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
. 2017 Dec 8;8(70):115164-115178.
doi: 10.18632/oncotarget.23046. eCollection 2017 Dec 29.

Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression

Se Hoon Hong #  1 , Dae-Hee Lee #  2   3 , Young-Sun Lee  1 , Min Jee Jo  2 , Yoon A Jeong  2 , William T Kwon  1 , Haroon A Choudry  1 , David L Bartlett  1 , Yong J Lee  1
Affiliations

Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression

Se Hoon Hong et al. Oncotarget. .

Erratum in

Abstract

Ferroptosis is a type of programmed cell death that depends on iron and is characterized by the accumulation of lipid peroxides. In the present study, we investigated the nature of the interplay between ferroptosis and other forms of cell death such as apoptosis. Human pancreatic cancer PANC-1 and BxPC-3 and human colorectal cancer HCT116 cells were treated with ferroptotic agents such as erastin and artesunate (ART) in combination with the apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We observed synergistic interaction of erastin or ART with TRAIL as determined by cell death assay, caspase activation, poly [ADP-ribose] polymerase 1 (PARP-1) cleavage, flow cytometry analysis, and lipid peroxidation assay. Moreover, erastin and ART induced endoplasmic reticulum (ER) stress and promoted p53 upregulated modulator of apoptosis (PUMA) expression via C/EBP-homologous protein (CHOP). Synergy of erastin/ART and TRAIL was abolished in PUMA-deficient HCT116 cells and CHOP-deficient mouse embryonic fibroblasts, but not in p53-deficient HCT116 cells. The results suggest the involvement of the p53-independent CHOP/PUMA axis in response to ferroptosis inducers, which may play a key role in ferroptotic agent-mediated sensitization to TRAIL-induced apoptosis.

Keywords: ER; PUMA; apoptosis; ferroptosis; p53.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Artesunate (ART) promotes TRAIL-induced apoptosis
(A-F) PANC-1 (A), BxPC-3 (B), and HCT116 (C) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (PANC-1, 100 ng/ml; BxPC-3, 2 ng/ml; HCT116, 1 ng/ml) for an additional 4 h. The cells were stained with propidium iodide (PI). Phase-contrast images or fluorescence images were visualized under a light or fluorescence microscope, respectively (upper panels). Representative images are shown (magnification, X200). Cell death was determined by counting PI-stained cells and plotted (lower panels). Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01; ***, 0.001. Cell lysates of PANC-1 (D), BxPC-3 (E), and HCT116 (F) cells were analyzed with immunoblotting assay using indicated antibodies.
Figure 2
Figure 2. Erastin promotes TRAIL-induced apoptosis
(A and B) HCT116 cells were treated with various doses of erastin for 24 h (A) or TRAIL for 4 h (B) and then the cell death rate was determined, respectively (upper panels). Cell lysates were analyzed with immunoblotting assay using indicated antibodies (lower panels). (C and D) HCT116 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined by counting and plotted. Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies (C). The cells were stained with annexin V and PI and then analyzed using flow cytometry (D). (E) BxPC-3 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (6 ng/ml) for an additional 4 h. Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies.
Figure 3
Figure 3. ART and erastin, but not TRAIL, induce lipid peroxidation
(A-C) HCT116 cells were treated with various doses of erastin for 24 h (A) or TRAIL for 4 h (B). Cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h (C). Lipid peroxidation (upper panels) and heme oxygenase (HO-1, lower panels) levels were analyzed by malondialdehyde (MDA) assay and immunoblotting assay, respectively. (D and E) HCT116 cells were treated with various doses of ART (D) or pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h (E). (F) BxPC-3 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h. MDA levels were determined and plotted. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01.
Figure 4
Figure 4. Ferroptotic agents induce ER stress in HCT116 cells
(A) Microarray assay for detection of ART-induced gene expression. Cells were treated with 50 μM ART for 24 h and triplicate Illumina gene expression microarrays were performed with BeadArray microarray technology. (B) Ferroptotic agents induce the unfolded protein response (UPR). Cells were treated with 50 μM ART or 50 μM erastin for 24 h in the presence/absence of 250 nM MG132. Cell lysates were subjected to immunoblotting analysis using FK2 antibody specific to ubiquitin-conjugated proteins. Actin was used to confirm equal amounts of proteins loaded in each lane. (C and D) Cells were treated with ART (50 μM, B) or erastin (50 μM, C) for various times (1-24 h). Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (E) Cells were treated with various doses of erastin for 24 h. Whole-cell extracts were analyzed using immunoblotting assay with indicated antibodies. (F) Cells were treated with erastin alone (10 or 50 μM) for 24 h, TRAIL (1 ng/ml) alone for 4 h, or pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Whole-cell lysates were analyzed using immunoblotting assay with indicated antibodies.
Figure 5
Figure 5. The combinatorial treatment of ferroptotic agent and TRAIL promotes apoptosis via ER stress, but not lipid peroxidation in HCT116 cells
(A and B) Cells were treated with 50 μM erastin (A) or ART (B) for 24 h in the absence/presence of DFO (100 μM), ferrostatin-1 (Fer-1, 10 μM), or liproxstatin-1 (Lip-1, 2 μM). Lipid peroxidation level was detected by MDA assay (left panels) and whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies (right panels). (C and D) Cells were pretreated with erastin (C) and ART (D) for 20 h in the absence/presence of Z-VAD (1 μM), DFO (100 μM), Fer-1 (10 μM), or Lip-1 (2 μM) and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted.
Figure 6
Figure 6. ART increases PUMA protein level
PANC-1 (A), BxPC-3 (B), and HCT116 (C) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (PANC-1, 100 ng/ml; BxPC-3, 2 ng/ml; HCT116, 1 ng/ml) for additional 4 h. Whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies. (D) HCT116 cells were treated with erastin (1-50 μM) or mitomycin C (1-20 μM, MMC) for 24 h. Whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies.
Figure 7
Figure 7. ART promotes TRAIL-induced apoptosis via the p53-independent CHOP/PUMA pathway
(A) Mouse embryonic fibroblasts (MEFs) wild-type (WT) or MEF CHOP knockout (KO) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to recombinant murine TRAIL (mTRAIL; 100 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted (left panel). Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies (right panel). (B) HCT116 WT or HCT116 PUMA KO cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h. Cell death was determined by counting and plotted (left panel). Whole-cell extracts were then analyzed with immunoblotting using indicated antibodies (right panel). (C) HCT116 WT or HCT116 p53 KO cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted (left panel). Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies (right panel).
Figure 8
Figure 8. The combinatorial treatment of ART and TRAIL synergistically inhibits tumor growth
Nude mice were subcutaneously inoculated with 1×106 HCT116-luc cells. When the tumor volume reached approximately 200 mm3, tumor bearing mice were treated with ART (A, 200 mg/kg, twice per week, oral gavage) alone, TRAIL (T, 100 μg/kg, twice per week, intra-tumoral) alone, or the combination of ART and TRAIL (A + T). (A) Mice were imaged using the NightOWL LB983 bioluminescence imaging (BLI) system. Representative images are shown on day 19. (B) Tumor tissues were harvested on day 19 and displayed. (C) Line graph illustrating the tumor volume (mm3) in HCT116-luc tumor-bearing mice treated with PBS alone, ART alone, TRAIL alone, or the combination from day 0 to day 19. Error bars represent the mean ± SD from five mice. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01; ***, 0.001. (D and E) Tumor tissues were harvested on day 19 and subjected to TUNEL assay and DAPI (4′,6-diamidino-2-phenylindole) staining. Cell nuclei were stained with DAPI. Apoptosis was detected using TUNEL assay (D) and percents of TUNEL-positive cells were plotted (E). (F) Line graph illustrating the body weight (gram) in HCT116-luc tumor-bearing mice treated with PBS alone, ART alone, TRAIL alone, or the combination from day 0 to day 19.
See this image and copyright information in PMC

References

    1. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun. 2005;73:1907–1916. - PMC - PubMed
    1. Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N. Crosstalk between apoptosis, necrosis and autophagy. Biochimica et Biophysica Acta. 2013;1833:3448–3459. - PubMed
    1. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B, 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149:1060–1072. - PMC - PubMed
    1. Magtanong L, Ko PJ, Dixon SJ. Emerging roles for lipids in non-apoptotic cell death. Cell Death Differ. 2016;23:1099–1109. - PMC - PubMed
    1. Shimada K, Skouta R, Kaplan A, Yang WS, Hayano M, Dixon SJ, Brown LM, Valenzuela CA, Wolpaw AJ, Stockwell BR. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis. Nat Chem Biol. 2016;12:497–503. - PMC - PubMed

LinkOut - more resources