Letter | Published:

Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin

Nature volume 547, pages 99103 (06 July 2017) | Download Citation

Abstract

Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide1,2,3. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity4,5,6,7,8,9. GSDMD belongs to a gasdermin family that shares the pore-forming domain4,6,10. The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5)11, can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme−/− (also known as Dfna5−/−) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

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Acknowledgements

We thank F. Wang and the NIBS transgenic facility for generating Gsdme−/− mice, E. Zhang and J. Sui for reagents, and K. Jiang, Z. Liu and L. Sun for technical assistance. This work was supported by the National Key Research and Development Project on Protein Machinery and its Control and Regulation of Biological Processes (2016YFA0501500) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202).

Author information

Author notes

    • Yupeng Wang
    •  & Wenqing Gao

    These authors contributed equally to this work.

Affiliations

  1. College of Biological Sciences, China Agricultural University, Beijing 100094, China

    • Yupeng Wang
    • , Wenqing Gao
    •  & Xuyan Shi
  2. National Institute of Biological Sciences, Beijing 102206, China

    • Yupeng Wang
    • , Wenqing Gao
    • , Xuyan Shi
    • , Jingjin Ding
    • , Wang Liu
    • , Huabin He
    • , Kun Wang
    •  & Feng Shao
  3. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

    • Jingjin Ding
  4. National Institute of Biological Sciences, Collaborative Innovation Center for Cancer Medicine, Beijing 102206, China

    • Feng Shao

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Contributions

Y.W., W.G. and F.S. conceived the study; Y.W. and W.G. performed cellular studies; W.G. and X.S. performed mouse experiments; X.S. and Y.W. performed in vitro experiments; W.L., J.D., H.H. and K.W. provided technical assistance; Y.W., W.G. and F.S. analysed the data; F.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Feng Shao.

Reviewer Information Nature thanks M. Albert, V. Hornung and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure

    This file contains the uncropped immunoblots for key data presented in the Main and Extended Data Figures.

Videos

  1. 1.

    GSDMA3 can not switch TNFα-induced apoptosis to pyroptosis

    Flag-tagged GSDMA3 was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 2.5 h after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm.

  2. 2.

    Wild-type GSDME can switch TNFα-induced apoptosis to pyroptosis

    Flag-tagged human GSDME was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm.

  3. 3.

    GSDME-D270A can not switch TNFα-induced apoptosis to pyroptosis

    Flag-tagged human GSDME-D270A mutant was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 2.5 h after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm

  4. 4.

    Wild-type GSDME1 can switch TNFα-induced apoptosis to pyroptosis

    Flag-tagged wild-type zebrafish GSDME1 was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm.

  5. 5.

    GSDME1-D256A can not switch TNFα-induced apoptosis to pyroptosis

    Flag-tagged zebrafish GSDME1-D256A mutant was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm.

  6. 6.

    TNFα-induced apoptosis in control HeLa cells

    GSDMD-/- HeLa cells expressing an empty vector, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm.

  7. 7.

    TNFα-induced pyroptosis in GSDMEhigh HeLa cells

    GSDMD-/- HeLa cells expressing a high level of GSDME, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm.

  8. 8.

    TNFα-induced secondary pyroptosis after apoptosis in GSDMElow HeLa cells

    GSDMD-/- HeLa cells expressing a low level of GSDME, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm.

  9. 9.

    Chemotherapy drug-induced apoptosis in GSDME-negative primary HUVEC cells

    Cells were treated with Doxorubicin for 2 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm.

  10. 10.

    Chemotherapy drug-induced pyroptosis in GSDME-positive primary NHEK cells

    Cells were treated with Doxorubicin for 6 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm.

  11. 11.

    Knockdown of GSDME switches chemotherapy drug-induced pyroptosis to apoptosis in primary NHEK cells

    Cells were transfected with GSDME-specific siRNAs. The knockdown cells were treated with doxorubicin for 6 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm.

  12. 12.

    Chemotherapy drug-induced pyroptosis in GSDME-positive primary HPlEpC cells

    Cells were treated with doxorubicin for 12 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm.

  13. 13.

    Knockdown of GSDME switches chemotherapy drug-induced pyroptosis to apoptosis in primary HPlEpC cells

    Cells were transfected with GSDME-specific siRNAs. The knockdown cells were treated with doxorubicin for 12 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm.

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https://doi.org/10.1038/nature22393

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