Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 28:1:210.
doi: 10.1038/s42003-018-0212-6. eCollection 2018.

Golgi stress mediates redox imbalance and ferroptosis in human cells

Hamed Alborzinia  1   2 , Tatiana I Ignashkova  1 , Francesca R Dejure  1 , Mathieu Gendarme  1 , Jannick Theobald  3 , Stefan Wölfl  3 , Ralph K Lindemann  4 , Jan H Reiling  1   5
Affiliations

Golgi stress mediates redox imbalance and ferroptosis in human cells

Hamed Alborzinia et al. Commun Biol. .

Abstract

Cytotoxic activities of several Golgi-dispersing compounds including AMF-26/M-COPA, brefeldin A and golgicide A have previously been shown to induce autophagy or apoptosis. Here, we demonstrate that these Golgi disruptors also trigger ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent oxidative degradation of lipids. Inhibitors of ferroptosis not only counteract cell death, but they also protect from Golgi dispersal and inhibition of protein secretion in response to several Golgi stress agents. Furthermore, the application of sublethal doses of ferroptosis-inducers such as erastin and sorafenib, low cystine growth conditions, or genetic knockdown of SLC7A11 and GPX4 all similarly protect cells from Golgi stress and lead to modulation of ACSL4, SLC7A5, SLC7A11 or GPX4 levels. Collectively, this study suggests a previously unrecognized function of the Golgi apparatus, which involves cellular redox control and prevents ferroptotic cell death.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ROS induction and depletion of glutathione in response to Golgi stress. a Analysis of intracellular reactive oxygen species (ROS) levels using dihydroethidium (DHE) staining and flow cytometry of HeLa cells treated for 48 h with the indicated compounds. CCCP was used as a positive control. b Quantification of the relative DHE mean fluorescence intensity (MFI) levels of samples shown in the FACS histogram in a; **P < 0.01; a.u. arbitrary unit. c Quantification of total intracellular glutathione (GSH) levels in HeLa cells treated with 60 nM BFA or 2 µM GCA for 48 h; **P < 0.01. d Relative viability (survival of compound-treated cells divided by survival of vehicle-treated cells) of HeLa cells after treatment with BFA (35 nM), AMF-26 (35 nM) or GCA (1.5 µM) for 72 h in the presence or absence of glutathione (GSH, 2 mM) or N-acetyl cysteine (NAC, 1 mM) as measured by the CellTiter-Blue (CTB) assay; *P < 0.05 and **P < 0.01. a–d) Shown are representative examples of at least three independent experiments, and three wells per treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. e Displayed is the relative mean cell viability ±SD of HeLa cells stably overexpressing Flag-γ-Tubulin, Flag-GSR or Flag-GSTA1 following treatment with 35 nM BFA for 72 h; **P < 0.01. Cell survival was measured by the CTB assay. Shown is a representative example of three independent experiments, and three wells per genotype and treatment condition were measured. Expression levels of overexpressed proteins are shown by immunoblotting. Protein lysates were run on the same gel, and dashed lines in blots indicate where irrelevant samples were cropped out. a–e Statistical analysis was performed using Student’s two-tailed t-test. Scanned images of unprocessed blots are shown in Supplementary Fig. 7
Fig. 2
Fig. 2
Golgi stress leads to ferroptosis. a Real-time lipid peroxidation analysis using the IncuCyte system and Liperfluo staining of HeLa cells treated with vehicle or the indicated concentrations of BFA and erastin (ERS), respectively. Immunofluorescence images of HeLa cells treated for 20 h with 40 nM BFA and stained with Liperfluo are shown on the right; scale bar: 50 µm. Shown is a representative example of three independent experiments. Bars and error bars represent the means and SD, respectively. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test, and the average fluorescence intensity for each condition was derived from four pictures taken per well from four separate wells. BFA treatment (both concentrations) caused significant differences in lipid peroxide production compared to vehicle treatment starting at ten hours of treatment; P < 0.01. b Schematic illustration of Cys2 uptake/metabolism and GSH biosynthesis and the role of iron and GSH in ferroptosis induction. A number of known ferroptosis/ROS inhibitors are shown (CPX, Fer-1, Lip-1, LOXi, Pran). c Relative viability of a panel of cancer cell lines as well as primary lung fibroblasts after treatment with BFA for 72 h in the presence or absence of 10 µM ferrostatin-1 (Fer-1); **P < 0.01; ***P < 0.001. BFA concentrations used: HeLa: 35 nM; A549, DU145, HT-29 and primary lung fibroblast: 60 nM; Panc-1: 120 nM. d Western blot analysis of HeLa cells that were either vehicle-treated, treated with 35 nM BFA or with 1.5 µM GCA in the presence or absence of 10 µM ferrostatin-1 (Fer-1) for 24 h. β-actin was used as loading control. e, f Relative viability of HeLa cells after treatment with 35 nM BFA or 1.5 µM GCA for 72 h in the presence or absence of 1 µM liproxstatin-1 (Lip-1) (e) or 1 µM CPX (f); ***P < 0.001. g Relative viability of HeLa cells following treatment with 35 nM BFA or 1.5 µM GCA for 72 h in the presence or absence of 10 µM PD-146176 (LOXi); *P < 0.05; ***P < 0.001. h Relative HeLa cell viability in response to 35 nM BFA for 72 h in the presence or absence of 100 µM Trolox, 4 µM NOX1 inhibitor (=GKT137831) or 10 µM pranlukast (Pran); **P < 0.01; ***P < 0.001. c, e–h Cell survival was determined using the CTB assay. Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of at least three independent experiments, and three wells per treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. i Western blot analysis of HeLa cells that were treated prior to lysis with the indicated BFA or GCA concentrations for 24 h. j Western blot analysis of HeLa cells incubated in Cys2-free or low-dose (2.5 µM) Cys2-culture medium for 24 h before protein extraction. d, i, j Shown is a representative western blot of three independent experiments. Scanned images of unprocessed blots are shown in Supplementary Fig. 7
Fig. 3
Fig. 3
Impact of ferroptosis inhibitors on Golgi morphology and protein secretion. a Immunofluorescence microscopic pictures of HeLa cells that were either vehicle-treated, treated with 30 nM BFA, 2 mM GSH or a combination thereof for 72 h before fixation and staining for the cis-Golgi marker GM130; scale bar: 20 µm. Quantification of Golgi area measurements is shown in the bar graphs on the right; **P < 0.01. b Immunofluorescence microscopic pictures of HeLa cells treated for 72 h with vehicle, 35 nM BFA, 10 µM ferrostatin-1 (Fer-1), or a combination thereof before fixation and staining for GM130; scale bar: 20 µm. Graphs on the right display the quantification of Golgi area measurements shown in the immunofluorescence images; **P < 0.01. Center bars indicate the median, and tails indicate the minimum and maximum. a, b Shown are representative images of two independent experiments each time quantifying Golgi area measurements of approximately 1000 cells derived from three wells per genotype and condition. c, d HeLa stably overexpressing Gaussia Luciferase (Gluc-flag) treated with 40 nM BFA alone or in combination with 2 mM GSH (c) or 10 μM Fer-1 (d) for 2 h. Before BFA addition, cells were pretreated for 24 with GSH or Fer-1, respectively. The secretion was determined as a ratio calculated by dividing the luminescence values of treated samples by the values of the corresponding vehicle control (after signal background subtraction). c, d Center bars indicate the mean, error bars indicate the SD. Shown is a representative example of two independent experiments each time measuring three wells per genotype and condition; *P < 0.05, **P < 0.01 (Student’s two-tailed test)
Fig. 4
Fig. 4
Loss of function studies of key ferroptosis regulators in the context of Golgi stress. a Relative survival of control (shLUC) and ACSL4 knockdown HeLa cells following treatment with 35 nM BFA for 72 h; **P < 0.01. ACSL4 knockdown levels are shown by immunoblotting. b Survival ratios of SLC7A11 knockdown and control HeLa cells after treatment for 72 h with either 30 nM BFA or 2 µM erastin; **P < 0.01; ***P < 0.001. SLC7A11 expression is shown by immunoblot analysis. c Relative viability of GPX4 knockdown or control HeLa cells after treatment with 35 nM BFA for 72 h; **P < 0.01. Expression levels of GPX4 are shown by western blot. Protein lysates were run on the same gel, and dashed lines in blots indicate where irrelevant samples were cropped out. a–c Cell survival of vehicle-treated and BFA-treated cells was analyzed using the CTB assay. Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of three independent experiments, and three wells per genotype and treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. d Representative immunoblot analysis (n = 2) of HeLa SLC7A11 knockdown (shSLC7A11#1) or control (shRFP) cells treated for 72 h with either vehicle, 40 nM and 50 nM BFA or 1.5 μM GCA. e Representative immunoblot analysis (n = 2) of GPX4-depleted (shGPX4#1) or control HeLa cells treated for 72 h with vehicle, 40 nM and 50 nM BFA or 1.5μM GCA. Note that the same shRFP protein lysates were loaded on separate gels in d and e. f HeLa knockdown cells with the indicated genotypes were treated for 72 h with 40 nM BFA before fixation, GM130 immunofluorescence staining and image acquisition. Graph shows quantification of Golgi area following vehicle or BFA treatment. Center bars indicate the median, and tails indicate the minimum and maximum; boxes extend from the 25th to 75th percentiles. shControls refers to shLUC and shRFP hairpins which were combined for the analysis. On average 1000 cells were analyzed for Golgi area quantification; ***P < 0.001. The corresponding immunofluorescence pictures are presented in Supplementary Fig. 4. g Real-time lipid peroxidation measurements using Liperfluo dye added to vehicle-treated HeLa knockdown cells of the genotypes shown for the indicated amount of time. Each point presented is the average fluorescence intensity derived from three separate wells, and four pictures per well were measured. Error bars indicate SD of the mean. h Relative lipid peroxide fold changes as calculated by dividing the average GFP values (derived from three wells) of HeLa knockdown cells of the indicated genotype treated with 40 nM BFA by their corresponding average GFP values of vehicle-treated cells. Error bars indicate SD of the mean. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test. GPX4 and SLC7A11 knockdown cell lines (at least one of the two hairpins targeting either GPX4 or SLC7A11) displayed significantly reduced lipid peroxide-fold changes compared to both shRFP and shLUC control knockdown cell lines starting approximately at 24 h (GPX4 knockdown cells) or 30 h (SLC7A11 knockdown cells) of BFA treatment (in comparison to shRFP control cells, significance is reached earlier); P < 0.05. Scanned images of unprocessed blots are shown in Supplementary Fig. 7
Fig. 5
Fig. 5
Cellular responses to low doses of ferroptosis inducers. a Schematic illustration of Cys2 uptake and GSH metabolism including known ferroptosis inducers such as erastin (ERS) and sorafenib (SRF). b–f Relative viability of HeLa cells treated with with BFA (35 nM) or AMF-26 (35 nM) for 72 h in the presence or absence of sublethal doses of erastin (ERS, 1 µM), sorafenib (SRF, 1 µM), sulfasalazine (SAS, 200 µM), L-buthionine sulfoximine (BSO, 50 µM); **P < 0.01; ***P < 0.001. g Shown is the relative survival of HeLa cells grown in medium supplemented with increasing concentrations of Cys2 for 72 h in the presence or absence of 35 nM BFA; **P < 0.01; ***P < 0.001. b–g Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of three independent experiments, and three wells per treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. h Real-time analysis of lipid peroxide formation of HeLa cells following BFA treatment in the presence or absence of 1 µM erastin (ERS) using Liperfluo staining. Shown is a representative example of two independent experiments each time measuring the average fluorescence intensity which was derived from four pictures taken per well from five wells. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test. [BFA + 1 μM ERS] treatment caused significant lower lipid peroxide generation compared to BFA-only treatment starting at 13 h of treatment, whereas 1 μM ERS by itself had no significant effect on oxidation of lipids; P < 0.01. i HeLa cells were treated for 72 h with 40 nM BFA in the presence or absence of ferrostatin-1 (Fer-1, 10 µM), low concentration of erastin (ERS, 1 µM), sorafenib (SRF, 1 µM) or medium containing 7 µM Cys2 (low Cys2) for 72 h. Shown is a representative western blot of three independent experiments. Protein lysates were run on the same gel, and dashed lines in scans indicate where irrelevant samples were cropped out. j Immunofluorescence microscopic pictures of HeLa cells treated for 72 h with vehicle, 1 µM erastin (ERS), 30 nM BFA or a combination thereof; scale bar: 20 µm. k Immunofluorescence microscopic pictures of HeLa cells vehicle-treated, treated with 30 nM BFA, 1 µM sorafenib or a combination thereof for 72 h; scale bar: 20 µm. j, k Quantification of Golgi areas of HeLa cells depicted in the immunofluorescence images on the left is shown in the right graphs. Center bars indicate the median, and tails indicate the minimum and maximum. On average 1000 cells per genotype and conditions were analyzed for the quantification; **P < 0.01. b–g Cell survival was determined using the CTB assay. Scanned images of unprocessed blots are shown in Supplementary Fig. 7
Fig. 6
Fig. 6
Pharmacological inhibition of the transsulfuration pathway counteracts the effects of sublethal erastin doses or cystine-deprived conditions in the presence of BFA. a Schematic illustration of Cys2 uptake/metabolism, GSH biosynthesis and the transsulfuration pathway. Propargylglycine (PPG) is a cystathionine-γ-lyase (CTH) inhibitor. b Relative viability of HeLa cells following treatment with 35 nM BFA and 1 µM ERS for 72 h in the presence or absence of 50 µM 2-Mercaptoethanol (2-ME); **P < 0.01, ***P < 0.001. c Relative viability of HeLa cells following treatment with 35 nM BFA and 1 µM ERS for 72 h in the presence or absence of 1 mM PPG, ***P < 0.001. d Relative viability of HeLa cells after treatment with 35 nM BFA and 1 mM PPG for 72 h in normal medium or medium with a low Cys2 concentration (7 µM); ***P < 0.01. b–d Cell survival was measured by the CTB assay; statistical analysis was performed using a one-way ANOVA test. Shown are representative examples of three independent experiments, and three wells per genotype and treatment condition were measured. Center bars indicate the mean, error bars indicate the SD
See this image and copyright information in PMC

References

    1. Arya R, White K. Cell death in development: signaling pathways and core mechanisms. Semin. Cell Dev. Biol. 2015;39:12–19. doi: 10.1016/j.semcdb.2015.02.001. - DOI - PMC - PubMed
    1. Shimada K, et al. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat. Chem. Biol. 2016;12:497–503. doi: 10.1038/nchembio.2079. - DOI - PMC - PubMed
    1. Dong T, Liao D, Liu X, Lei X. Using small molecules to dissect non-apoptotic programmed cell death: Necroptosis, ferroptosis, and pyroptosis. Chembiochem. 2015;16:2557–2561. doi: 10.1002/cbic.201500422. - DOI - PubMed
    1. Conrad M, Angeli JPF, Vandenabeele P, Stockwell BR. Regulated necrosis: disease relevance and therapeutic opportunities. Nat. Rev. Drug. Discov. 2016;15:348–366. doi: 10.1038/nrd.2015.6. - DOI - PMC - PubMed
    1. Laster SM, Wood JG, Gooding LR. Tumor necrosis factor can induce both apoptotic and necrotic forms of cell lysis. J. Immunol. 1988;141:2629–2634. - PubMed