Caspase-3-mediated GSDME activation contributes to cisplatin- and doxorubicin-induced secondary necrosis in mouse macrophages
Caspase-3-mediated GSDME activation contributes to cisplatin- and doxorubicin-induced secondary necrosis in mouse macrophages
复制标题
Caspase-3 介导的 GSDME 激活导致顺铂和阿霉素诱导的小鼠巨噬细胞继发性坏死
DOI：
10.1111/cpr.12663
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发表时间：
2019-07-26
影响因子：
8.5
通讯作者：
Hu, Bo
中科院分区：
文献类型：
--
作者：
Mai, Feng-Yi;He, Pengyan;Hu, Bo
目的诱导继发性坏死/焦亡是化疗药物毒性的重要组成部分，其中气凝胶蛋白E （GSDME）在其中起着至关重要的作用。本研究旨在探讨GSDME是否参与介导顺铂和阿霉素对小鼠巨噬细胞的细胞毒性作用。方法采用顺铂或阿霉素治疗RAW 264.7细胞和骨髓源性巨噬细胞（bmmdms）。碘化丙啶染色检测坏死，免疫印迹检测蛋白表达。用小干扰RNA敲除GSDME。小鼠腹腔注射，观察其对体内巨噬细胞的毒性。采用流式细胞术和免疫荧光显微镜对腹膜细胞进行表型分析。细胞因子水平通过细胞细胞阵列检测。结果顺铂和阿霉素剂量依赖性诱导小鼠RAW 264.7巨噬细胞和bmdm坏死。与此同时，多个半胱天冬酶被激活，并伴随poly （adp -核糖）聚合酶的裂解。与caspase-3激活一致，GSDME被裂解生成其n端片段（GSDME- nt），从而导致继发性坏死/焦亡。抑制caspase-3可显著减少巨噬细胞中GSDME-NT的产生，同时减少坏死。GSDME敲除也能明显减少RAW 264.7和BMDMs的坏死。此外，顺铂使小鼠腹腔巨噬细胞减少，这与caspase-3激活和GSDME-NT生成有关。与巨噬细胞耗损一致，顺铂给药显著降低细菌感染小鼠的存活率。结论顺铂和阿霉素对巨噬细胞的毒性作用部分是通过诱导caspase-3/ gsdme介导的继发性坏死发挥的。
Objective Induction 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. Methods RAW 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. Results Both 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. Conclusion Chemotherapeutic cisplatin and doxorubicin exerted their cytotoxicity on macrophages partly by inducing caspase-3/GSDME-mediated secondary necrosis.