Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis
- Columbia University, United States
- Brain Science Institute, Johns Hopkins Medicine, United States
- Johns Hopkins Medicine, United States
- Howard Hughes Medical Institute, Columbia University, United States
Figures
Figure 1
Cell death is triggered by erastin and related compounds in different cell lines under a variety of physiological conditions.
(A and B) Modulatory effect (Me) profiles of erastin- and SAS-induced death in five different cell lines (143B, BJeHLT, BJeLR, Calu-1, and HT-1080) in response to six different cell death inhibitors …
Figure 2 with 1 supplement
Erastin inhibits system xc− function potently and specifically.
(A and B) Na+-independent uptake of 14C-cystine (A) and 14C-L-phenylalanine (Phe) (B) over 5 min in HT-1080 and Calu-1 cells treated with erastin or SAS. D-Phe was included as a positive control in B…
Figure 2—figure supplement 1
Monitoring system xc− activity by following glutamate release.
(A) Overview of the assay design. Glutamate released by the cell is detected by an enzyme-linked reaction. Erastin and SAS inhibit glutamate release from system xc−, but not other transporters. …
Figure 3
Structure activity relationship (SAR) analysis of erastin.
(A) Structures of 20 erastin analogs. (B) Lethal EC50 for each analog determined in HT-1080 cells in a 10-point, twofold dilution assay, starting at a high concentration of 20 μM, ±β-ME (18 μM). …
Figure 4 with 1 supplement
Analysis of erastin effects using RNA-Seq.
(A and B) List of genes upregulated (A) and downregulated (B) by erastin treatment, as detected in HT-1080 cells using RNA Seq. The number of fragments per kilobase of exon per megabase of sequence …
Figure 4—figure supplement 1
Analysis of ER stress in response to erastin treatment.
(A) Western blot analysis of erastin (5 μM, 7 hr)—sorafenib (10 μM, 12 hr)—and thapsigargin (10 ng/ml, 6 hr)-treated cells reveals changes associated with ER stress including phosphorylation of …
Figure 5 with 1 supplement
Identification of sorafenib as an inhibitor of system xc−.
(A and B) Modulatory profiling of (A) A549 and (B) HCT-116 cells in response to either buthionine sulfoximine (BSO) or erastin ±20 different lethal compounds (see ‘Materials and methods’ for the …
Figure 5—figure supplement 1
Effect of sorafenib on cell viability.
(A) Glutamate release from same cell lines in response to erastin or sorafenib. (B) Viability of five cell lines (HT-1080, TC-32, 143B, Calu-1 and U2OS) in response to 48 hr treatment with …
Figure 6
Analysis of sorafenib analog function.
(A and B) Sorafenib and 87 sorafenib analogs were prepared and tested in HT-1080 cells for the induction of cell death (EC50) and the suppression of this cell death by β-ME (18 μM) and Fer-1 (1 μM) …
Figure 7 with 1 supplement
Summary of adverse events reported with sorafenib and other kinase inhibitors.
Analysis of adverse events across 20 physiological system categories associated with kinase inhibitor treatment.
Figure 7—figure supplement 1
Test of the ability of various kinase inhibitors to induce ferroptosis and inhibit system xc−.
(A) Viability of HT-1080 cells in response to 48 hr treatment with erastin (as a control) or various kinase inhibitors ± β-ME and Fer-1. (B) Ability of kinase inhibitors to inhibit glutamate release …
Figure 8 with 1 supplement
Isolation and analysis of erastin-resistant clones identifies AKR1C genes as mediators of resistance to system xc− inhibition.
(A) Outline of the isolation of DU-145 erastin-resistant clones. (B–H) Comparison of the parental DU-145 cell line and five erastin-resistant clonal lines to the indicated lethal compounds: (B–E), …
Figure 8—figure supplement 1
Test of the ability of additional ferroptosis inducers to cause death in parental DU-145 and erastin-resistant DU-145 cell lines.
Dose-response curves for parental DU-145 and erastin-resistant DU-145 clones treated with active (3, 20, 21, 22) and inactive (14, 16) erastin analogs and 1S,3R-RSL3. The color coding for all graphs …
Additional files
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Supplementary file 1
Extended materials and methods. Description of chemical synthesis and characterization. Data also available as ‘Extended Materials and Methods’ from Dryad data Repository: http://dx.doi.org/10.5061/dryad.jp43c.
- https://doi.org/10.7554/eLife.02523.016
