicated concentrations of erastin, RSL3, ferrostatin-1, Z-VAD-FMK, chloroquine diphosphate salt, bafilomycin A1, and necrostatin-1. After incubation, 10 µL MTT solution (0.5 mg/mL) dissolved in distilled water and 20 µL medium was added to each well. After 2 h of additional incubation, the MTT solution was discarded and DMSO was added. Absorbance was measured at 570 nm using a microplate reader (SpectraMax Plus 384, CA, USA). Cells were counted to measure cell proliferation. Cells were seeded in 60 mm dishes at a density of 2 × 10 5 cells/dish. The following day, cells were treated with either 100 nM RSL3 or 1 μM ferrostatin-1 for 24 h. The cells were counted using a hemocytometer after trypan blue staining (Sigma-Aldrich). Cells were imaged using an inverted light microscope (Olympus IX71, Olympus Corp., Japan).  www.nature.com/scientificreports/ Immunoblotting. Cells were lysed in RIPA buffer that contained a protease/phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min and used for immunoblotting. The cytosolic fraction for cathepsin B detection was prepared as previously described 45

Lipid peroxidation and ROS analysis. Lipid peroxidation activity in the cells was analyzed using
BODIPY 581/591 C11 (Invitrogen, D3861, MA, USA). For live cell imaging, cells were seeded in a confocal dish at a density of 1 × 10 4 . The following day, the cells were treated with either RSL3 (100 nM) or ferrostatin-1 (1 μM) for 24 h. Next, the cells were incubated in fresh medium with BODIPY 581/591 C11 (2 μM) and covered with a sterile coverslip before incubation at 37 °C for 30 min. BODIPY fluorescence was measured at 20 °C using the LSM 780 NLO system (Carl Zeiss, Baden-Württemberg, Germany). Fluorescence intensity was analyzed using ZEN software (Carl Zeiss, Baden-Württemberg, Germany). Cellular ROS levels were analyzed using a DCFA/ H2DCFDA-cellular ROS assay kit (ab11385, Abcam) after treatment with RSL3 for 6 h following the manufacturer's instructions. Absorbance was measured at excitation and emission wavelengths of 543 and 580 nm, respectively, using an Enspire Multi Plate Reader (PerkinElmer, MA, USA). ROS generation in live cells treated with DCFDA was measured using an LSM 780 NLO (Carl Zeiss, Baden-Württemberg, Germany).
GSH/GSSG analysis. The GSH concentration and GSH/GSSG ratio were calculated using the EZ-Glutathione Assay Kit (Dogenbio, Seoul, South Korea) following the manufacturer's instructions. Cells were seeded at a density of 5 × 10 5 cells/dish in 60 mm dishes. The following day, the cells were treated with RSL3 and ferrostatin-1 for 24 h. Cells were harvested and sonicated with PBS, and cell debris was removed. Absorbance was measured at 412 nm using a microplate reader (SpectraMax Plus 384). Each experiment was performed in triplicate and repeated three times.
Autophagic flux assessment. Autophagic flux was assessed in live cells by mRFP-GFP-LC3 expressing plasmid, which was kindly provided by Professor T. Yoshimori of the Department of Genetics, Graduate School of Medicine, Osaka University, Japan 46 . Cells cultured on confocal dishes were transfected with mRFP-GFP-LC3 plasmid for 24 h and then treated with or without RSL3 for 24 h. Fluorescence images were obtained using a Laser Scanning Confocal Microscope (LSM800, Carl Zeiss) and visualized with a 63 × water immersion objective lens.
Immunofluorescence staining. The cells were fixed and stained with primary antibodies against LAMP-1 (sc-20011, Santa Cruz Biotechnology) and cathepsin B, followed by a secondary antibody conjugated with Alexa Fluor 488 or 546. Nuclei were counterstained with (DAPI; 10236276001; Roche, Mannheim, Germany). Live cells were stained with 5 μg/mL acridine orange at 37 ℃ for 30 min. Fluorescent images were obtained using a laser-scanning confocal microscope (LSM800; Carl Zeiss, Oberkochen, Germany).
In vivo zebrafish tumor model. Zebrafish (Danio rerio) and embryos were bred and maintained according to standard procedures. All animal experimental protocols were approved by the Committee for Ethics of Animal Experimentation of Sookmyung Women's University (SMWU-IACUC-1712-036-03) and were performed in accordance with the relevant guidelines and regulation of Sookmyung Women's University as previously described 47  www.nature.com/scientificreports/ accordance with ARRIVE guidelines. Approximately 50 fluorescent cell tracker CM-Dil-labeled H1299 or H23 cells were injected into the yolk sac of zebrafish embryos at 2 days post-fertilization (dpf). One day post injection (dpi), the zebrafish larvae were treated with RSL3 (100 nM) and maintained at 34 °C for 5 days. Fluorescence images were acquired using a Zeiss LSM700 confocal microscope (Carl Zeiss AG). The area penetrated by the CM-Dil-labeled cancer cells was quantified using the ImageJ software (version 1.52n, NIH) and normalized to cancer cells in untreated zebrafish embryos for each group.
Statistical analysis. SPSS Statistics version 25 software (IBM, NY, USA) and GraphPad Prism version 5 software (GraphPad, CA, USA) were used for statistical analyses. Experimental results were presented as the mean ± SD from at least two independent experiments. T-tests were performed to compare two independent groups. Comparisons of nultiple groups were analyzed using ANOVA. Significance was defined as: # = 0.05 < p (No significance); * = 0.01 < p < 0.05; ** = 0.001 < p < 0.01; *** = p < 0.001.

Data availability
The raw scanned bot images used in the figures are provided in Supplementary Data. Other datasets used and/ or analyzed in the current study are available from the corresponding author upon request.