Caspase-3-mediated GSDME activation contributes to cisplatin- and doxorubicin-induced secondary necrosis in mouse macrophages.

Feng‐Yi Mai,Chen‐Guang Li,Pengyan He,Bo Hu,Xian‐Hui He,Qiong‐Zhen Zeng,Li‐Hui Xu,Dong‐Yun Ouyang,Jie‐Zhou Ye,Cheng‐Cheng Zhang,Chen‐Ying Zeng

doi:10.1111/cpr.12663

Feng‐Yi Mai, Chen‐Guang Li + Show 9 more

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https://doi.org/10.1111/cpr.12663

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Abstract

ObjectiveInduction of secondary necrosis/pyroptosis contributes to the toxicity of chemotherapeutic drugs, in which gasdermin E (GSDME) plays critical roles. This study aimed to explore whether GSDME is involved in mediating the cytotoxic effects of cisplatin and doxorubicin on mouse macrophages.MethodsRAW 264.7 cells and bone marrow‐derived macrophages (BMDMs) were treated with cisplatin or doxorubicin. Propidium iodide staining was used to assay necrosis, and immunoblotting was performed to detect protein expression. GSDME was knocked down by using small interfering RNA. Mice were injected intraperitoneally to evaluate toxicity to macrophages in vivo. Flow cytometry and immunofluorescence microscopy were adopted to analyse phenotypes of peritoneal cells. Cytokine levels were assayed by cytometric bead array.ResultsBoth cisplatin and doxorubicin dose‐dependently induced necrosis in mouse RAW 264.7 macrophages and BMDMs. Accompanying this, multiple caspases were activated, concomitant with the cleavage of poly (ADP‐ribose) polymerase. Consistent with caspase‐3 activation, GSDME was cleaved to generate its N‐terminal fragment (GSDME‐NT), thus leading to secondary necrosis/pyroptosis. Inhibition of caspase‐3 significantly attenuated the generation of GSDME‐NT concurrently with decreased necrosis in macrophages. GSDME knockdown also evidently decreased the necrosis in RAW 264.7 and BMDMs. Besides, cisplatin administration depleted peritoneal macrophages in mice, which was associated with caspase‐3 activation and GSDME‐NT generation. Consistent with the macrophage depletion, cisplatin administration significantly decreased survival of mice with bacterial infection.ConclusionChemotherapeutic cisplatin and doxorubicin exerted their cytotoxicity on macrophages partly by inducing caspase‐3/GSDME‐mediated secondary necrosis.

Highlights

DNA‐damaging agents, including cisplatin and doxorubicin, have been used for cancer chemotherapy for decades and are currently still the mainstays of chemotherapeutic drugs
The levels of gasdermin E (GSDME)‐NT were correlated with the percentages of necrosis revealed by Propidium io‐ dide (PI) stain‐ ing (Figure 1 A‐F). These results suggested that cisplatin‐ and doxorubicin‐induced secondary necrosis in mouse macrophages was probably mediated by the caspase‐3/GSDME axis
GSDMD cleavage and production of the fragment (48‐50 kDa) were reduced by DEVD in RAW 264.7 cells, suggesting that caspase‐3 had been involved in this process; no GSDMD‐NT was observed either in the presence or in the absence of DEVD. These results indicated that active caspase‐3 was responsible for the production of GSDME‐NT, which correlated with secondary necrosis in mouse macrophages

Summary

| INTRODUCTION

DNA‐damaging agents, including cisplatin and doxorubicin, have been used for cancer chemotherapy for decades and are currently still the mainstays of chemotherapeutic drugs. We found in this study that cisplatin and doxorubicin induced secondary necro‐ sis in mouse macrophages in vitro and in vivo, in part through the caspase‐3/GSDME axis. Such cytotoxicity was involved in depleting peritoneal macrophages with recruitment of inflammatory mono‐ cytes. Mice administered with cisplatin had drastically increased susceptibility to bacterial infection These results suggest that che‐ motherapeutic agents exert adverse toxicity on macrophages partly by inducing GSDME‐mediated secondary necrosis, compromis‐ ing the innate immunity against infections

| MATERIALS AND METHODS

Findings

| DISCUSSION