Skip to main content
NCBI home page
Redox Biology logoLink to Redox Biology
. 2023 Feb 28;61:102650. doi: 10.1016/j.redox.2023.102650

Redox phospholipidomics discovers pro-ferroptotic death signals in A375 melanoma cells in vitro and in vivo

Yulia Y Tyurina a,b,∗∗∗, Alexandr A Kapralov a,b, Vladimir A Tyurin a,b, Galina Shurin a,d, Andrew A Amoscato a,b, Dhivyaa Rajasundaram e, Hua Tian a,b, Yuri L Bunimovich a,c, Yulia Nefedova f, William G Herrick g, Ralph E Parchment g, James H Doroshow h, Hulya Bayir a,e, Apurva K Srivastava g,∗∗, Valerian E Kagan a,b,∗
PMCID: PMC9996109  PMID: 36870109

Abstract

Growing cancer cells effectively evade most programs of regulated cell death, particularly apoptosis. This necessitates a search for alternative therapeutic modalities to cause cancer cell's demise, among them – ferroptosis. One of the obstacles to using pro-ferroptotic agents to treat cancer is the lack of adequate biomarkers of ferroptosis. Ferroptosis is accompanied by peroxidation of polyunsaturated species of phosphatidylethanolamine (PE) to hydroperoxy- (-OOH) derivatives, which act as death signals. We demonstrate that RSL3-induced death of A375 melanoma cells in vitro was fully preventable by ferrostatin-1, suggesting their high susceptibility to ferroptosis. Treatment of A375 cells with RSL3 caused a significant accumulation of PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH), the biomarkers of ferroptosis, as well as oxidatively truncated products - PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA) and PC-(18:0/HOOA). A significant suppressive effect of RSL3 on melanoma growth was observed in vivo (utilizing a xenograft model of inoculation of GFP-labeled A375 cells into immune-deficient athymic nude mice). Redox phospholipidomics revealed elevated levels of 18:0/20:4-OOH in RSL3-treated group vs controls. In addition, PE-(18:0/20:4-OOH) species were identified as major contributors to the separation of control and RSL3-treated groups, with the highest variable importance in projection predictive score. Pearson correlation analysis revealed an association between tumor weight and contents of PE-(18:0/20:4-OOH) (r = −0.505), PE-18:0/HOOA (r = −0.547) and PE 16:0-HOOA (r = −0.503). Thus, LC-MS/MS based redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid biomarkers of ferroptosis induced in cancer cells by radio- and chemotherapy.

Keywords: Ferroptosis, Redox lipidomics, Hydroperoxy-phosphatidylethanolamine, Biomarkers, A375 melanoma cells

1. Introduction

Characteristic to cancer cells is their ability to evade most common programs of regulated cell death, particularly apoptosis and pyroptosis [1,2]. This is due, at least in part, to the engagement of the caspase-associated machinery in the execution of these death pathways – a feature that can be overcome by the malignant cells. Therefore, significant research focus has been placed on a newly discovered necrotic program of regulated cell death, ferroptosis, which is executed in an entirely caspase independent manner.

A major player in the ferroptotic program is the aberrantly enhanced selective process of phospholipid peroxidation initiated enzymatically or chemically and driven by a free radical mechanism of abstraction of H-atoms from “weak” bis-allylic positions in penta-dienyl fragments of polyunsaturated-PL molecules [3]. The generation of intermediary carbon-centered radicals and peroxyl-radicals is followed by the production of primary molecular products, hydroperoxy-phospholipids. The latter are unstable and can readily undergo – in the presence of catalytic amounts of transition metals, particularly Fe, – β-scission yielding oxidatively-truncated electrophilic derivatives as the secondary peroxidation products [[3], [4], [5]]. Electrophilic oxidatively-truncated derivatives readily form adducts with proteins by attacking nucleophilic amino acids [4,6]. These protein-lipid adducts may play a role in ferroptotic death-gateway mechanism(s). Dozens of carbonylated protein adducts accumulating during ferroptosis have been detected, yet none of them have been identified as a specific executioner of plasma membrane rupture and cell death [6]. Both adducts of proteins with oxidatively-modified lipids as well as clusters composed of exclusively oxidized lipids (without lipid/protein adducts) may function as a mechanism of plasma membrane damage and rupture culminating in cell death. While the formation of the adducts of peroxidized lipids with proteins have been documented, identification of the adducts specifically identified as a cell death mechanism has not been achieved. Similarly, the formation of clusters of oxidatively modified lipids has been hypothesized and somewhat confirmed by indirect methods, their role in cell death has not been specifically characterized. A comprehensive characterization of phospholipid peroxidation products of both types, primary and secondary as well as adducts of proteins with oxidatively-modified lipids, represents a necessary, yet still lacking, evidence for the relevance of ferroptosis in cancer therapy.

The ferroptotic death induction has been considered in an attempt to overcome the enhanced resistance of tumor cells to apoptosis and pyroptosis [1]. A well-known propensity of cancer cells for continuous growth and division depends on a sufficient supply and high content of iron [7]. An elevated level of iron is also required to induce lipid peroxidation, making cancer cells potentially vulnerable to ferroptotic demise, particularly in the context of insufficient thiol regulation of hydroperoxy-phosphatidylethanolamines (PE-OOH) by GPx4 [[8], [9], [10]]. Massive and highly diversified molecular speciation of peroxidized phospholipids is a hallmark of ferroptosis [11].

The utilization of a pro-ferroptotic approach for cancer therapy has generated significant enthusiasm and has stimulated a broader research effort in the field (reviewed in Ref. [12]). Despite of the sharp focus of ferroptosis research (>2200 entries out of total 4121 on ferroptosis) and lipid peroxidation (>350 entries) in cancer, published reports assessing peroxidized lipids in cancer utilized indirect, non-specific protocols, (reviewed in Ref. [13]). LC-MS studies of oxidatively-modified lipids in cancer cells undergoing ferroptosis are lacking [14,15].

Melanoma is a highly aggressive cancer arising from the melanocyte lineage [16], and is highly refractory to treatment once it metastasizes, with a 5-year overall survival of 25–50% for stage IV disease [17]. We employed the well-studied A375 human melanoma cell line as a model for redox lipidomics characterization of phospholipid peroxidation products in the context of pro-/anti-ferroptotic environments created in vitro and in vivo. In this work, we performed detailed redox lipidomics analyses of peroxidized and oxidatively-truncated phospholipids generated during RSL3-induced ferroptosis in A375 melanoma cells.

2. Materials and methods

2.1. Animals

Six-week-old male Nude mice (Crl: NU(NCR)-Fox1nu, Charles River) were housed under the specific pathogen-free conditions with food and water available at libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee (protocol #20097961) and performed in accordance with the U.S. Public Health Service policy.

2.2. Cell cultures

A375 melanoma cells (ATCC) and GFP-expressing A375 human melanoma cells (Imanis Life Sciences; authenticated) were maintained in complete DMEM medium (ATCC) supplemented with 10% heat inactivated FBS (Gemini Bio-Products), 1% penicillin/streptomycin (ATCC), and 1 μg/mL puromycin to maintain high GFP expression (GIBCO).

2.3. In vitro treatment

A375 or GFP-expressing A375 melanoma cells were plated in 24-well plates (12,500 cells per well). After attachment, cells were treated with RSL3 (0.125 μM, 0.5 μM), erastin (2.0 μM, 5.0 μM), ML-162 (0.25 μM,0.5 μM, 1 μM) and imidazole ketone erastin (IKE) (0.5 μM, 2.5 μM, 5 μM) for 16 h in the absence or in the presence of ferrostatin-1 (1 μM). For lipidomics experiments, A375 cells were seeded in 150 mm cell culture dishes (1x106 cells per dish) and treated with RSL3 (0.5 μM) for different times (3–16 h). After treatment, cells were trypsinized, centrifuged at 3000 g for 30 min and pellets were stored at −80 °C. To assess the effects of oleic acid (OA) and arachidonic acid (AA) on ferroptosis, A375 cells were preincubated with OA-BSA or AA-BSA complexes for 3 h, after which RSL3 (0.5 μM) was added and cells were incubated for additional 16 h at 37 °C. To prepare fatty-acid-BSA complexes, fatty acid free albumin was diluted in PBS, sterilized by filtration through 0.22 μm filter and mixed with AA or OA dissolved in DMSO. The molar fatty acid:BSA ratio was 2:1 and DMSO concentration in stock solution was 1%.

2.4. In vivo pro-ferroptotic treatment

GFP-expressing A375 melanoma cells were maintained in complete DMEM medium (ATCC) supplemented with 10% heat inactivated FBS (Gemini Bio-Products), 1% penicillin/streptomycin (ATCC), and 1 μg/mL puromycin to maintain high GFP expression (GIBCO). Before treatment, cells were washed twice with PBS and resuspended in saline. Cells (5 × 106/200 μl) were inoculated subcutaneously in the right flank of mice. Mice were divided in two groups: (i) control (tumor cells), (ii) tumor cells + RSL3 (R&D system, 50 mg/kg), peritumoral injection, Day 8 and Day 10 after tumor injection). RSL3 was dissolved in 5% DMSO+ 45% PEG400 + 50% NaCl (0.9%) and delivered via intra-tumoral injection. The same solvent was used for the control group. Tumor growth was monitoring using caliper measurement of the width and length of the tumor. The tumor size was expressed as the tumor area (mm2). Tumor weight was also determined at Day 14 after tumor injection, when the animals were sacrificed. All studies consisted of 7 mice per group and were repeated two times.

2.5. Ferroptosis assay

Cell death was determined by measuring the release of lactate dehydrogenase (LDH). LDH activity was measured using the CytoTox-ONE™ Cyto-toxicity Detection Kit (Promega) according to the manufacturer's instructions. In addition, assessment of cell death was performed by propidium iodide (PI) uptake. Aliquots of cells were resuspended in PBS containing PI (2 μg/ml) for 5 min on ice and then PI uptake was measured by flow cytometry. To estimate ferroptotic cell death, PI uptake and LDH release were measured after incubation of cells in the absence and in the presence of ferroptosis inhibitor, ferrostatin-1.

2.6. Assessment of BODIPY581/591 oxidation related to lipid peroxidation in cells

Fluorescence assay using BODIPY581/591 c11 was used to characterize the ability of cells/tissues to trigger lipid peroxidation. While this protocol does not directly assess lipid peroxidation products, the oxidation of the butadienyl-portion of the probe's fluorophore conjugated to undecanoic acid and consequent shift of the fluorescence emission peak from 590 nm to 510 nm made it a popular measure of the oxidizability of the probe by a tentative catalytic peroxidation mechanism [13,18,19]. A375 cells were incubated with 5 μM BODIPY581/592 c11 (Invitrogen, D3861) for 25–30 min at 37 °C, washed with HBSS (2X) and resuspended in HBSS followed by flow cytometric analysis. A BD Canto-II flow cytometer (BD Biosciences) was used for flow cytometry and FlowJo software was used for data analysis.

2.7. GSH measurements

Cells were trypsinized, centrifuged at 700 g for 6 min, resuspended in 80 μl of PBS and then lysed by using a freeze-thaw procedure. Aliquots (10 μl) in triplicate were incubated with 10 μM Thiol fluorescent probe IV (Millipore) in PBS for 5 min and then fluorescence was measured using the Cytation 5 imaging reader (BioTek) (excitation and emission wavelengths of 400 nm and 465 nm, respectively). To exclude the contribution of other small molecular weight thiols, samples were treated with Glutathione peroxidase from bovine erythrocytes (Sigma Aldrich) in the presence of cumene hydroperoxide (Sigma-Aldrich). GSH content was normalized by protein and presented as pmol/μg of protein. Protein was measured by the BCA method.

2.8. Lipid extraction

Lipids were extracted using the Folch procedure, and phosphorus was determined by a micro-method as described previously [20]. Briefly, Cells (1.5x106) were resuspended in 0.9% KCl, pieces of tumor (2–10 mg of protein) tissue were homogenized in PBS containing DTPA (100 μM) and lipids were extracted using a chloroform methanol mixture of 2:1 (v/v). To prevent oxidation of lipids during extraction and sample preparation for LC/MS analysis, a chloroform-methanol mixture containing 0.01% butylated hydroxytoluene was used.

3. Redox phospholipidomics

LC/ESI-MS analyses of lipids, oxygenated lipids as well as oxidatively truncated species were performed on a Thermo HPLC system coupled to an Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer. Phospholipids were separated on a normal phase column (Luna 3 μm Silica (2) 100 A, 150 × 1.0 mm, (Phenomenex)) at a flow rate of 0.065 mL/min. The column was maintained at 35 °C. The analysis was performed using gradient solvents (A and B) containing 10 mM ammonium formate as previously described [20]. Solvent A contained isopropanol/hexane/water (285:215:5, v/v/v), and solvent B contained isopropanol/hexane/water (285:215:40, v/v/v). All solvents were LC/MS-grade. The gradient was as follows:0–3 min, 10–37 %B; 3–15 min, hold at 37 %B; 15–23 min, 37–100 %B; 23–75 min, hold at 100 %B; 75–76 min, 100-10 %B; 76–90 min, equilibrate at 10 %B. For normal phase analysis, lipids were analyzed in negative ion mode using the following parameters: capillary voltage, 3500; sheath, aux and sweep gases (35, 17, 0, respectively); ion transfer tube temperature, 300 deg. C; orbitrap resolution, 120,000; scan range 400–1800 m/z; Rf lens, 40; injection time, 100 ms; intensity threshold set at 4e2. For data dependent MS2, an isolation window of 1.2 m/z was used. Collision energy (HCD) was static at 24 with an orbitrap resolution of 15,000 with an injection time of 22 ms. To assess oxygenated and oxidatively-truncated phospholipid molecular species, separation on a C30 reverse phase column (Accucore 2.6 μm, 2.1 mm × 25 cm, Thermo Scientific) was employed. Solvent A: acetonitrile/water (50/50); Solvent B: 2-propanol/acetonitrile/water (85/10/5). Both A and B solvents contained 5 mM ammonium formate and 0.1% formic acid as modifiers. Gradient method was as follows: 0–40 min, 15%–50% B (linear, 5); 40–130 min, 50–100% B (linear, 5); 130–135 min, hold at 100% B; 135–140 min, 15% B (linear, 5); 140–150 min, 15% B for equilibration. The flow was maintained at 100 μl/min. Column temperature was set at 35 °C. Solvent A: acetonitrile/water (50/50); Solvent B: 2-propanol/acetonitrile/water (85/10/5). For C30 analysis, lipids were analyzed in negative ion mode using the following parameters: capillary voltage, 3900; sheath, aux and sweep gases (30, 23, 1, respectively); ion transfer tube temperature, 300 deg. C; orbitrap resolution, 120,000; scan range 150–1800 m/z; Rf lens, 40; injection time, 100 ms; intensity threshold set at 4e2. For data dependent MS2, an isolation window of 1.2 m/z was used. Collision energy (HCD) was static at 24 with an orbitrap resolution of 15,000 with an injection time of 22 ms. Low abundance oxygenated phospholipids were assessed by using a targeted selected ion monitoring (tSIM) method with inclusion lists and an increased injection time to 500 ms for MS/MS analysis. The injection volume was 5 μL that was equivalent to 5 nmol of total phospholipids. Targeted (tSIM) and MS2 analysis of specific oxidized masses was performed on a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo) in negative ion mode (profile) at a resolution of 120,000. The maximum injection time of 128 ms with 1 microscan and a normalized AGC target of 400% using the orbitrap as the detector. MS2 analysis was performed with a 500 ms injection time using high energy collisional dissociation (HCD) with collision energy set to 24 with an isolation window of 1.2 m/z and a resolution of 30,000, also using the orbitrap as the detector. Capillary spray voltage was set at 3900 V, and ion transfer tube temperature was 300 °C. Sheath, auxiliary and sweep gasses were set to 30, 23 and 1 (arbitrary units), respectively.

3.1. Identification of phospholipid oxygenated species

Comprehensive analyses and identification of lipids and their oxygenated metabolites was performed with high accuracy by exact masses using Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer. Compound Discoverer™ software package (ThermoFisher Scientific) with an in-house generated analysis workflow and oxidized phospholipid database was used to evaluate LC/MS data. Peaks with a signal/noise ratio of >3 were identified and searched against the database of oxidized phospholipids. Lipid signals were further filtered by retention time and values for m/z were matched within 5 ppm to identify the lipid species. Under conditions used, the number of points required per peak was from 10 to 15. Commercially available 1-stearoyl-2-15(S)-HpETE-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-15(S)-HETE-sn-glycero-3-phospho-ethanolamine, 1-stearoyl-2-15(S)-HpETE-sn-glycero-3-phosphocholine, 1-stearoyl-2-15(S)-HETE-sn-glycero-3-phosphocholine (Cayman Chemicals) were used as reference standards for oxidized phospholipids. In addition, we have biosynthesized and purified (>95%) in the lab several additional hydroperoxy-phospholipids, such as mono- and di-HOO-PI, mono- and di-HOO-CL and HOO-PS as well as individual lyso-PUFA-PL and their oxidation products (such as lyso-HOO-LPE and lyso-HOO-LPC). 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanol-amine, 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-inositol (ammonium salt), 1,1′,2,2′-tetralinoleyl-cardiolipin (sodium salt) (Avanti Polar Lipids) were used as reference standards to build the calibration curve for non-oxidized phospholipids. Deuterated phospholipids: 1-hexadecanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-ethanolamine (PE(16:0D31/18:1)), 1-hexadecanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0D31/18:1)), 1-hexadecanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoserine (PS(16:0D31/18:1)), 1-hexa-decanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphate (PA(16:0D31/18:1)), 1-hexadecanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoglycerol (PG(16:0D31/18:1)), 1-hexadecanoyl(d31)-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-myo-inositol) (PI(16:0D31/18:1)) and 1,1′,2,2′-tetramyristoyl-cardiolipin (sodium salt) (Avanti Polar Lipids) were used as internal standards. Internal standards were added directly to the MS sample to a final concentration of 1 μM. Phospholipids were separated by solid phase extraction (SPE) using a sequential combination of silica gel/aminopropyl-silica gel SPE cartridges as described previously [21]. Fractions were collected, solvent was evaporated under N2 and 20 μL of 100% of mobile phase B was added (see above). Targeted selected ion monitoring (tSIM) with an inclusion list and MS/MS analysis were used to identify oxygenated phospholipids.

3.2. Statistical analysis

Statistical analyses were performed by either unpaired t-test or one-way ANOVA, Tukey's multiple comparisons test, using GraphPad Prism 9 software. The data are presented as mean ± S.D. Principal component analysis (PCA) and orthogonal projection of latent structures - discriminant analysis (OPLS-DA) of total and redox lipidomes data were performed by using SIMCA 16.0 software (Sartorius).

4. Results

4.1. Modulation of A375 cells survival by pro- and anti-ferroptotic agents in vitro

To induce ferroptosis, we used two canonical prototypical ferroptotic cell death inducers – RSL3 (a GPX4 inhibitor) [22] and erastin (a GSH depleting agent inhibiting the cystine/glutamate anti-porter [23]). Both agents caused significant death of A375 cells (Fig. 1A), which was fully preventable by a typical ferroptosis inhibitor, ferrostatin-1 (Fer-1). Similar results were obtained with an inhibitor of GPx4, ML-162, and with an inhibitor of cystine/glutamate anti-porter, imidazole-keto-erastin (IKE) (Supplementary Fig. S1A). Depletion of GSH by an alternative mechanism, treatment with l-buthionine sulfoximine (BSO), also caused significant death of A375 cells (Supplementary Fig. S1B). These data are indicative of cell death by ferroptosis. In line with this, inhibitors of other cell death programs, zVAD-fmk (apoptosis), necrostatin-1S (necroptosis), MCC950 (pyroptosis) and chloroquine (autophagy) were ineffective in suppressing cell death induced by RSL3 (Fig. 1B).

Fig. 1.

Fig. 1

Open in a new tab

Effect of different cell death inducers on survival of A375 melanoma cells. (A) Erastin (left panel) and RSL3 (right panel) induce death of A375 melanoma cells. Cells were incubated with either RSL3 (0.125 μM, 0.5 μM) or erastin (2.3 μM, 5 μM) in the absence and in the presence of Fer-1 (1 μM) for 20 h at 37 °C. (B) Effect of different cell inhibitors on RSL3 (0.5 μM) induced death of A375 melanoma cells. Fer-1 (1 μM; ferroptosis), zVAD-fmk (20 μM, apoptosis), necrostatin-1S (20 μM, necroptosis), MCC950 (0.3 μM; pyroptosis) and chloroquine (40 μM; autophagy). Cells were incubated for 20 h at 37 °C. Effects of oleic acid (OA) (C) and arachidonic acid (AA) (E) on ferroptosis induced by RSL3 in A375 melanoma cells. Cells were preincubated with AA or OA conjugated with BSA for 3 h before treatment with RSL3 (20 h at 37 °C). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Content of PE molecular species in A375 melanoma obtained from control cells and cells treated with AA or OA. Data are expressed as pmol/μmol of phospholipids and presented as heat maps auto-scaled to z scores and coded blue (low values) to red (high values). (F) Score plot of PCA shows the differences in lipidomes of control cells and cells treated with AA or OA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To further confirm the engagement of ferroptotic pathways in RSL3-induced death of A375 cells, we tested the effects of mono-unsaturated oleic acid, which is known to block ferroptosis by decreasing the amounts of peroxidizable polyunsaturated fatty acid (PUFA) residues in phospholipids [24,25]. Oleic acid significantly suppressed ferroptotic death of A375 cells (Fig. 1C) by modulating the cell phospholipid composition (Fig. 1D). Expectedly, the content of OA-containing PE species in OA-treated cells was increased at the expense of species with 4 and more double bonds (Fig. 1D). In contrast, supplementing cell media with the readily oxidizable arachidonic acid (AA) significantly enhanced melanoma cell sensitivity to ferroptosis (Fig. 1E). The lipidome of the AA treated cells was enriched with PE molecular species containing AA (Fig. 1D). The principal component analysis (PCA) of lipidome profiles obtained from control (untreated) cells and cells exposed to AA or OA showed three distinct clusters of the samples in the score plot of the two principal components (t [1] and t [2]) which accounted for 0.40 and 0.31 of total variance, respectively (Fig. 1F). These results emphasize the importance of peroxidation substrates, PUFA-phospholipids, in the execution of ferroptosis [26].

Next, we performed a time-course study of cell death caused by RSL3 (0.5 μM) and observed an almost linear increase in the number of dead cells from 3 to 40% of RSL3 treatment over 3–16 h (Fig. 2A). Given the importance of lipid peroxidation in the execution of ferroptosis [11], we correlated cell death with the time-course of the peroxidation process. One of the most commonly used and technically simple techniques is based on measurements of BODIPY581/591 c11 fluorescence [27]. The fluorescence response from BODIPY581/591 c11 is due to the oxidation of the polyunsaturated butadienyl portion of the dye, which does not directly relate to lipid peroxidation. However, we were eager to compare this indirect protocol with our LC-MS redox lipidomics assessment of different phospholipid peroxidation products. The BODIPY581/591 assay showed a different time-dependence of the response: the number of BODIPY581/591 c11-positive cells increased to its maximum at 3 h and did not substantially change in the course of further incubation (Fig. 2B). Spearman's rank correlation was computed to assess the relationship between cell death and lipid peroxidation (fluorescence of BODIPY581/591 c11). There was an insignificant positive correlation between the two variables r(15) = 0.33, p = 0.190 (Table 1). We further performed detailed LC-MS based time-course analyses of phospholipid peroxidation.

Fig. 2.

Fig. 2

Open in a new tab

RSL3 induces cell death, BODIPY581/591 c11 oxidation, accumulation of PE-derived ferroptotic cell death signals and oxidatively truncated PE and PC species in A375 melanoma cells. (A) RSL3 induces cell death in a time-dependent manner as evidenced by PI positive cells. (B) Oxidation of BODIPY581/591 c11 in cells exposed to RSL3 (0.5 μM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Accumulation of PE-derived ferroptotic cell death signals - PE-38:4-OOH (C), PE-40:4-OOH (D) and oxidatively truncated species originated from PE-38:4-OOH (E) and PC-38:4-OOH species (F) in cells exposed to RSL3.

Table 1.

Spearman's rank correlation between the levels of oxygenated phospholipids and RSL3-induced cell death with corresponding p values.

Oxidative metabolites Spearman's rank correlation (r) with corresponding p values
Total PEox r(17) = .51, p = 0.026
PE-(18:0/20:4-OOH) r(17) = .65, p = 0.002
PE-(18:0/22:4-OOH) r(17) = .85, p = 0.000
PE-(18:0/HOOA) r(17) = .53, p = 0.020
Total PCox r(17) = .45, p = 0.054
PC-(18:0/HOOA) r(17) = .55, p = 0.014
BODIPY581/591 c11 r(15) = .33, p = 0.190
Open in a new tab

PEox – oxidized phosphatidylethanolamine; PCox – oxidized phosphatidylcholine.

18:0 – stearic acid; 20:4-OOH – hydroperoxy-arachidonic acid; 22:4-OOH – hydroperoxy-adrenic acid; HOOA – hydroxy-8-oxooct-6-enoic acid.

4.2. Redox phospholipidomics analysis of ferroptosis in A375 cells in vitro

The phospholipidome of control, untreated, A375 melanoma cells was represented by 597 phospholipid species, including 378 non-oxidized phospholipids, 70 lyso-phospholipids and 149 oxygenated phospholipids (including oxidatively-truncated species) (Supplementary Figs. S2A and S2B). The oxy-phospholipidome of A375 cells was highly diversified and contained oxygenated species in all major classes of phospholipids: phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), phosphatidylglycerol (PG) and bis-monoacylglycero-phosphate (BMP). Levels of oxygenated species were upregulated in different phospholipid classes of RSL3-treated cells (Supplementary Fig. S3). The contents of 79 oxidized species were significantly increased in RSL3 treated cells, including 21 PEox, 19 PCox, 9 PIox, 23 PSox and 7 CLox species. For the majority of these oxygenated metabolites, the structures were identified by exact mass, retention time and MS2 (Supplementary Figs. S4–S10). We also noted the presence of secondary peroxidation products, oxidatively truncated electrophilic species generated via the β-scission process of hyrdoperoxy-phospholipids. These products were present in the two most-abundant classes, PE and PC. Lyso-phospholipids, intermediaries of the phospholipid redox metabolism and remodeling, were also broadly represented in essentially all major classes (Supplementary Fig. S2B).

Next, we assessed the changes in the redox phospholipidome caused by the treatment of A375 cells with RSL3. While PC was the major phospholipid in A375 cells (58.8 ± 6.1% of total phospholipids), PE species were predominantly oxidized in cells exposed to RSL3 (Supplementary Fig. 3). We found a positive correlation (r(15) = 0.51 with p value of 0.026) between total oxygenated PE levels and the extent of cell death (Table 1). Focusing on cell death, we noted that RSL3 induced a time-dependent accumulation of two PL species that have been previously identified as biomarkers of ferroptosis, PE-(18:0/20:4-OOH) and PE-(18:0/22:4-OOH) (Fig. 2C and D). Using exact mass, retention time and MS/MS analysis, we verified the structure of these PE molecular species as 1-steraoyl-2-15-HpETE-PE and 1-stearoyl-2-HpDTE-PE species (Supplementary Fig. S4A, S4B and S4C). Strong positive Spearman's rank correlations between the contents of these species and RSL3-induced cell death were found (Table 1). Furthermore, elevated levels of an oxidatively truncated product formed from PE-(18:0/20:4-OOH) species, PE-(18:0/hydroxy-8-oxo-oct-6-enoic acid (HOOA)), were detected (Fig. 2E). Of note, a significant accumulation of oxidatively-truncated product originated from PC-(18:0/20:4-OOH), PC-(18:0/HOOA), was also detected (Fig. 2F). The levels of PE-(18:0/HOOA) and PC-(18:0/HOOA) were positively correlated with cell death (Table 1). Structures of PE-(18:0/HOOA) and PC-(18:0/HOOA) were confirmed by MS2 and exact mass, respectively (Supplementary Fig. S4D).

Principal component analysis (PCA) using all identified phospholipids (non-oxygenated plus oxygenated phospholipids) revealed that control and RSL3-treated cells (3 h–16 h time points) clustered into two well-separated groups on the PCA score plot (Fig. 3A). Orthogonal projection of latent structures - discriminant analysis (OPLS-DA) was utilized for the detailed characterization of phospholipid peroxidation, which confirmed that RSL3 treatment had a profound impact on the separation of samples (Fig. 3A). OPLS-DA showed that control samples grouped tightly and were well separated from RSL3-treated cells (Fig. 3A). To identify phospholipid species responsible for group separation, we generated S-plots and VIP predictive score plots (with a threshold of 1) from OPLS-DA (Supplementary Figs. S11A and S11B). Among those, PE-(18:0/20:4-OOH), PE-(18:0/22:4-OOH), PE-(18:0/HOOA) and PC-(18:0/HOOA) were identified as biochemically significant metabolites associated with RSL3 treatment, and exhibited high VIP values of 1.291, 1.477, 1.430 and 1.322, respectively. We further performed multivariant analysis of the peroxidation products, ie the oxy-lipidome, (Fig. 3B and C, Supplementary Figs. S11C and S11D). As shown on OPLS-DA plots, control group clustered tightly, while RSL3-treated cells (3–16 h) were broadly separated, demonstrating a heterogeneity of lipids associated with cell death induced by RSL3 (Fig. 3B). LC/MS/MS analysis revealed that species with a high VIP score were predominantly represented by PE species containing hydroxy- or hydoperoxy groups in linoleic (18:2), arachidonic (C20:4), adrenic (C22:4), docosapentaenoic (C22:5) and docosahexaenoic (C22:6) acid residues as evidenced by MS2 analysis (Supplementary Figs. S5–S10). More importantly, this analysis confirmed that PE-(18:0/20:4-OOH), PE-(18:0/22:4-OOH), PE-(18:0/HOOA) and PC-(18:0/HOOA) species were biochemically significant metabolites associated with RSL3 treatment with high VIP predicative scores (Fig. 3C and Supplementary Figs. S11C and S11D). Consequently, these four species have a major impact on the discrimination between untreated and RSL3 treated group of samples and could be considered as potential biomarkers of ferroptosis associated with RSL3 treatment.

Fig. 3.

Fig. 3

Open in a new tab

RSL3 induces changes in the oxylipidome of A375 melanoma cells in vitro. Score plots of PCA (upper panel) and OPLS-DA (lower panel) show the differences in lipidomes (A) and oxylipidome (B) of control and RSL3 (0.5 μM) treated cells. (C) Content of oxygenated phospholipids in A375 cells exposed to RSL3. Data are expressed as pmol/μmol of phospholipids; Variable importance in projection (VIP) score plots reflecting the significance of variables for the OPLS-DA models (bar graphs on the right); N = 3. PE, phosphatidylethanolamine, PC, phosphatidylcholine, PI, phosphatidylinositol, PS, phosphatidylserine. PEp, PE plasmalogen.

To uncover oxidized phospholipid metabolites which may be ferroptosis driving forces in the RSL3-treated group, we compared samples at different time points of RSL3 treatment (3 h vs 6 h, 6 h vs 9, 9 h vs 12 h and 12 vs 16 h) (Supplementary Fig. S12). The samples obtained at different time points of the treatment with RSL3 were well separated from each other (Supplementary Fig. S12, upper panels). Using VIP scores which reflected significant variable contributions in the OPLS-DA model, we demonstrated that oxygenated PE and PC species along with their two oxidatively-truncated species were mainly responsible for the differences between 3 h and 6 h treatment groups (Supplementary Fig. S12A, lower panel). In addition to PE and PC, oxidized CL species were responsible for the differences between 6 h and 9 h treatment groups (Supplementary Fig. S12B, lower panel). Interestingly, seven CL oxidized species were among the top 10 phospholipid metabolites responsible for the difference between 9 h and 12 h treatment groups (Supplementary Fig. S12C, lower panel). Furthermore, oxygenated PE, CL and PI produced the separation between 12 h and 16 h treatment groups (Supplementary Fig. S12D, lower panel). Thus, OPLS-DA analyses suggest that different oxygenated phospholipid metabolites contributed to the separation between RSL3-treated samples at different time points.

4.3. Redox phospholipidomics analysis of ferroptosis in GFP-A375 cells in vitro

In our in vivo experiments, we employed fluorescently labeled GFP-A375 cells, permitting their flow-cytometry-based separation from the non-tumor cells. We first examined whether the response of GFP-A375 cells to RSL3 in vitro is similar to that of the non-labeled A375 cells. GFP-A375 cells treated with RSL3 (0.5 μM) for 16 h displayed a cell death which was preventable by Fer-1 (Supplementary Fig. S13A). In addition, decreased GSH levels were detected in RSL3-treated cells. No changes in GSH levels were detectable in the presence of Fer-1 (Supplementary Fig. S13B). Importantly, redox lipidomics analysis of RSL3-treated GFP-A375 cells revealed an accumulation of PE species with oxygenated arachidonic acid - PE-(16:0/20:4-OOH), PE-(18:0/20:4-OOH), and adrenic acid PE-(18:0/22:4-OOH) that were identified as ferroptotic cell death signals (Supplementary Fig. S13C). Thus, major features of RSL3-induced ferroptosis were retained by the GFP-A375 melanoma cells.

4.4. Redox phospholipidomics analysis of ferroptosis in GFP-A375 cells isolated from the tumor in vivo

To determine whether ferroptosis-associated oxygenated phospholipid species relate to melanoma ferroptosis in vivo, we employed an allogeneic model of subcutaneous inoculation of GFP- A375 cells into immune-deficient athymic nude mice. Based on the time-course studies of tumor growth, we evaluated the maximally tolerated dose of A375 cells and chose the dose of 5x106 cells for further experiments. Assessment of tumor weight and size on day 14 after the inoculation showed a significant suppressive effect of RSL3 on tumor growth. At the dose of 50 mg/kg of body weight, RSL3 significantly reduced the weights (Fig. 4A) and sizes of GFP-A375 tumors (Fig. 4B).

Fig. 4.

Fig. 4

Open in a new tab

RSL3 suppresses the tumor growth and induces accumulation of PE-derived ferroptotic cell death signals and oxidatively truncated PE species in GFP-A375 melanoma in vivo. Effect of RSL3 on tumor weight (A) and size (B) on day 14 after the GFP-A375 melanoma inoculation. N = 5, **p < 0.01; ***p < 0.001. (C) Volcano plot showing the changes in the levels of oxygenated phospholipids in tumors isolated from RSL3 treated mice. (D) Content of ferroptotic cell death signal, PE-38:4-OOH (D), oxidatively truncated PE-16:0-HOOA (F) and PE-18:0-HOOA (H) in tumors obtained from control (untreated) group of mice and animals treated with RSL3. N = 5, **p < 0.01. The Pearson Correlation Coefficient demonstrate the strong relationship between the content of PE-38:4-OOH (E), PE-18:0-HOOA (G), PE-16:0-HOOA (I) and tumor weight.

To assess whether RSL3 causes melanoma ferroptosis in vivo, we performed redox phospholipidomics assessments of peroxidation products in GFP-A375 cells isolated from the tumors. We identified 552 phospholipid species, including 108 oxygenated and 8 oxidatively-truncated phospholipid species, as well as 54 lyso-phospholipids, (Supplementary Figs. S14A and S14B). We found that 10 oxygenated phospholipid species were up-regulated, and 4 oxygenated phospholipid species were down-regulated in melanoma cells from the tumors of RSL3-treated mice (Fig. 4C). Elevated levels of one of the major pro-ferroptotic PE oxidation products, 18:0/20:4-OOH identified as 1-sn-stearoyl-2-sn-15-HpETE-PE (Supplementary Fig. S15A), were detected in the melanoma cells isolated from RSL3-treated tumors (Fig. 4D). Next, to reveal the relationship between oxidatively modified lipids and tumor regression, and anti-melanoma role of oxidized metabolites, we conducted correlation analyses (using the Pearson correlation coefficient) and found a negative association between tumor weight and the content of PE-(18:0/20:4-OOH) (r = −0.503) (Fig. 4E). Tumor weights also negatively correlated with the levels of oxidatively truncated PE species PE-16:0-HOOA (Fig. 4F and G) and PE 18:0-HOOA (Fig. 4H and I, Supplementary Fig. S15B) in the melanoma cells of RSL3-treated tumors. The Pearson correlation coefficients were estimated as r = −0.547 and r = −0.505 for PE-18:0-HOOA and PE 16:0-HOOA, respectively.

OPLS-DA analysis revealed that the phospholipidomes of GFP-A375 cells isolated from tumors of untreated mice and those from tumors of RSL3-treated mice were significantly different (Fig. 5A). PE-(18:0/20:4-OOH), a ferroptosis death signal that was significantly elevated in melanoma cells isolated from tumors of RSL3-treated mice (Fig. 4D), had the highest VIP predictive score (Supplementary Figs. S16A and S16B). Two additional PE species containing oxygenated arachidonic acid – PE-(18:1/20:4-OOH) and PE-(20:4/20:4-OOH), as well as two arachidonoyl-PE derived oxidatively truncated species - PE-(16:0/HOOA) and PE-(18:0/HOOA), had high VIP scores (>1.7) (Supplementary Fig. S16B). When only the oxy-lipidomes were subjected to OPLS-DA analysis (Fig. 5B, and Supplementary Figs. S16C and S16D), oxygenated (PE-(18:0/20:4-OOH), PE-(18:1/20:4-OOH), PE-(20:4/20:4-OOH)) and oxidatively-truncated (PE-(16:0/HOOA) and PE-(18:0/HOOA)) PE species were identified as the metabolites (biomarkers of ferroptosis) with a statistically significant increase in melanoma cells from the tumors of the RSL3-treated mice (Fig. 5C, Supplementary Figs. S16C and S16D). Furthermore, PE-(18:0/20:4-OOH) species was identified as the major contributor to the separation of the melanoma cells from control tumors versus RSL-3 treated tumors, with the highest VIP (>1.7) predictive score (Supplementary Fig. S16D).

Fig. 5.

Fig. 5

Open in a new tab

RSL3 induced changes in the phospholipidome and oxylipidome of GFP-A375 melanoma in vivo. OPLS-DA score plots showing the differences in phospholipidome (A) and oxylipidome (B) of RSL3-treated cells. (C) Content of oxygenated phospholipids in A375 melanoma obtained from control mice (C1–C5) and mice exposed to RSL3 (R1-R5). Data are expressed as pmol/μmol of phospholipids and presented as heat maps auto-scaled to z scores and coded blue (low values) to red (high values); Variable importance in projection (VIP) score plots reflecting the significance of variables for the OPLS-DA models (bar graphs on the right); PE, phosphatidylethanolamine, PI, phosphatidylinositol. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

5. Discussion

The incidence of melanoma has been steadily increasing and it remains one of the most aggressive and treatment-resistant malignancies [28]. While targeted therapies and immune checkpoint inhibitors have improved survival of patients with advanced melanomas, a significant number of individuals either do not benefit from these therapies or relapse after the initial response [29]. Thus, there is an urgent need to identify new therapeutic targets in melanoma. One new approach involves the induction of ferroptotic death in the melanoma cells. Ferroptosis-inducing drugs have been considered as an orthogonal therapeutic regimen to target the differentiation plasticity of melanoma cells, enhance the efficacy of and overcome the resistance to targeted and immune checkpoint therapies [29].

Our previous work has documented the presence of characteristic pro-ferroptotic signals – peroxidized derivatives of PUFA-PE – in several types of cancer cells such as human fibrosarcoma HT-1080, human SH-SY5Y neuroblastoma, MDA-MB and BT-20 breast cancer cells [20,30,31]. We also performed LC-MS analyses of peroxidized PE species in non-cancerous cells: human airway epithelial cells, human kidney epithelial cells, human HK2 kidney cells, H109 human fibroblasts, placental trophoblasts, mouse embryonic fibroblasts, mouse hippocampal HT22 neuronal cells, mouse cardiomyocytes, murine macrophages and neutrophils [9,20,[32], [33], [34], [35], [36]] exposed to different ferroptosis inducing agents, including a GPX4 inhibitor, RSL3. In the current work, our goal was to characterize, in a comparative way, the time-course of in vitro and in vivo (using a xenograft mouse model) generation of PUFA-PL oxidation products in A375 human melanoma cells. For this reason, we limited the study to a single tumor cell line and performed detailed redox lipidomics analysis of several types of non-truncated peroxidation products (hydroperoxy-, hydroxy-, oxo-) as well as oxidatively-truncated species generated during RSL3-induced ferroptosis in A375 melanoma cells in vitro and in vivo. In this work, we demonstrate: i) significantly elevated levels of hydroperoxy-PE species originating from arachidonoyl- and adrenoyl-PEs as well as oxidatively-truncated PE species during ferroptosis triggered in vitro and in vivo; ii) a strong positive correlation between the contents of PE-derived oxidation products acting as pro-ferroptotic signals and ferroptotic death in vitro; iii) a strong negative correlation between tumor weight and the levels of PE-derived ferroptotic cell death signals and oxidatively truncated PE species in vivo. Thus, redox lipidomics is a sensitive and precise approach for the detection and characterization of phospholipid-derived biomarkers of ferroptosis in cancer cells.

The redox phospholipidomics results indicate that naïve, actively proliferating A375 cells contain more than 145 species of peroxidized phospholipids species, including both hydroperoxy-products and oxidatively truncated species. In vitro treatment of A375 cells with the pro-ferroptotic inducer, RSL3, increased several oxygenated phospholipid species, including hydroperoxy-species and oxidatively-truncated molecules. Among them we detected hydroperoxy-PE species that have been associated with the execution of the ferroptotic program – PE-18:0/20:4-OOH and PE-18:0/22:4-OOH [9]. The contents of oxidatively-truncated species derived from these hydroperoxy-products were also elevated in RSL3-treated cells. A commonly used anti-ferroptotic agent, ferrostatin-1, suppressed the generation of pro-ferroptotic signals and cell death. These data strongly support the engagement of the ferroptotic mechanism in melanoma demise.

A variety of other oxidatively modified phospholipids of different classes (PC, CL, PI, PS) were also elevated in RSL3-treated cells. While ferroptosis is accompanied by the peroxidation of several other classes of phospholipids [37], their roles in ferroptosis or other (patho)physiological responses to RSL3 has to be further explored and may relate to pro-inflammatory responses, immunosuppression, reactivation of dormant tumor cells, stimulation of proliferation, etc [33,[38], [39], [40], [41], [42]].

LC-MS measurements revealed that the intracellular accumulation of lipid peroxidation products was time-dependent, steadily increasing over the course of 16 h of RSL3 treatment. There was a strong correlation between increased levels of hydroperoxy-arachidonoyl-PE-derivatives and oxidatively-truncated products and the extent of cell death (Table 1). These correlations were insignificant when lipid peroxidation was assessed by a protocol utilizing BODIPY581/592 c11. This finding is not surprising if one considers that the latter protocol does not measure the levels of lipid peroxidation products [43]. Of note, correlations between cell death and non-PE hydroperoxy-phospholipids were relatively weaker vs. peroxidized PE, indicating an important role of oxidized PE as the driving species of ferroptotic death.

Ferroptosis is a non-cell autonomous process: initiated in one cell it propagates to the closest neighbors [44]. It is likely that peroxidized phospholipids, including oxidized PE molecules, are responsible, at least in part, for this wave-like cell-to-cell spread of ferroptosis [45]. A recent report documented that melanoma cells in lymph experience less oxidative stress, and thus may metastasize more easily in that environment, than melanoma cells in blood [25]. The report demonstrated that the immunocompromised mice bearing patient-derived melanomas, and immunocompetent mice bearing syngeneic tumors, had more melanoma cells per microliter in tumor-draining lymph than in tumor-draining blood. This was also associated with higher levels of OA (along with GSH) and less free iron in lymph versus blood. In line with this, our data demonstrate that OA strongly protects A375 cells from ferroptosis.

Our previous work established that in lipid-enriched TME PMN-myeloid-derived suppressor cells (PMN-MDSC) are an important source of peroxidized phospholipids affecting anti-tumor immune responses [46,47], including i) the immunosuppression by PGE2 (free or esterified into phospholipids and releasable by specialized Ca2+independent phospholipases A2) [47], ii) the inhibition of antigen cross-presentation by the dendritic cells [46], and iii) the immunosuppression by M2 macrophages [48]. In addition, CD36-dependent uptake of peroxidized lipids has recently been described which promotes CD8+ T cell dysfunction and immunosuppression [49]. Therefore, there are concerns that therapies aimed at inducing ferroptosis in the malignant cells may have pro-tumor effects through their deleterious actions on anti-tumor immune responses. Appreciation of the reported cases of failures of ferroptosis targeting in immunocompetent animals and vice versa, effective tumor eradication in immunocompromised animals [50,51] dictates the demand for new concepts in designing anticancer strategies based on ferroptosis. It is conceivable that a an aggressive pro-ferroptotic targeting of cancer cells combined with a precise and reliable anti-ferroptotic targeting of regulatory and effector immune cells represents a novel optimized and balanced differential approach.

Authors’ disclosures

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Author contributions

Conception and design – VEK, AKS, HB.

Acquisition, analysis and interpretation of data – YYT, AAK, VAT, GS, DR, WGH, REP, HT.

Writing or edition of the manuscript – YYT, YLB, YN, WGH, JHD, HB, AKS, VEK.

Administrative, technical and material support – AKS, JHD, VEK.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

This work has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N910D00024, RO1 CA266342, RO1 CA243142, U01 AI156924 and U01AI156923.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2023.102650.

Contributor Information

Yulia Y. Tyurina, Email: yyt1@pitt.edu.

Apurva K. Srivastava, Email: srivastavaa4@mail.nih.gov.

Valerian E. Kagan, Email: kagan@pitt.edu.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.pptx (2.4MB, pptx)

Data availability

Data will be made available on request.

References

  • 1.Okada H., Mak T.W. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat. Rev. Cancer. 2004;4(8):592–603. doi: 10.1038/nrc1412. [DOI] [PubMed] [Google Scholar]
  • 2.Xia X., Wang X., Cheng Z., Qin W., Lei L., Jiang J., Hu J. The role of pyroptosis in cancer: pro-cancer or pro-"host". Cell Death Dis. 2019;10(9):650. doi: 10.1038/s41419-019-1883-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Yin H., Porter N.A. New insights regarding the autoxidation of polyunsaturated fatty acids. Antioxidants Redox Signal. 2005;7(1–2):170–184. doi: 10.1089/ars.2005.7.170. [DOI] [PubMed] [Google Scholar]
  • 4.Kagan V.E., Tyurina Y.Y., Vlasova, Kapralov A.A., Amoscato A.A., Anthonymuthu T.S., Tyurin V.A., Shrivastava I.H., Cinemre F.B., Lamade A., Epperly M.W., Greenberger J.S., Beezhold D.H., Mallampalli R.K., Srivastava A.K., Bayir H., Shvedova A.A. Redox epiphospholipidome in programmed cell death signaling: catalytic mechanisms and regulation. Front. Endocrinol. 2020;11 doi: 10.3389/fendo.2020.628079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Stoyanovsky D.A., Tyurina Y.Y., Shrivastava I., Bahar I., Tyurin V.A., Protchenko O., Jadhav S., Bolevich S.B., Kozlov A.V., Vladimirov Y.A., Shvedova A.A., Philpott C.C., Bayir H., Kagan V.E. Iron catalysis of lipid peroxidation in ferroptosis: regulated enzymatic or random free radical reaction? Free Radic. Biol. Med. 2019;133:153–161. doi: 10.1016/j.freeradbiomed.2018.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chen Y., Liu Y., Lan T., Qin W., Zhu Y., Qin K., Gao J., Wang H., Hou X., Chen N., Friedmann Angeli J.P., Conrad M., Wang C. Quantitative profiling of protein carbonylations in ferroptosis by an aniline-derived probe. J. Am. Chem. Soc. 2018;140(13):4712–4720. doi: 10.1021/jacs.8b01462. [DOI] [PubMed] [Google Scholar]
  • 7.Hassannia B., Vandenabeele P., Vanden Berghe T. Targeting ferroptosis to iron out cancer. Cancer Cell. 2019;35(6):830–849. doi: 10.1016/j.ccell.2019.04.002. [DOI] [PubMed] [Google Scholar]
  • 8.Seiler A., Schneider M., Forster H., Roth S., Wirth E.K., Culmsee C., Plesnila N., Kremmer E., Radmark O., Wurst W., Bornkamm G.W., Schweizer U., Conrad M. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metabol. 2008;8(3):237–248. doi: 10.1016/j.cmet.2008.07.005. [DOI] [PubMed] [Google Scholar]
  • 9.Kagan V.E., Mao G., Qu F., Angeli J.P., Doll S., Croix C.S., Dar H.H., Liu B., Tyurin V.A., Ritov V.B., Kapralov A.A., Amoscato A.A., Jiang J., Anthonymuthu T., Mohammadyani D., Yang Q., Proneth B., Klein-Seetharaman J., Watkins S., Bahar I., Greenberger J., Mallampalli R.K., Stockwell B.R., Tyurina Y.Y., Conrad M., Bayir H. Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat. Chem. Biol. 2017;13(1):81–90. doi: 10.1038/nchembio.2238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yang W.S., SriRamaratnam R., Welsch M.E., Shimada K., Skouta R., Viswanathan V.S., Cheah J.H., Clemons P.A., Shamji A.F., Clish C.B., Brown L.M., Girotti A.W., Cornish V.W., Schreiber S.L., Stockwell B.R. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014;156(1–2):317–331. doi: 10.1016/j.cell.2013.12.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Yang W.S., Stockwell B.R. Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 2016;26(3):165–176. doi: 10.1016/j.tcb.2015.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wang H., Cheng Y., Mao C., Liu S., Xiao D., Huang J., Tao Y. Emerging mechanisms and targeted therapy of ferroptosis in cancer. Mol. Ther. 2021;29(7):2185–2208. doi: 10.1016/j.ymthe.2021.03.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Bayir H., Anthonymuthu T.S., Tyurina Y.Y., Patel S.J., Amoscato A.A., Lamade A.M., Yang Q., Vladimirov G.K., Philpott C.C., Kagan V.E. Achieving Life through death: redox biology of lipid peroxidation in ferroptosis. Cell Chem Biol. 2020;27(4):387–408. doi: 10.1016/j.chembiol.2020.03.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ghoochani A., Hsu E.C., Aslan M., Rice M.A., Nguyen H.M., Brooks J.D., Corey E., Paulmurugan R., Stoyanova T. Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer. Cancer Res. 2021;81(6):1583–1594. doi: 10.1158/0008-5472.CAN-20-3477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Liu J., Kang R., Tang D. The art of war: ferroptosis and pancreatic cancer. Front. Pharmacol. 2021;12 doi: 10.3389/fphar.2021.773909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Liao Y., Jia X., Ren Y., Deji Z., Gesang Y., Ning N., Feng H., Yu H., Wei A. Suppressive role of microRNA-130b-3p in ferroptosis in melanoma cells correlates with DKK1 inhibition and Nrf2-HO-1 pathway activation. Hum. Cell. 2021;34(5):1532–1544. doi: 10.1007/s13577-021-00557-5. [DOI] [PubMed] [Google Scholar]
  • 17.Ugurel S., Rohmel J., Ascierto P.A., Becker J.C., Flaherty K.T., Grob J.J., Hauschild A., Larkin J., Livingstone E., Long G.V., Lorigan P., McArthur G.A., Ribas A., Robert C., Zimmer L., Schadendorf D., Garbe C. Survival of patients with advanced metastatic melanoma: the impact of MAP kinase pathway inhibition and immune checkpoint inhibition - update 2019. Eur. J. Cancer. 2020;130:126–138. doi: 10.1016/j.ejca.2020.02.021. [DOI] [PubMed] [Google Scholar]
  • 18.MacDonald M.L., Murray I.V., Axelsen P.H. Mass spectrometric analysis demonstrates that BODIPY 581/591 C11 overestimates and inhibits oxidative lipid damage. Free Radic. Biol. Med. 2007;42(9):1392–1397. doi: 10.1016/j.freeradbiomed.2007.01.038. [DOI] [PubMed] [Google Scholar]
  • 19.Murphy M.P., Bayir H., Belousov V., Chang C.J., Davies K.J.A., Davies M.J., Dick T.P., Finkel T., Forman H.J., Janssen-Heininger Y., Gems D., Kagan V.E., Kalyanaraman B., Larsson N.G., Milne G.L., Nystrom T., Poulsen H.E., Radi R., Van Remmen H., Schumacker P.T., Thornalley P.J., Toyokuni S., Winterbourn C.C., Yin H., Halliwell B. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 2022;4(6):651–662. doi: 10.1038/s42255-022-00591-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Sun W.Y., Tyurin V.A., Mikulska-Ruminska K., Shrivastava I.H., Anthonymuthu T.S., Zhai Y.J., Pan M.H., Gong H.B., Lu D.H., Sun J., Duan W.J., Korolev S., Abramov A.Y., Angelova P.R., Miller I., Beharier O., Mao G.W., Dar H.H., Kapralov A.A., Amoscato A.A., Hastings T.G., Greenamyre T.J., Chu C.T., Sadovsky Y., Bahar I., Bayir H., Tyurina Y.Y., He R.R., Kagan V.E. Phospholipase iPLA2beta averts ferroptosis by eliminating a redox lipid death signal. Nat. Chem. Biol. 2021;17(4):465–476. doi: 10.1038/s41589-020-00734-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Fauland A., Trotzmuller M., Eberl A., Afiuni-Zadeh S., Kofeler H., Guo X., Lankmayr E. An improved SPE method for fractionation and identification of phospholipids. J. Separ. Sci. 2013;36(4):744–751. doi: 10.1002/jssc.201200708. [DOI] [PubMed] [Google Scholar]
  • 22.Yang W.S., Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 2008;15(3):234–245. doi: 10.1016/j.chembiol.2008.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Dixon S.J., Lemberg K.M., Lamprecht M.R., Skouta R., Zaitsev E.M., Gleason C.E., Patel D.N., Bauer A.J., Cantley A.M., Yang W.S., Morrison B., 3rd, Stockwell B.R. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–1072. doi: 10.1016/j.cell.2012.03.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yang W.S., Kim K.J., Gaschler M.M., Patel M., Shchepinov M.S., Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl. Acad. Sci. U. S. A. 2016;113(34):E4966–E4975. doi: 10.1073/pnas.1603244113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ubellacker J.M., Tasdogan A., Ramesh V., Shen B., Mitchell E.C., Martin-Sandoval M.S., Gu Z., McCormick M.L., Durham A.B., Spitz D.R., Zhao Z., Mathews T.P., Morrison S.J. Lymph protects metastasizing melanoma cells from ferroptosis. Nature. 2020;585(7823):113–118. doi: 10.1038/s41586-020-2623-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Doll S., Proneth B., Tyurina Y.Y., Panzilius E., Kobayashi S., Ingold I., Irmler M., Beckers J., Aichler M., Walch A., Prokisch H., Trumbach D., Mao G., Qu F., Bayir H., Fullekrug J., Scheel C.H., Wurst W., Schick J.A., Kagan V.E., Angeli J.P., Conrad M. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat. Chem. Biol. 2017;13(1):91–98. doi: 10.1038/nchembio.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Drummen G.P., van Liebergen L.C., Op den Kamp J.A., Post J.A. C11-BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic. Biol. Med. 2002;33(4):473–490. doi: 10.1016/s0891-5849(02)00848-1. [DOI] [PubMed] [Google Scholar]
  • 28.Gagliardi M., Saverio V., Monzani R., Ferrari E., Piacentini M., Corazzari M. Ferroptosis: a new unexpected chance to treat metastatic melanoma? Cell Cycle. 2020;19(19):2411–2425. doi: 10.1080/15384101.2020.1806426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Talty R., Bosenberg M. The role of ferroptosis in melanoma. Pigment Cell Melanoma Res. 2022;35(1):18–25. doi: 10.1111/pcmr.13009. [DOI] [PubMed] [Google Scholar]
  • 30.Gaschler M.M., Andia A.A., Liu H., Csuka J.M., Hurlocker B., Vaiana C.A., Heindel D.W., Zuckerman D.S., Bos P.H., Reznik E., Ye L.F., Tyurina Y.Y., Lin A.J., Shchepinov M.S., Chan A.Y., Peguero-Pereira E., Fomich M.A., Daniels J.D., Bekish A.V., Shmanai V.V., Kagan V.E., Mahal L.K., Woerpel K.A., Stockwell B.R. FINO(2) initiates ferroptosis through GPX4 inactivation and iron oxidation. Nat. Chem. Biol. 2018;14(5):507–515. doi: 10.1038/s41589-018-0031-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Beatty A., Singh T., Tyurina Y.Y., Tyurin V.A., Samovich S., Nicolas E., Maslar K., Zhou Y., Cai K.Q., Tan Y., Doll S., Conrad M., Subramanian A., Bayir H., Kagan V.E., Rennefahrt U., Peterson J.R. Ferroptotic cell death triggered by conjugated linolenic acids is mediated by ACSL1. Nat. Commun. 2021;12(1):2244. doi: 10.1038/s41467-021-22471-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Jang S., Chapa-Dubocq X.R., Tyurina Y.Y., St Croix C.M., Kapralov A.A., Tyurin V.A., Bayir H., Kagan V.E., Javadov S. Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes. Redox Biol. 2021;45 doi: 10.1016/j.redox.2021.102021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kim R., Hashimoto A., Markosyan N., Tyurin V.A., Tyurina Y.Y., Kar G., Fu S., Sehgal M., Garcia-Gerique L., Kossenkov A., Gebregziabher B.A., Tobias J.W., Hicks K., Halpin R.A., Cvetesic N., Deng H., Donthireddy L., Greenberg A., Nam B., Vonderheide R.H., Nefedova Y., Kagan V.E., Gabrilovich D.I. Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature. 2022;612(7939):338–346. doi: 10.1038/s41586-022-05443-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kapralov A.A., Yang Q., Dar H.H., Tyurina Y.Y., Anthonymuthu T.S., Kim R., St Croix C.M., Mikulska-Ruminska K., Liu B., Shrivastava I.H., Tyurin V.A., Ting H.C., Wu Y.L., Gao Y., Shurin G.V., Artyukhova M.A., Ponomareva L.A., Timashev P.S., Domingues R.M., Stoyanovsky D.A., Greenberger J.S., Mallampalli R.K., Bahar I., Gabrilovich D.I., Bayir H., Kagan V.E. Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death. Nat. Chem. Biol. 2020;16(3):278–290. doi: 10.1038/s41589-019-0462-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wenzel S.E., Tyurina Y.Y., Zhao J., St Croix C.M., Dar H.H., Mao G., Tyurin V.A., Anthonymuthu T.S., Kapralov A.A., Amoscato A.A., Mikulska-Ruminska K., Shrivastava I.H., Kenny E.M., Yang Q., Rosenbaum J.C., Sparvero L.J., Emlet D.R., Wen X., Minami Y., Qu F., Watkins S.C., Holman T.R., VanDemark A.P., Kellum J.A., Bahar I., Bayir H., Kagan V.E. PEBP1 wardens ferroptosis by enabling lipoxygenase generation of lipid death signals. Cell. 2017;171(3):628–641 e26. doi: 10.1016/j.cell.2017.09.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Beharier O., Tyurin V.A., Goff J.P., Guerrero-Santoro J., Kajiwara K., Chu T., Tyurina Y.Y., St Croix C.M., Wallace C.T., Parry S., Parks W.T., Kagan V.E., Sadovsky Y. PLA2G6 guards placental trophoblasts against ferroptotic injury. Proc. Natl. Acad. Sci. U. S. A. 2020;117(44):27319–27328. doi: 10.1073/pnas.2009201117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wiernicki B., Dubois H., Tyurina Y.Y., Hassannia B., Bayir H., Kagan V.E., Vandenabeele P., Wullaert A., Vanden Berghe T. Excessive phospholipid peroxidation distinguishes ferroptosis from other cell death modes including pyroptosis. Cell Death Dis. 2020;11(10):922. doi: 10.1038/s41419-020-03118-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Perego M., Tyurin V.A., Tyurina Y.Y., Yellets J., Nacarelli T., Lin C., Nefedova Y., Kossenkov A., Liu Q., Sreedhar S., Pass H., Roth J., Vogl T., Feldser D., Zhang R., Kagan V.E., Gabrilovich D.I. Reactivation of dormant tumor cells by modified lipids derived from stress-activated neutrophils. Sci. Transl. Med. 2020;12(572) doi: 10.1126/scitranslmed.abb5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Johnstone S.R., Ross J., Rizzo M.J., Straub A.C., Lampe P.D., Leitinger N., Isakson B.E. Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation. Am. J. Pathol. 2009;175(2):916–924. doi: 10.2353/ajpath.2009.090160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Greig F.H., Hutchison L., Spickett C.M., Kennedy S. Differential effects of chlorinated and oxidized phospholipids in vascular tissue: implications for neointima formation. Clin. Sci. (Lond.) 2015;128(9):579–592. doi: 10.1042/CS20140578. [DOI] [PubMed] [Google Scholar]
  • 41.Bochkov V.N. Inflammatory profile of oxidized phospholipids. Thromb. Haemostasis. 2007;97(3):348–354. [PubMed] [Google Scholar]
  • 42.Xu S., Chaudhary O., Rodriguez-Morales P., Sun X., Chen D., Zappasodi R., Xu Z., Pinto A.F.M., Williams A., Schulze I., Farsakoglu Y., Varanasi S.K., Low J.S., Tang W., Wang H., McDonald B., Tripple V., Downes M., Evans R.M., Abumrad N.A., Merghoub T., Wolchok J.D., Shokhirev M.N., Ho P.C., Witztum J.L., Emu B., Cui G., Kaech S.M. Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8(+) T cells in tumors. Immunity. 2021;54(7):1561–1577 e7. doi: 10.1016/j.immuni.2021.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Drummen G.P., Gadella B.M., Post J.A., Brouwers J.F. Mass spectrometric characterization of the oxidation of the fluorescent lipid peroxidation reporter molecule C11-BODIPY(581/591) Free Radic. Biol. Med. 2004;36(12):1635–1644. doi: 10.1016/j.freeradbiomed.2004.03.014. [DOI] [PubMed] [Google Scholar]
  • 44.Linkermann A., Skouta R., Himmerkus N., Mulay S.R., Dewitz C., De Zen F., Prokai A., Zuchtriegel G., Krombach F., Welz P.S., Weinlich R., Vanden Berghe T., Vandenabeele P., Pasparakis M., Bleich M., Weinberg J.M., Reichel C.A., Brasen J.H., Kunzendorf U., Anders H.J., Stockwell B.R., Green D.R., Krautwald S. Synchronized renal tubular cell death involves ferroptosis. Proc. Natl. Acad. Sci. U. S. A. 2014;111(47):16836–16841. doi: 10.1073/pnas.1415518111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Dar H.H., Tyurina Y.Y., Mikulska-Ruminska K., Shrivastava I., Ting H.C., Tyurin V.A., Krieger J., St Croix C.M., Watkins S., Bayir E., Mao G., Armbruster C.R., Kapralov A., Wang H., Parsek M.R., Anthonymuthu T.S., Ogunsola A.F., Flitter B.A., Freedman C.J., Gaston J.R., Holman T.R., Pilewski J.M., Greenberger J.S., Mallampalli R.K., Doi Y., Lee J.S., Bahar I., Bomberger J.M., Bayir H., Kagan V.E. Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium. J. Clin. Invest. 2018;128(10):4639–4653. doi: 10.1172/JCI99490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ugolini A., Tyurin V.A., Tyurina Y.Y., Tcyganov E.N., Donthireddy L., Kagan V.E., Gabrilovich D.I., Veglia F. Polymorphonuclear myeloid-derived suppressor cells limit antigen cross-presentation by dendritic cells in cancer. JCI Insight. 2020;5(15) doi: 10.1172/jci.insight.138581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Veglia F., Tyurin V.A., Blasi M., De Leo A., Kossenkov A.V., Donthireddy L., To T.K.J., Schug Z., Basu S., Wang F., Ricciotti E., DiRusso C., Murphy M.E., Vonderheide R.H., Lieberman P.M., Mulligan C., Nam B., Hockstein N., Masters G., Guarino M., Lin C., Nefedova Y., Black P., Kagan V.E., Gabrilovich D.I. Fatty acid transport protein 2 reprograms neutrophils in cancer. Nature. 2019;569(7754):73–78. doi: 10.1038/s41586-019-1118-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Kwak T., Wang F., Deng H., Condamine T., Kumar V., Perego M., Kossenkov A., Montaner L.J., Xu X., Xu W., Zheng C., Schuchter L.M., Amaravadi R.K., Mitchell T.C., Karakousis G.C., Mulligan C., Nam B., Masters G., Hockstein N., Bennett J., Nefedova Y., Gabrilovich D.I. Distinct populations of immune-suppressive macrophages differentiate from monocytic myeloid-derived suppressor cells in cancer. Cell Rep. 2020;33(13) doi: 10.1016/j.celrep.2020.108571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Ma X., Xiao L., Liu L., Ye L., Su P., Bi E., Wang Q., Yang M., Qian J., Yi Q. CD36-mediated ferroptosis dampens intratumoral CD8(+) T cell effector function and impairs their antitumor ability. Cell Metabol. 2021;33(5):1001–1012 e5. doi: 10.1016/j.cmet.2021.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Schreiber R.D., Old L.J., Smyth M.J. Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science. 2011;331(6024):1565–1570. doi: 10.1126/science.1203486. [DOI] [PubMed] [Google Scholar]
  • 51.Basel M.T., Narayanan S., Ganta C., Shreshta T.B., Marquez A., Pyle M., Hill J., Bossmann S.H., Troyer D.L. Developing a xenograft human tumor model in immunocompetent mice. Cancer Lett. 2018;412:256–263. doi: 10.1016/j.canlet.2017.10.009. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.pptx (2.4MB, pptx)

Data Availability Statement

Data will be made available on request.

Articles from Redox Biology are provided here courtesy of Elsevier

RESOURCES