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. 2020 Feb;30(2):146-162.
doi: 10.1038/s41422-019-0263-3. Epub 2020 Jan 16.

The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression

Guang Lei #  1   2   3 , Yilei Zhang #  2 , Pranavi Koppula  2   4 , Xiaoguang Liu  2 , Jie Zhang  2 , Steven H Lin  2   5 , Jaffer A Ajani  6 , Qin Xiao  1   3 , Zhongxing Liao  5 , Hui Wang  7   8 , Boyi Gan  9   10
Affiliations

The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression (VSports)

Guang Lei et al. Cell Res. 2020 Feb.

Abstract

Ferroptosis, a form of regulated cell death caused by lipid peroxidation, was recently identified as a natural tumor suppression mechanism. Here, we show that ionizing radiation (IR) induces ferroptosis in cancer cells. Mechanistically, IR induces not only reactive oxygen species (ROS) but also the expression of ACSL4, a lipid metabolism enzyme required for ferroptosis, resulting in elevated lipid peroxidation and ferroptosis. ACSL4 ablation largely abolishes IR-induced ferroptosis and promotes radioresistance. IR also induces the expression of ferroptosis inhibitors, including SLC7A11 and GPX4, as an adaptive response VSports手机版. IR- or KEAP1 deficiency-induced SLC7A11 expression promotes radioresistance through inhibiting ferroptosis. Inactivating SLC7A11 or GPX4 with ferroptosis inducers (FINs) sensitizes radioresistant cancer cells and xenograft tumors to IR. Furthermore, radiotherapy induces ferroptosis in cancer patients, and increased ferroptosis correlates with better response and longer survival to radiotherapy in cancer patients. Our study reveals a previously unrecognized link between IR and ferroptosis and indicates that further exploration of the combination of radiotherapy and FINs in cancer treatment is warranted. .

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Conflict of interest statement

S. H V体育安卓版. L. receives grant funding from Hitachi Chemical Diagnostics, Genentech, Beyond Spring Pharmaceuticals, New River Labs; honorarium from Varian Medical Systems; and serves in Advisory Board for AstraZeneca and Beyond Spring Pharmaceuticals. Other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Ionizing radiation induces ferroptosis in cancer cells.
a Lipid peroxidation assessment in H460, A549, and H1299 cell lines at 24 h after exposure to 6 Gy of IR. Bar graph showing relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. b qRT-PCR analysis of PTGS2 expression in H460, A549, and H1299 cell lines at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c Transmission electron microscopy images of H460 cells without radiation (control) or at 24 h after exposure to 6 Gy of IR (post-IR). Nu, nucleus; red arrows, mitochondria; yellow arrows, autophagosomes; black arrows, necrosis-related vacuoles. Scale bars: left, 2 µm; right, 500 nm. d Clonogenic survival assay in H460, A549, and H1299 cell lines that were pretreated with 2 μM necrostatin-1s, 5 μM Z-VAD-fmk, 5 μM ferrostatin-1, 5 mM N-acetyl-L-cysteine, or DMSO for 24 h followed by exposure to 6 Gy of IR. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. e Clonogenic survival curves for H460, A549, and H1299 cell lines that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to IR at doses from 0 to 6 Gy. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 2
Fig. 2. Ionizing radiation induces ferroptosis partly through upregulating ACSL4.
a qRT-PCR analysis of GPX4, SLC7A11, and ACSL4 expression in A549 and H460 cell lines at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. b Western blotting analysis of ACSL4, SLC7A11, and GPX4 expression in A549 and H460 cell lines at 2 h, 24 h, and 48 h after exposure to 6 Gy of IR or no radiation. c Western blotting analysis of ACSL4 expression in sg Control (sg C), sg ACSL4-1, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines. d Cell viability measurements in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines treated with or without 10 μM erastin for 24 h. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. e, f Lipid peroxidation assessment in sg C, sg ACSL4-2, and sg ACSL4-3 A549 (e) and H460 (f) cell lines at 24 h after exposure to 6 Gy of IR. Bar graph showing IR-induced relative fold changes of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. g qRT-PCR analysis of PTGS2 expression in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. h Clonogenic survival assay in sg C, sg ACSL4-2, and sg ACSL4-3 A549 and H460 cell lines that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy of IR. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 3
Fig. 3. SLC7A11 overexpression or KEAP1 deficiency promotes radioresistance largely through inhibiting ferroptosis.
a Western blotting analysis showing SLC7A11 levels in H1299 and H23 cell lines with stable expression of empty vector (EV) and SLC7A11. b Lipid peroxidation assessment in EV- and SLC7A11-expressing H1299 or H23 cells at 24 h after exposure to 6 Gy or 2 Gy of IR, respectively. Bar graph showing the relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c qRT-PCR analysis of PTGS2 expression in EV- and SLC7A11-expressing H1299 or H23 cells at 24 h after exposure to 6 Gy or 2 Gy of IR, respectively. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. d Representative images of clonogenic survival assays in EV- and SLC7A11-expressing H1299 or H23 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy or 2 Gy of IR, respectively. e The quantified clonogenic survival assay in EV- and SLC7A11-expressing H1299 or H23 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to IR at a dose of 6 Gy or 2 Gy, respectively. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. f The quantified clonogenic survival curve in EV- and SLC7A11-expressing H1299 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to IR at the indicated doses. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using multiple t-test. g Western blotting analysis of KEAP1, SLC7A11 and ACSL4 expression in sg Control (sg C), sg KEAP1-2, and sg KEAP1-2-sg SLC7A11 (sg SLC) H1299 cell lines. h, i Lipid peroxidation assessment in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells at 24 h after exposure to 6 Gy of IR h. Bar graph showing IR-induced relative fold changes of lipid peroxidation by C11-BODIPY staining in the indicated cells (i). Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. j qRT-PCR analysis of PTGS2 expression in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells at 24 h after exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. k Clonogenic survival assays in sg C, sg KEAP1-2, and sg KEAP1-2-sg SLC H1299 cells that were pretreated with 5 μM ferrostatin-1 or DMSO for 24 h followed by exposure to 6 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 4
Fig. 4. Ferroptosis is not involved in IR-induced DNA damage and repair.
a Western blotting analysis of phospho-H2AX levels in H460, H1299, and H23 cell lines pretreated with DMSO (control) or 5 μM ferrostatin-1 for 24 h followed by exposure to IR at a dose of 6 Gy (for H460 and H1299 cells) or 2 Gy (for H23 cells). b The numbers of phospho-H2AX foci per nucleus were counted on the basis of phospho-H2AX immunofluorescence at 30 min or 24 h after exposure to 6 Gy of IR in cells that had been pretreated with DMSO or 5 μM ferrostatin-1 for 24 h. Error bars are means ± SD, n = 20 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c Representative immunofluorescence images showing staining of phospho-H2AX foci (red) and nuclei counterstained with DAPI (blue) in the indicated cells. d Western blotting analysis of phospho-Chk2 and phospho-p53 levels in H460 cells pretreated with DMSO or 5 μM ferrostatin-1 for 24 h followed by exposure to 6 Gy of IR. e, f Lipid peroxidation assessment in H460 (e) and H1299 cells (f) pretreated with DMSO or 5 μM ferrostatin-1 for 24 h followed by exposure to 6 Gy of IR. Bar graph showing relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. g Representative immunofluorescence images showing staining of phospho-H2AX foci (red) and nuclei counterstained with DAPI (blue) in the indicated cells with or without exposure to IR. h The numbers of phospho-H2AX foci per nucleus were counted on the basis of immunofluorescence at 30 min or 24 h after exposure to 6 Gy of IR in H1299 cells with stable expression of empty vector (EV) or SLC7A11 (SLC). Error bars are means ± SD, n = 50 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 5
Fig. 5. FINs sensitize cancer cells to IR.
a Lipid peroxidation assessment in H460 and A549 cells pretreated with DMSO or 10 μM erastin for 24 h followed by exposure to 4 Gy of IR. Bar graph showing relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. b qRT-PCR analysis of PTGS2 expression in H460 and A549 cells pretreated with DMSO or 10 μM erastin for 24 h followed by exposure to 4 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c Clonogenic survival curves in H460 and A549 cells pretreated with DMSO or 10 μM erastin for 24 h followed by exposure to 4 Gy of IR. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. d Lipid peroxidation assessment in A549 cells pretreated with DMSO, 500 mM sulfasalazine, 10 μM RSL3, 10 μM ML162, or 10 μM FIN56 for 24 h followed by exposure to 4 Gy of IR. Bar graphs showing relative levels of lipid peroxidation by C11-BODIPY staining in the indicated cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. e qRT-PCR analysis of PTGS2 expression in A549 cells pretreated with DMSO, 500 mM sulfasalazine, 10 μM RSL3, 10 μM ML162, or 10 μM FIN56 for 24 h followed by exposure to 4 Gy of IR. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. f Clonogenic survival curves for A549 cells pretreated with DMSO, 500 mM sulfasalazine, 10 μM RSL3, 10 μM ML162, or 10 μM FIN56 for 24 h followed by exposure to 4 Gy of IR. The survival data were normalized to those of unirradiated control cells. Error bars are means ± SD, n = 3 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 6
Fig. 6. Ferroptosis is involved in IR-induced tumor suppression in vivo.
a Volumes of H460 xenograft tumors with the indicated genotypes and treatments at different time points (days) after exposure to 10 Gy of IR. Error bars are means ± SD, n = 8 independent repeats. P values were determined using 2-way ANOVA. b Individual value plot showing the weights of H460 tumor xenografts in the indicated genotype and treatment groups. Error bars are means ± SD, n = 8 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. c Volumes of A549 xenograft tumors with the indicated treatments at different time points (days) after exposure to 10 Gy of IR. Error bars are means ± SD, n = 8 independent repeats. P values determined using 2-way ANOVA. d Individual value plot showing the weights of A549 tumor xenografts from the indicted treatments. Error bars are means ± SD, n = 8 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. e Volumes of lung cancer patient-derived xenograft tumors with the indicated treatments at different time points (days) after exposure to 8 Gy of IR. Error bars are means ± SD, n = 4-5 independent repeats. P values were determined using 2-way ANOVA. f Individual value plot showing the weights of lung cancer patient-derived xenograft tumor xenografts from the indicted treatments. Error bars are means ± SD, n = 4-5 independent repeats. P values were calculated using two-tailed unpaired Student’s t-test. g Representative images of hematoxylin and eosin and immunohistochemical staining (Ki67, phospho-H2AX, cleaved caspase-3 and 4-HNE) of A549 xenograft tumors with the indicated treatments. Scale bars, 50 µm/20 µm (inset). h–k Immunochemistry scoring of Ki67, phospho-H2AX, cleaved caspase-3 and 4-HNE staining. Error bars are means ± SD, n = 6 randomly selected magnification fields. P values were calculated using two-tailed unpaired Student’s t-test. l Representative images of hematoxylin and eosin and immunohistochemical staining (Ki67, phospho-H2AX, cleaved caspase-3 and 4-HNE) of lung cancer patient-derived xenograft tumors with the indicated treatments. Scale bars, 50 µm/20 µm (inset). m–p Immunochemistry scoring of Ki67, phospho-H2AX, cleaved caspase-3 and 4-HNE staining. Error bars are means ± SD, n = 6 randomly selected magnification fields. P values were calculated using two-tailed unpaired Student’s t-test.
Fig. 7
Fig. 7. Ferroptosis induction correlates with cancer patient response to radiotherapy.
a Representative images of 4-HNE immunohistochemical staining of matched esophageal tumor samples from the same patients before and after radiotherapy. Scale bars, 50 µm/20 µm (inset). b Immunochemistry scoring of 4-HNE staining of matched esophageal tumor samples before and after radiotherapy from 8 cancer patients. Error bars are means ± SD, n = 6 randomly selected magnification fields. P values calculated using two-tailed unpaired Student’s t-test. c Immunochemistry scoring of 4-HNE staining of post-radiotherapy esophageal tumor samples from 30 esophageal cancer patients. Error bars are means ± SD, n = 6 randomly selected magnification fields. P values were calculated using two-tailed unpaired Student’s t-test. d Correlations between radiotherapy response (or recurrence) and 4-HNE staining scores in post-radiotherapy esophageal tumor samples from 30 esophageal cancer patients. SD: stable disease; PR: partial response; P values were calculated using χ2 test. e Representative images of 4-HNE immunohistochemical staining of post-radiotherapy esophageal tumor samples in 4-HNE mild/moderate group or 4-HNE strongly positive group with the indicated status of patient response and recurrence. Scale bars, 20 µm (inset). f Disease-free survival Kaplan–Meier curves of esophageal cancer patients treated with radiotherapy from 4-HNE mild/moderate group and 4 -HNE strongly positive group. P values were calculated using log-rank test. g–i The working model depicting the roles and mechanisms of ferroptosis in IR response and radioresistance. See Discussion for detailed description.
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References

    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. - DOI - PubMed
    1. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat. Rev. Cancer. 2002;2:277–288. doi: 10.1038/nrc776. - DOI - PubMed
    1. Green DR, Evan GI. A matter of life and death. Cancer Cell. 2002;1:19–30. doi: 10.1016/S1535-6108(02)00024-7. - DOI (V体育安卓版) - PubMed
    1. Dixon SJ, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149:1060–1072. doi: 10.1016/j.cell.2012.03.042. - "V体育平台登录" DOI - PMC - PubMed
    1. Stockwell BR, et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell. 2017;171:273–285. doi: 10.1016/j.cell.2017.09.021. - DOI - PMC - PubMed

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