Phospholipids with two polyunsaturated fatty acyl tails promote ferroptosis

Abstract

Phospholipids containing a single polyunsaturated fatty acyl tail (PL-PUFA₁s) are considered the driving force behind ferroptosis, whereas phospholipids with diacyl-PUFA tails (PL-PUFA₂s) have been rarely characterized. Dietary lipids modulate ferroptosis, but the mechanisms governing lipid metabolism and ferroptosis sensitivity are not well understood. Our research revealed a significant accumulation of diacyl-PUFA phosphatidylcholines (PC-PUFA₂s) following fatty acid or phospholipid treatments, correlating with cancer cell sensitivity to ferroptosis. Depletion of PC-PUFA₂s occurred in aging and Huntington's disease brain tissue, linking it to ferroptosis. Notably, PC-PUFA₂s interacted with the mitochondrial electron transport chain, generating reactive oxygen species (ROS) for initiating lipid peroxidation. Mitochondria-targeted antioxidants protected cells from PC-PUFA₂-induced mitochondrial ROS (mtROS), lipid peroxidation, and cell death. These findings reveal a critical role for PC-PUFA₂s in controlling mitochondria homeostasis and ferroptosis in various contexts and explain the ferroptosis-modulating mechanisms of free fatty acids. PC-PUFA₂s may serve as diagnostic and therapeutic targets for modulating ferroptosis.

Keywords: PUFA; ROS; complex I; diacyl-PUFA phosphatidylcholine; electron transport chain; ferroptosis; lipids; mitochondria; phospholipid; polyunsaturated fatty acid.

Figure 1.

PC-PUFA₂s induce ferroptosis. A, Viability of A549 cells (left) and IGROV-1 cells (right) treated with 50 μM phospholipids with different head groups and fatty acyl tails with or without 10 μM ferrostatin-1 (Fer-1) for 24 hr. B, Viability of IGROV-1 cells co-treated with 50 μM PC(22:6, 22:6) and 10 μM Fer-1, 100 μM deferoxamine (DFO), 20 μM Z-VAD-FMK, 20 μM necrostatin-1s (Nec-1s), or 100 nM bafilomycin A1 (Baf-A1) for 24 hr. C, IC₅₀ of PC-PUFA₂ with two C22:6 or C20:4 tails versus PC-PUFA₁ with one C18:0 tail and one of the C22:6 or C20:4 PUFA tail in multiple cancer cell lines. Each color represents a cell line. D, IGROV-1 cells treated with vehicle, 10 μM Fer-1 alone, or 100 μM PC(22:6, 22:6) with or without 10 μM Fer-1 for 4 hr were stained with TfR1 antibody. Immunofluorescent images showing nucleus DAPI and TfR1 stain. Scale bar is shown as 20 μm. The relative mean fluorescence intensity of TfR1 membrane staining compared to vehicle is plotted as mean ± SD. Each dot represents a cell. n=50–164 for all groups. E, Lipid peroxidation measured by C11-BODIPY^581/591 in IGROV-1 cells treated with 100 μM PC(22:6, 22:6) with or without 20 μM ferrostatin-1 for 4 hr. The relative mean fluorescence intensity of oxidized C11-BODIPY^581/591 compared to vehicle is plotted as mean ± SD of n=3 biological replicates. F, HT-1080 cells were co-treated with 12.5 μM PC(22:6, 22:6) and ferroptosis inducers (RSL3 15.6 nM, IKE 0.63 μM, FIN56 10 μM, media cystine concentration 6.25 μM, CoQ₁₀ complete depletion) for 24 hr. Data plotted as mean ± SD of n=2 technical replicates. G, RSL3 IC₅₀ in 20 cancer cell lines. Data plotted as mean ± SD of n=2 biological replicates. H, The signal intensity of PCs normalized to protein concentration of each cell sample is plotted as mean ± SD of n=3 biological replicates. I, Statistical analysis of PC abundances between ferroptosis-sensitive and resistant cells. Data plotted as mean ± SD of n=10 for each group. One-way and two-way ANOVA: p<0.03 (*), p<0.002 (**), p<0.0002 (***), p<0.0001 (****). See also Figure S1 and Table S1.

Figure 2.

Dietary PC-PUFA₂s are remodeled into the cell lipidome. A, A498 cells were treated with 25 μM PC(18:0, 20:4) or PC(20:4, 20:4) for 6 hr. The relative abundance of PCs and lysoPCs compared to untreated group is plotted as mean ± SD of n=3 biological replicates. B, Viability of IGROV-1 cells co-treated with 6.25 μM PC(22:6, 22:6) and 30 μM PLA2 inhibitor, ONO-RS-082 for 24 hr. Data plotted as mean ± SD of n=2 technical replicates. C, Structures of deuterated PCs. D, IGROV-1 cells were treated with 25 μM [d11]-PC(18:0, 20:4) or [d22]-PC(20:4, 20:4) for 6 hr. The relative abundance of original and remodeled deuterated lipid species compared to [d11]-PC(18:0, 20:4)-treated group is plotted as mean ± SD of n=4 biological replicates. Two-way ANOVA: p<0.03 (*), p<0.002 (**), p<0.0002 (***), p<0.0001 (****). See also Figure S2.

Figure 3.

PC-PUFA₂ is involved in dietary fatty acid remodeling. A, Viability of IGROV-1 cells treated with 50 μM DHA or PC(22:6, 22:6) with or without 10 μM Fer-1 for 24 hr. Data plotted as mean ± SD of n=2 technical replicates. B, Viability of IGROV-1 cells pre-treated with 25 μM DHA, or 50 μM oleic acid, or both DHA and oleic acid for 24 hr and then treated with 31.3 nM RSL3 for 24 hr. Data plotted as mean ± SD of n=4 technical replicates. C, Viability of Calu-1 cells pre-treated with 25 μM DHA, or 50 μM oleic acid for 24 hr and then treated with 25 μM PC for 24 hr. Data plotted as mean ± SD of n=2 technical replicates. D, IGROV-1 cells were treated with 25 μM DHA for 6 hr. The relative abundance of lipids compared to untreated group is plotted as mean ± SD of n=3 biological replicates. E, IGROV-1 cells were treated with 25 μM DHA, or 50 μM oleic acid, or both DHA and oleic acid for 6 hr. The relative abundance of lipids compared to untreated group is plotted as mean ± SD of n=3 biological replicates. F, Calu-1 cells were knockdown using pooled siRNA containing 4 different sequences targeting ACSL4 and then treated with 50 μM PCs for 24 hr. Viability is compared to vehicle-treated group and is plotted as mean ± SD of n=4 technical replicates. The knockdown efficiency was confirmed by qPCR and western blot. G, IGROV-1 cells were treated with PCs or vehicle for 5 hr and whole-cell lysate was analyzed for western blot detection of GPX4 and ACSL4. Two-way ANOVA: p<0.03 (*), p<0.002 (**), p<0.0002 (***), p<0.0001 (****). See also Figure S3.

Figure 4.

PL-PUFA₂s exhibit interaction with mitochondrial electron transport chain. A, Structures of biotinylated phospholipids. B, HT-1080 whole-cell lysate was incubated with biotinylated phospholipids, vehicle, or biotin, and phospholipid-bound proteins were identified by MS-based proteomics. 2-D plot of principle component analysis (PCA) of proteomic data of all groups. C, Volcano plot of genes differentially enriched in PL(22:6, 22:6) vs PL(18:0, 22:6) groups. Data cutoff at fold change >4 and FDR adjusted p <0.05. Significantly enriched mitochondrial complex I genes are labeled. D, Western blot analysis of mitochondrial complex proteins in pull-down samples. Empty lanes are cut and shown by the dotted line. E, HT-1080 cells were treated with 50 μM biotinylated PL(22:6, 22:6) or PL(18:0, 22:6) for 6 hr and immunostained with anti-COX IV, anti-Calnexin, anti-Giantin, and anti-LAMP1 antibodies. Composite fluorescent images are shown as PL in red and each antibody in pseudo-cyan. Scale bar is shown as 20 μm. Colocalization between PL and each subcellular organelle is represented by Pearson’s coefficient plotted as mean ± SD of n=6–8 images for all groups. One-way ANOVA: p<0.03 (*), p<0.002 (**), p<0.0002 (***), p<0.0001 (****). See also Figure S4.

Figure 5.

PC-PUFA₂s induces mitochondrial stress. A, Mitochondrial superoxide accumulation measured by MitoSOX red in IGROV-1 cells treated with 100 μM PC or 5 μM antimycin A for 4 hr. The relative mean fluorescence intensity of MitoSOX compared to vehicle is plotted as mean ± SD of 2 biological replicates. B, Mitochondrial membrane potential measured by rhodamine123 in IGROV-1 cells treated with 100 μM PC or 10 μM antimycin A for 4 hr. Scale bar is shown as 20 μm. The relative ratio of rhodamine123 to MitoTracker compared to vehicle is plotted as mean ± SD of n=9–10 images. C, Oxygen consumption rate was measured kinetically for 4 hr in HT-1080 pre-treated with 100 μM PC(22:6, 22:6) with or without 20 μM Fer-1, or 1 μM RSL3 for 2 hr. D, Viability of IGROV-1 cells co-treated with 100 μM PC(22:6, 22:6) and 0.5 μM mitoquinone (MitoQ) or 0.5 μM decylubiquinone (DecylQ) for 24 hr. E, Mitochondrial superoxide accumulation measured by MitoSOX green in IGROV-1 cells treated with 100 μM PC(22:6, 22:6) with or without 10 μM Fer-1 or 0.2 μM mitoQ for 4 hr. Relative ratio of MitoSOX to MitoTracker compared to vehicle is plotted as mean ± SD. Each dot represents a cell. n=67–124 for all groups. F, Lipid peroxidation measured by C11-BODIPY^581/591 in IGROV-1 cells co-treated with 100 μM PC(22:6, 22:6) and 0.5 μM mitoQ for 4 hr. One-way and two-way ANOVA: p<0.03 (*), p<0.002 (**), p<0.0002 (***), p<0.0001 (****). See also Figure S5.

Figure 6.

PC-PUFA₂s induce lipid peroxidation, but not an unfolded protein response in ER. A, Lipid peroxidation measured by Liperfluo in IGROV-1 cells treated with 100 μM PC(22:6, 22:6) for 4 hr. Cells were co-stained with ER-Tracker and MitoTracker. Composite fluorescent images are shown as Liperfluo in green and ER-Tracker or MitoTracker in pseudo-magenta. Scale bar is shown as 20 μm. Colocalization between Liperfluo and mitochondria or ER is represented by Pearson’s coefficient plotted as mean ± SD of n=9–11 images. B, Relative mean intensity of Liperfluo compared to vehicle is plotted as mean ± SD of n=10–14 images. C, Western blot analysis of UPR mediators, XBP1s and GADD in IGROV-1 cells treated with 100 μM PC, 10 μM CCCP, or 1 μM Brefeldin A for 4 hr. The transcript levels of XBP1s and CHOP were measured in same cells used for western blot. Data plotted as mean ± SD of n=3 technical triplicates. D, Schematics of the mechanism of PC-PUFA₂-induced ferroptosis. See also Figure S6.