Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling path...Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.展开更多
Objective:To reveal GSDME-executed pyroptosis in cancer cells induced by the Chinese traditional herbal medicine plant Lithospermum erythrorhizon(L.erythrorhizon,Zi Cao)and to investigate the potential mechanism.Metho...Objective:To reveal GSDME-executed pyroptosis in cancer cells induced by the Chinese traditional herbal medicine plant Lithospermum erythrorhizon(L.erythrorhizon,Zi Cao)and to investigate the potential mechanism.Methods:L.erythrorhizon was extracted by ultrasonication in 95%ethanol,and determined using high-performance liquid chromatography(HPLC).He La,A549,SW620,HEK-293 T,THP-1,K562,Raw264.7 and MDA-MB-231 cell lines were used to investigate the morphology and mechanism of pyroptosis induced by L.erythrorhizon.The lactate dehydrogenase(LDH)release,propidium iodide(PI)/Hoechst double-staining,and pyroptosis reconstitution experiments were performed to study L.erythrorhizon-induced cell pyroptosis.Results:Compared with the death inhibitor,PI/Hoechst and LDH release experiments,we found that L.erythrorhizon induced pyroptosis.Recombination and western blot experiments confired that L.erythrorhizon induced GSDME cleavage,which drives pyroptosis.This phenomenon is conserved in several cancer cell lines that might be triggered by caspase family proteases.The mechanism of L.erythrorhizon inducing pyroptosis is widely found in tumor cells.Conclusion:Our findings not only explain how L.erythrorhizon triggers cancer cell pyroptosis,but also provide mechanistic insights to guide its clinical application in the future.展开更多
文摘目的检测PP1A与GSDME在结直肠癌组织中的表达与CD8^(+)T淋巴细胞丰度,探讨PP1A与GSDME介导焦亡的相关性和临床意义。方法应用GEPIA数据库分析PP1A与GSDME在结直肠癌组织与正常组织中mRNA的表达。采用Western blot法检测结直肠癌组织与对应癌旁正常黏膜中PP1A蛋白表达水平,运用免疫组化法检测107例结直肠癌与癌旁正常黏膜中PP1A、GSDME蛋白的表达和CD8^(+)T淋巴细胞丰度。利用Spearman等级相关性分析PP1A、GSDME和CD8^(+)T淋巴细胞丰度的相关性。结果GEPIA数据库检索显示,PP1A与GSDME的mRNA在结直肠癌组织和正常组织中的表达差异有统计学意义(P<0.05)。Western blot法检测结果显示,结直肠癌组织中PP1A相对表达量明显高于癌旁组织(0.937 vs 0.643,P<0.001)。免疫组化结果显示,结直肠癌组织中PP1A的表达明显高于正常黏膜,而GSDME的表达明显低于正常黏膜(P<0.05),GSDME表达与结直肠癌患者年龄、临床分期和错配修复蛋白密切相关(P<0.05);CD8^(+)T细胞在癌浸润前沿的分布明显高于癌旁正常黏膜,且CD8^(+)T细胞在癌组织中的分布与pT分期、临床分期及淋巴结转移相关。Spearman相关性分析显示,PP1A与GSDME表达呈负相关(r=-0.196,P<0.05)。PP1A阳性结直肠癌患者的总生存期低于PP1A阴性患者(P<0.05),患者预后与分化程度、淋巴结转移、pT分期和临床分期相关。PP1A表达、肿瘤分化程度、临床分期、pT分期和淋巴结转移均是影响结直肠癌患者预后的独立危险因素。结论PP1A在结直肠癌中高表达,与GSDME介导的细胞焦亡呈负相关,两者表达差异性与结直肠癌的发生、发展及预后均密切相关,可作为判断结直肠癌患者预后的潜在指标,CD8^(+)T细胞的差异性分布可能与GSDME介导的细胞焦亡及肿瘤的发展相关。
基金supported by grants from startup fund program at Beijing University of Chinese Medicine(90011451310011)key research fund for drug discovery in Chinese medicine at Beijing University of Chinese Medicine(1000061223476)startup fund program at Beijing University of Chinese Medicine(90020361220006).
文摘Objective To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.
基金supported by Program for the research startup fund program at Beijing University of Chinese Medicine(90011451310011)the key research fund for drug discovery in Chinese medicine at Beijing University of Chinese Medicine(1000061223740)+1 种基金the experimental technology standardization research project at Beijing University of Chinese Medicine(2021-SYJS-009)the fundamental research funds for the central universities of Beijing University of Chinese Medicine(2020-JYBZDGG-057)。
文摘Objective:To reveal GSDME-executed pyroptosis in cancer cells induced by the Chinese traditional herbal medicine plant Lithospermum erythrorhizon(L.erythrorhizon,Zi Cao)and to investigate the potential mechanism.Methods:L.erythrorhizon was extracted by ultrasonication in 95%ethanol,and determined using high-performance liquid chromatography(HPLC).He La,A549,SW620,HEK-293 T,THP-1,K562,Raw264.7 and MDA-MB-231 cell lines were used to investigate the morphology and mechanism of pyroptosis induced by L.erythrorhizon.The lactate dehydrogenase(LDH)release,propidium iodide(PI)/Hoechst double-staining,and pyroptosis reconstitution experiments were performed to study L.erythrorhizon-induced cell pyroptosis.Results:Compared with the death inhibitor,PI/Hoechst and LDH release experiments,we found that L.erythrorhizon induced pyroptosis.Recombination and western blot experiments confired that L.erythrorhizon induced GSDME cleavage,which drives pyroptosis.This phenomenon is conserved in several cancer cell lines that might be triggered by caspase family proteases.The mechanism of L.erythrorhizon inducing pyroptosis is widely found in tumor cells.Conclusion:Our findings not only explain how L.erythrorhizon triggers cancer cell pyroptosis,but also provide mechanistic insights to guide its clinical application in the future.
