Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel type of cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.
Cell death plays an important role in the normal development and homoeostasis of living individuals, and is also implicated in abnormal activities of these processes in several diseases. Cells possess multiple cell death programmes including apoptosis and necrosis, which are mutually connected and can occur in response to defects in different pathways . Studies involving genetics and chemical biology have revealed the existence of several regulated types of necrosis [2,3], with iron-related cell death, or ferroptosis, being proposed by Stockwell and colleagues . Ferroptosis is induced in oncogenic Ras-expressing cells by a number of small compounds referred to as ferroptosis inducers (FINs), which include erastin and RSL3 [3–6]. Ferroptosis can be characterized at a morphological level by the presence of shrunken mitochondria, and is functionally independent of caspases, ATP depletion or mitochondrial outer membrane permeabilization. Ferroptosis requires intracellular iron that may induce reactive oxygen species (ROS) production and lipid peroxidation [3–7].
The ferroptotic cell death pathway has been classified into two types where: (i) glutathione (GSH) depletion results from inhibition of system xc− by class-I FINs (e.g. erastin), and (ii) glutathione peroxidase 4 (GPx4) activity is directly inhibited by class-II FINs (e.g. RSL3) . Erastin has three main target proteins: the cell-surface cystine/glutamate antiporter (system xc−: SLC7A11/SLC3A2 complex), the large neutral amino acid transporter (system L: SLC7A5/SLC3A2 complex), and mitochondrial voltage-dependent anion channels [4,5]. However, ferroptotic cell death requires a remarkable reduction in GSH that is triggered by direct impairment of cystine uptake by system xc− . GSH depletion in turn inhibits GSH-dependent peroxidases (GPxs), particularly GPx4, which is a unique enzyme that reduces lipid hydroperoxides in cell membranes . Pronounced accumulation of lipid ROS via GPx4 suppression is thought to be critical for the execution of ferroptotic cell death, because compounds related to the membrane antioxidant vitamin E (e.g. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid: Trolox) have been shown to prevent both FIN-induced and GPx4 knockdown-induced cell death [6,7]. Moreover, (1S,3R)-RSL3, a typical class-II FIN, interacts with GPx4 directly and prevents its activity, whereas GPx4 overexpression and inhibition suppresses and enhances FIN-induced ferroptosis, respectively, which indicates that GPx4 is a central regulator of ferroptotic cell death .
GPx4 is thought to be the limiting GSH-dependent enzyme, because GPx4-knockout mice die at 7.5 days, which is the same day on which mice lacking GSH-synthesizing enzymes die [9–11]. Inducible genetic deletion of GPx4 in mouse embryonic fibroblasts leads to cell death in response to 12/15-lipoxygenase (12/15-LOX)-dependent lipid peroxidation . Similar signalling pathways have been suggested to be important for neuronal oxidative toxicity caused by acute cerebral ischaemia. In cell model systems, excess glutamate triggers cell death of neurons by a decrease in GSH and an associated activation of 12/15-LOX . Furthermore, glutamate-induced cell death in brain slices can be prevented by the ferroptosis inhibitor ferrostatin 1 (Fer-1) that serves as an antioxidant in membranes, suggesting that neuronal cell death and ferroptosis share several common molecules and pathways . More recently, acute renal injury in mice was shown to involve cell death that has several characteristics of ferroptosis, including lipid peroxidation, and treatment with Fer-1 or its related agents can prevent this type of cell death [14,15]. These observations indicate that ferroptosis occurs not only in cells or tissues but also in vivo.
The susceptibility of cancer cells to ferroptosis appears to be related to constitutive activation of Ras-MEK signalling [3–5], which can contribute to iron abundance in tumours by altering the expression levels of the transferrin receptor and ferritin . Generally, iron enters cells by internalization of transferrin receptors bound to transferrin–Fe(III)2 . The ferric ion that is released from transferrin is reduced by an endosomal reductase activity (e.g. six-transmembrane epithelial antigen of the prostate 3) prior to export of ferrous ion by a transporter such as divalent metal transporter 1 (DMT1). Most cellular iron is stored as a component of ferritin in the Fe(III) form. The labile iron pool is presumably used to control intracellular iron homoeostasis through its own redox activity or to provide iron constituents for functional proteins such as haem enzymes. Several ROS-generating enzymes, including LOX and NADPH oxidases (NOXs) require iron or its derivatives for their activity. Interestingly, the pentose phosphate and NOX pathway may contribute to erastin-induced ferroptotic cell death , whereas oncogenic Ras reportedly induces rapid production of ROS partly by up-regulating levels of NOX1 [17,18]. In contrast, redox-active iron catalyses Fenton-type reactions for the direct production of ROS, such as the hydroxyl radical, which is thought to oxidize cell constituents that in turn cause damage to cell structures and integrity. In vitro, the initiation of lipid peroxidation requires both ferric and ferrous ions, and the ratio of Fe(III) to Fe(II) is important in determining its rate . However, how iron contributes to ferroptosis in conjunction with ROS is unclear.
In the present study, we used N-Ras-mutant HT1080 cells to examine how specific organelles and spatial dynamics of ROS contribute to ferroptosis. We evaluated various chemical reagents for their ability to protect tumour cells from death, and found that lysosome inhibitors repressed cell death by reducing basal ROS generation in these compartments. Our data suggest that endosomes/lysosomes are essential for the iron metabolism in ferroptosis-sensitive cells.
Erastin and the aspartic protease inhibitor pepstatin A-methyl ester (PepA-Me) were purchased from Calbiochem. Bafilomycin A1 (Baf A1), an inhibitor of vacuolar H+-ATPase, was purchased from Wako. The iron chelator deferoxamine (DFO) was purchased from Sigma.
Synthesis of RSL3
RSL3 was synthesized from L-tryptophan methyl ester hydrochloride (HCl·H-Trp-OMe, Kokusan Chemical) according to a previously described method . The corresponding RSL3 intermediate (mixture) was dissolved in chloroform and potassium carbonate (1.1 equiv.) was added. Chloroacetyl chloride (1.0 equiv.) was then added dropwise to the stirred solution at 0°C. The reaction mixture was stirred overnight at room temperature and filtered. The filtrate was extracted by water and the organic layer washed with brine before drying over anhydrous sodium sulfate. After filtering the sodium sulfate, the filtrate was concentrated in vacuo to yeild the crude product, which was revealed by reverse-phase (RP)-HPLC analysis to contain mainly two products (Supplementary Figure S1a). These two compounds were both identified as RSL3 by ESI–MS that were acquired with an Applied Biosystems API-2000 mass spectrometer (positive ion mode). The former product identified by RP-HPLC (Supplementary Figure S1b) was used for the following experiment with MS (ESI, m/z): [M-H]+ C23H21ClN2O5, calculated, 440.1; actual 440.9.
Cell culture and ferroptosis induction
HT1080 (human fibrosarcoma) and Calu-1 (human non-small-cell lung cancer) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and Eagle's minimum essential medium with Earle's salts, respectively, which both contained 10% FBS. For ferroptosis induction, cells were plated at 1.0×105 cells per well on six-well plates and cultured for 36 h. Culture medium was replaced with 1 ml of medium containing 5 or 10 μM erastin or 1 μM RSL3 with or without inhibitors. Cell viability was determined by Trypan Blue dye-exclusion assay. HT1080 or Calu-1 cells were collected at the indicated time and incubated with 0.4% Trypan Blue, and observed under a microscope where stained and unstained cells were counted separately with a haemocytometer. Because cells that had undergone ferroptosis were sometimes lost during the assay procedure, the cell survival rate was calculated according to the following formula: cell survival (%)= unstained cells number (drug treated/untreated) × 100. For the lactate dehydrogenase (LDH) assay, medium was collected at the indicated time and each LDH activity was measured by a CytoTox 96 cytotoxicity assay (Promega).
A vector expressing EGFP-tagged Atg4B-C74A was constructed as described previously . cDNAs of cation-dependent mannose 6-phosphate receptor (M6PR)  and immature colon carcinoma cell transcript 1 (ICT1) were subcloned into pTagBFP2 vector (Evrogen). The siRNA against human transferrin receptor 1 (TFR1) was designed and synthesized by Integrated DNA Technologies (code number HSC.RNAI.N001128148.12.3). Transfections were performed with HilyMax reagent (Dojindo Molecular Technologies), and 75% transfection efficiency was observed with a reporter gene (results not shown).
Fluorescence detection of ROS
HT1080 cells were incubated on poly-L-lysine (Sigma)-coated coverslips in the dark at 37°C with 5 μM BODIPY-(581/591)-C11 (Invitrogen) or with 5 μM 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H2TMRos) (Invitrogen) for 30 min. The cells were mounted on a glass slide with Hank's buffer. Dye oxidation, indicated by PF-TMRos red fluorescence and a shift in BODIPY fluorescence from red to green, was observed with a Keyence BZ-9000 microscope or with an epifluorescence microscope (Olympus BX-50) equipped with a SenSys™ charge-coupled device camera (Photometrics) as described previously . Co-localization with organelle markers was observed with an LSM5 PASCAL confocal microscope (Carl Zeiss) or FV10i-DOC confocal microscope (Olympus). To avoid indicator dye photo-oxidation, fluorescence images were collected by a single rapid scan with identical parameters (e.g. contrast and brightness) for all samples. For BODIPY-C11, fluorescence images detected with appropriate cut-off filters were simultaneously collected to compare oxidized (green) and unoxidized dye (red). Fluorescence intensity was quantified using ImageJ 1.40g (NIH).
Western blot analysis
Immunoblotting was performed as described previously . Blotted membranes were blocked with 5% non-fat dried skimmed milk powder for 30 min, and incubated with primary antibodies (1:1000 dilution of rabbit anti-ferritin, Abcam, or 1:10000 dilution of mouse anti-biotin, Jackson ImmunoResearch).
Transferrin uptake assay
Prior to transferrin loading, cells were treated with each inhibitor (DFO, ammonium chloride, Baf A1, PepA-Me) for 1 h and then loaded with biotin–transferrin (10 μg/ml; Invitrogen) or Alexa Fluor 488–transferrin (50 μg/ml; Invitrogen) for cumulative uptake at 37°C for 30 and 20 min respectively. Cells containing biotin–transferrin were washed and extracted in lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EGTA, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 10 μg/ml pepstatin). Cell extracts (5 μg) were then analysed by immunoblotting with anti-biotin antibody. Immunoreactive band intensity was measured by densitometry, quantified using ImageJ and normalized relative to the control sample (no inhibitors) in the same membrane.
Intracellular iron measurement
HT1080 cells seeded on two 100-mm-diameter dishes were incubated with or without inhibitors for 5 h. For evaluation of transferrin receptor, siRNA-transfected cells were pre-incubated in DMEM without serum for 30 min and then treated with 0.01 mg/ml human holo-transferrin (Sigma) for 8 h. Cells were washed with PBS and the collected cells were lysed in 80% HNO3/20% H2O2 for 24 h at room temperature. The total iron content in each diluted sample was determined using a Z-5300 flame atomic absorbance spectrophotometer (FAAS: Hitachi).
Data are presented as means ± S.E.M. Non-parametric data were compared using the Kruskal–Wallis test, followed by the Steel test.
We first examined the effects of the ferroptosis-inducing reagents erastin and RSL3 on human fibrosarcoma HT1080 cells. To avoid the loss of dead cells, the number of live cells was determined by Trypan Blue dye-exclusion assays. Treatment with erastin (5 and 10 μM) had little effect on cell survival within 6 h, but by 8 h most of the cells had died (Supplementary Figure S2A). RSL3 was the more potent FIN reagent and even at a low dose (1 μM) induced rapid cell death in HT1080 cells (Supplementary Figure S2A) and another cancer cell line (results not shown). The potency of these reagents is consistent with their target proteins and the signal pathways that were demonstrated by the Stockwell group [3,6]. As previously reported, the free iron chelator DFO prevented erastin-induced cell death (Supplementary Figure S2B) even when it was added after erastin treatment. Baf A1 (0.4 μM), which is a specific vacuolar ATPase inhibitor, also protected HT1080 cells from erastin toxicity (Supplementary Figure S2B and Figure 1A), although earlier studies had suggested that this inhibitor was ineffective . In the present study, we found that Baf A1 (>1 μM) alone induced some cell death (presumably apoptosis) when incubated with the cells for longer time periods (>16 h) (results not shown). In addition to Baf A1, ammonium chloride (10 mM), which acts to neutralize acidic organelles such as the lysosome, and the lysosomal aspartic protease inhibitor PepA-Me (25 μM) also blocked erastin-induced cell death completely at 8 h (Figure 1A). Similarly, the viability of lung cancer Calu-1 cells began to decline at 10 h after erastin treatment, and all of the above-described inhibitors also prevented ferroptosis even at the 24 h time point (Figure 1B). Cell damage was further assessed by a LDH release assay. Baf A1, ammonium chloride and PepA-Me all significantly protected cells from erastin- or RSL3-induced cell death (Figures 1C and 1D), suggesting that basal lysosomal activities are involved in ferroptotic cell death in these cells.
Lysosome/autophagy inhibitors suppress FIN-induced ferroptosis
Next, the contribution of autophagy to ferroptosis was examined in the presence of 3-methyladenine (3-MA), which is generally used as a macroautophagy inhibitor. When the number of viable cells was calculated at the 8 h time point, a significant reduction in cell death was observed in the presence of 3-MA, although the inhibitory effect of this compound was lower than that seen for the lysosomal inhibitors (Figure 1E). We next used an inactive mutant of Atg4B protease (Atg4B-C74A), which sequesters the LC3 protein and blocks formation of the Atg7-LC3 intermediate . Overexpression of EGFP-tagged Atg4B-C74A induced some levels of apoptosis in untreated cells (results not shown), suggesting that basal autophagic activity is required for HT1080 cell survival. Importantly, this mutant protease significantly reduced erastin-induced cell death compared with control cells (Figure 1F). These observations suggest that both autophagic activity and lysosomal function are involved in FIN-induced ferroptosis in Ras tumour cell lines.
Because erastin-induced ferroptosis is accompanied by an accumulation of ROS and an associated lipid peroxidation [3,6,7], we next used the fluorescent probe PF-H2TMRos to examine whether ROS levels were increased in erastin-treated HT1080 cells. As with the widely available probe 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), the general ROS sensor PF-H2TMRos can detect cell-death-associated ROS bursts . Microscopic applications using the PF-H2TMRos probe are also useful in observing the intracellular localization of ROS, because this probe is less sensitive to photo-oxidation than is CM-H2DCFDA. As shown in Figure 2A, erastin treatment evoked intense PF-TMRos red fluorescence, which became prominent at approximately 7 h. Consistent with earlier reports, the erastin-induced ROS burst was detected largely in the cytosol [3,6,7]. The finding that fluorescence intensity remained low in cells treated with Baf A1, ammonium chloride or PepA-Me (Figure 2B) suggests the involvement of lysosomes in the ROS burst. We then assessed the effects of inhibitors on lipid ROS production using the membrane peroxidation sensor BODIPY-C11, and confirmed that lipid ROS levels did not increase in these cells that had modified lysosome activity (Figure 2C). Similar observations were made in cells treated with the autophagy inhibitors 3-MA and chloroquine (results not shown). Therefore, inhibitors of autophagy and lysosomal activity both reduced erastin-induced ferroptosis, which is probably due to the activity of the agents in preventing cytosolic and lipid ROS production.
ROS generation is blocked by lysosome inhibitors
Because acidic organelles such as endosomes and lysosomes are required for iron uptake and release, we examined whether these lysosome-related inhibitors could alter iron equilibria. The total cellular iron concentration was first determined using a FAAS. FAAS analyses revealed that PepA-Me treatment caused a significant decrease in the cellular iron content that was comparable to the effect produced by DFO treatment (Figure 3A). Meanwhile, other reagents (Baf A1 and ammonium chloride) did not induce any significant decrease in iron levels. Cytosolic ferritin is known to be degraded within lysosomes via a specific autophagic pathway [24,25] that releases free iron for use. Thus, ferritin protein levels were analysed by immunoblotting with an anti-ferritin antibody. An obvious up-regulation of ferritin protein was detected in PepA-Me-treated cells (Figure 3B), suggesting that PepA-Me induces the iron content reduction by inhibiting ferritin degradation in lysosomes. Transferrin uptake was next examined by incubating cells with Alexa Fluor 488-conjugated transferrin. In control cells the endocytosed fluorescent transferrin was distributed sparsely around the perinuclear region, whereas Baf A1 and PepA-Me-treated cells displayed reduced punctate signals and puncta that were concentrated in regions with microtubule-organizing centres (Figure 3C). Meanwhile, transferrin fluorescence signals apparently declined in cells treated with ammonium chloride, although the overall distribution pattern did not change. The rate of transferrin uptake was then quantified by measuring the extent of biotin-labelled transferrin using immunoblotting. Ammonium chloride-treated cells consistently exhibited a lower rate of biotin–transferrin uptake relative to untreated cells (Figure 3C, histogram), suggesting that acidic organelles are important for iron homoeostasis. We then employed siRNA to suppress TFR1 expression. Silencing of TFR1 efficiently suppressed fluorescent transferrin uptake and also reduced iron uptake from transferrin (Figure 3D), whereas some TFR1-independent iron-uptake pathways were suggested . TFR1 knockdown led to a marked decrease in the cell death rate in cells incubated with erastin (Figure 3D, histogram), suggesting that transferrin-mediated iron uptake is essential for ferroptotic cell death.
Effects of lysosome inhibitors on intracellular iron transport and storage
Free iron released in the endosomes and lysosomes is thought to be involved in local ROS production through the catalysis of Fenton-type reactions . Notably, under basal conditions, intense punctate fluorescent signals produced by PF-TMRos were detected in HT1080 cells (Figures 2A and 4A). These signals overlapped with those produced by LysoSensor dye, a detector of lysosomes, whereas weak membrane-associated signals that were seen behind the spots co-localized with fluorescence produced by MitoTracker Green (Figure 4A). In addition, when HT1080 cells were treated with erastin, increased PF-TMRos signals were partly detected in lysosomes (Figure 4B). These observations suggest that certain reactive oxidants are generated in acidic organelles such as lysosomes as well as in mitochondria. We further analysed the effects of the lysosomal activity inhibitors on basal PF-TMRos signals, and observed that punctate signals were specifically altered in cells with modified lysosome activity (Figure 4C). Baf A1 or PepA-Me treatment completely eliminated punctate signals, with only a mitochondrial pattern remaining in these cells. In contrast, large punctate signals were concentrated in cells treated with ammonium chloride. Densitometric measurements revealed that Baf A1, PepA-Me and ammonium chloride all significantly reduced total cellular fluorescence intensity relative to untreated cells (Figure 4C, histogram). This result suggests that these inhibitors decreased intracellular ROS levels by altering lysosomal activity.
Analysis of intracellular localization of ROS by the redox sensor PF-H2TMRos
Because massive ROS production in ferroptotic cells was observed mostly in the cytosol (Figures 2A and 4B), lysosomal ROS are probably involved in oxidative cytotoxicity but are not the final determinant. In contrast with the cytosolic ROS burst, lipid-ROS detected in cells undergoing ferroptosis was widely distributed over both organellar and plasma membranes (Figures 5A and 5B, bottom panels). Very low signals from oxidized BODIPY-C11 were detected under basal conditions, whereas its distribution shown by red fluorescence was mostly observed in intracellular compartments such as the mitochondria and the endoplasmic reticulum (Figures 5A and 5B, top panels). When cells were treated with erastin or RSL3 for 6 and 2 h, respectively, oxidized signals gradually increased, with their strength varying between individual cells. In erastin-treated cells, membrane peroxidation was detected on both the plasma membrane and the perinuclear compartments shortly after treatment (Figure 5A, middle panels). In contrast, punctate signals appeared around the perinuclear region in RSL3-treated cells before the intensive lipids peroxidation occurred (Figure 5B, middle panels). To investigate the proper localization of oxidized signals, we used marker proteins with a blue fluorescent protein (BFP) tag. The cation-dependent M6PR is localized at the trans-Golgi network and subsequent endosomal compartments , and the ICT1 is a component of mitochondrial ribosome . The transient transfection analyses revealed that punctate signals observed in early phase of ferroptosis were co-localized with M6PR–BFP2 (Figure 5C, upper panels). This observation suggests that lipid ROS in ferroptotic cells is first generated around Golgi compartments that include endosomes. Meanwhile, increased oxidized signals in late-ferroptotic cells were found in mitochondria, with a ring-like structure surrounding the fragments (Figure 5C, lower panels).
Lipid ROS accumulates in ferroptotic cells
Ferroptosis is a novel form of cell death that is dependent on intracellular iron. In primary studies, erastin-induced ferroptosis was predominantly observed in oncogenic Ras-expressed cells [3–5]. This Ras-dependent selectivity could be explained by the finding that these cells accumulate iron by modulating expression levels of ferritin and the transferrin receptor . Iron that is taken up by the transferrin receptor is routed into the endosome/lysosome pathway where ferritin is degraded to release active iron via a specific autophagy pathway. Thus, autophagy and functional lysosomes probably contribute to ferroptosis through the provision of iron, which would seem to be at odds with the earlier finding that 3-MA and Baf A1 were ineffective at preventing erastin-induced ferroptosis . However, in the present study we demonstrated that treatment with autophagy inhibitors or compounds that alter lysosomal function indeed diminished FIN (e.g. erastin and RSL3)-induced oxidative toxicity in HT1080 and Calu-1 cells (Figure 1 and Supplementary Figure S2). The difference in the results obtained in these two studies may be due to the different time of FIN treatments and inhibitor concentrations. In the present study, we used <1 μg/ml Baf A1, which was sufficient to prevent the effects of FINs, and also found that treatment with a higher dose induced apoptotic cell death in these cells (results not shown). In contrast, the inhibitory activities of 3-MA and PepA-Me were relatively low, given that they induced cell death in 50% and 40% of HT1080 cells, respectively, at the 24 h point (results not shown). However, these reagents could efficiently block ferroptosis following a short-term incubation even in the presence of higher doses of erastin or RSL3 (Figure 1A and results not shown).
We further showed that punctate fluorescence signals produced by the ROS indicator PF-H2TMRos were present around perinuclear regions in proliferating HT1080 cells, and these signals were specifically reduced in the presence of lysosomal inhibitors (Figure 4C). This result suggests that certain oxidants are localized at acidic compartments such as endosomes/lysosomes. Recent studies have shown that selective autophagic degradation of ferritin promotes iron release within lysosomes, and this event, ferritinophagy, is important for cellular iron homoeostasis [24,25]. The observation that treatment of cells with PepA-Me caused both an increase in ferritin protein levels and a decrease in iron content (Figures 3A and 3B) indicated that this inhibitor may diminish ferroptosis by preventing autophagic degradation of ferritin. On the other hand, ammonium chloride promoted a significant reduction in iron uptake by transferrin (Figure 3C). Moreover, TFR1 knockdown attenuated basal ROS (results not shown) and inhibited erastin-induced ferroptosis (Figure 3D). These results indicate that redox-active iron from ferritin or transferrin is presumably involved in local ROS generation within endosomes/lysosomes. Labile iron was shown to be consistently observed in endosomes or lysosomes in primary hepatocytes or hepatocellular carcinoma by iron-specific fluorescent probes [30,31]. Lysosomal ROS or some ferrous ion [Fe(II)] may relocate to the cytoplasm, which in turn could induce the ROS burst during the FIN-induced oxidative toxicity. In addition, lysosomal ROS and iron may contribute to lipid ROS production because oxidized membranes were observed at the perinuclear compartments during the early phase of ferroptosis (Figure 5). We thus presume that lipid ROS within the endolysosomal membrane triggers iron-mediated chain lipid peroxidation.
Our data suggest that autophagy contributes to ferroptosis through the generation of lysosomal ROS in N-Ras-mutated HT1080 cells. Autophagy appears to have a dual role in cancer progression wherein during the early stages of transformation it suppresses cancer progression, but in later stages it maintains the cancer phenotype . Autophagy is reportedly up-regulated in Ras cancer cells and affects both mitochondrial activity and ROS production [33–36]. ROS generated in lysosomes is included in the total amount of cellular ROS in HT1080 cells (Figure 4C), which is in agreement with previous evidence demonstrating that autophagy elevates cellular ROS levels via the active functions of both mitochondria and lysosomes. Therefore, increased autophagy and ROS production in Ras cancers may provide modest selectivity against ferroptosis. Oncogenic Ras expression has been reported to repress toxic ROS levels by promoting the activity of the NRF2 antioxidant pathway , whereas elevated glutathione levels produced by NRF2 pathway activation are also critical for cancer cell proliferation [38,39]. Treatment with lysosomal inhibitors blocks lysosomal ROS production, which may lead to a relative increase in the cellular detoxification activity that is mainly contributed by intracellular GSH.
In the present study, we have shown that pharmacological inhibition of autophagy or lysosomes attenuates drug-induced ferroptosis in tumour cells. Our results suggest that endosomes/lysosomes contribute to this form of cell death by modulating iron equilibria and ROS expression. We further suggest the possibility that lipid ROS are generated at these organelles, which is consistent with a recent observation that in GPx4-deficient cells lipid peroxidation is initiated outside the mitochondria . Further localization analysis with additional specific probes will be required to determine the site of ROS triggers and the critical membrane break involved in ferroptosis.
Seiji Torii, Ryosuke Shintoku, Chisato Kubota and Keiichi Yamada designed and performed the experiments. Makoto Yaegashi, Ryoko Torii, Toshinobu Suzuki and Masaya Sasaki participated in the experiments. Masanobu Mori, Yuhei Yoshimoto and Toshiyuki Takeuchi supervised the present study and critically read the paper. Seiji Torii analysed the data and wrote the paper.
We thank Dr M. Naito (National Institute of Health Sciences), Dr N. Nameki, Dr T. Oda (Gunma University), Dr Y. Kato, Dr K. Nakayama and Dr Y. Takeda (Kyoto University) for technical advice and helpful discussion. We also thank Ms E. Tajima and Ms M. Hosoi for their generous support.
This work was supported by the Japanese Society for the Promotion of Science (JSPS KAKENHI) [grant number 24390050]; and the Institute for Molecular and Cellular Regulation, Gunma University [grant number 14036].
- Baf A1
blue fluorescent protein
Dulbecco's modified Eagle's medium
flame atomic absorbance spectrophotometer
glutathione peroxidase 4
immature colon carcinoma cell transcript 1
mannose 6-phosphate receptor
pepstatin A-methyl ester
reactive oxygen species
transferrin receptor 1
These authors contributed equally to this work.