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 investigate the potential anti-tumor mechanisms of naphthoquinone compound shikonin(SKN)extracted from the root of Chinese herbal medicine plant lithospermum(Lithospermum erythrorhizon Sieb.&Zucc.).Me...Objective:To investigate the potential anti-tumor mechanisms of naphthoquinone compound shikonin(SKN)extracted from the root of Chinese herbal medicine plant lithospermum(Lithospermum erythrorhizon Sieb.&Zucc.).Methods:We first observed that SKN treatment led to swelling and bubbles in HeLa cells that were similar to the phenotype of cell pyroptosis.Subsequently,the HeLa cells experienced a pyroptotic process with SKN,and this was then assessed using lactate dehydrogenase(LDH)release and propidium iodide(PI)/Hoechst double staining experiments.Pyroptosis is defined as gasdermin-mediated programmed necroptosis.To identify the potential pyroptosis machinery,two strategies were utilized that included a genome-wide clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9 screening experiment and a pyroptosis reconstitution assay executed by each of the five known gasdermins(GSDMA-E).Moreover,endogenous cleavage was also detected in a panel of tumor cell lines.Results:Compared with the control,both the LDH release and PI/Hoechst double-staining experiments suggested that SKN induced perforation and enhancement of the permeability of the cell membranes that resulted in pyroptosis in HeLa cells(P=.028 and P=.032,respectively).In addition,the reconstitution assays in human embryonic kidney 293T(HEK-293T)cells and endogenous cleavage assays in HeLa cells indicated that the pyroptosis was controlled by GSDME.In addition,we also found SKN could trigger pyroptosis in a panel of tumor cell lines in which the cellular morphologies were proportional to the GSDME expression levels.Additionally,the cleavage of GSDME was also detected,and this was indicative of a similar GSDME-mediated mechanism.Conclusion:Our study not only explained the molecular mechanism of cytotoxicity of SKN to various tumor cells,but also provided additional information for the potential clinical application of natural naphthoquinone compounds against cancer.展开更多
Via an insufficient coat protein complex I(COPI)retrieval signal,the majority of SARSCo V-2 spike(S)is resident in host early secretory organelles and a tiny amount is leaked out in cell surface.Only surface-exposed S...Via an insufficient coat protein complex I(COPI)retrieval signal,the majority of SARSCo V-2 spike(S)is resident in host early secretory organelles and a tiny amount is leaked out in cell surface.Only surface-exposed S can be recognized by B cell receptor(BCR)or anti-S therapeutic monoclonal antibodies(m Abs)that is the trigger step for B cell activation after S m RNA vaccination or infected cell clearance by S m Abs.Now,a drug strategy to promote S host surface exposure is absent.Here,we first combined structural and biochemical analysis to characterize S COPI sorting signals.A potent S COPI sorting inhibitor was then invented,evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity(ADCC).Importantly,with the inhibitor as a probe,we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations,possibly corresponding to its ER chaperone association.Our findings not only suggest COPI is a druggable target against COVID-19,but also highlight SARS-Co V-2 evolution mechanism driven by S folding and trafficking mutations.展开更多
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This research was supported by the Program for the Research Startup Fund Program at the Beijing University of Chinese Medicine(90011451310011)the Key Research Fund for Drug Discovery in Chinese Medicine at the Beijing University of Chinese Medicine(1000061223740)+1 种基金the Experimental Technology Standardization Research Project at the Beijing University of Chinese Medicine(2021-SYJS-009)the Fundamental Research Funds for the Central Universities of the Beijing University of Chinese Medicine(2020-JYB-ZDGG-057).
文摘Objective:To investigate the potential anti-tumor mechanisms of naphthoquinone compound shikonin(SKN)extracted from the root of Chinese herbal medicine plant lithospermum(Lithospermum erythrorhizon Sieb.&Zucc.).Methods:We first observed that SKN treatment led to swelling and bubbles in HeLa cells that were similar to the phenotype of cell pyroptosis.Subsequently,the HeLa cells experienced a pyroptotic process with SKN,and this was then assessed using lactate dehydrogenase(LDH)release and propidium iodide(PI)/Hoechst double staining experiments.Pyroptosis is defined as gasdermin-mediated programmed necroptosis.To identify the potential pyroptosis machinery,two strategies were utilized that included a genome-wide clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9 screening experiment and a pyroptosis reconstitution assay executed by each of the five known gasdermins(GSDMA-E).Moreover,endogenous cleavage was also detected in a panel of tumor cell lines.Results:Compared with the control,both the LDH release and PI/Hoechst double-staining experiments suggested that SKN induced perforation and enhancement of the permeability of the cell membranes that resulted in pyroptosis in HeLa cells(P=.028 and P=.032,respectively).In addition,the reconstitution assays in human embryonic kidney 293T(HEK-293T)cells and endogenous cleavage assays in HeLa cells indicated that the pyroptosis was controlled by GSDME.In addition,we also found SKN could trigger pyroptosis in a panel of tumor cell lines in which the cellular morphologies were proportional to the GSDME expression levels.Additionally,the cleavage of GSDME was also detected,and this was indicative of a similar GSDME-mediated mechanism.Conclusion:Our study not only explained the molecular mechanism of cytotoxicity of SKN to various tumor cells,but also provided additional information for the potential clinical application of natural naphthoquinone compounds against cancer.
基金supported by Startup fund program at Beijing University of Chinese Medicine(BUCM)(90011451310011,China)to Wenfu Mathe emergency fund against COVID-19 program at BUCM(1000061223476,China)to Wenfu Mathe innovation team and talents cultivation program of national administration of traditional Chinese medicine(ZYYCXTD-C202006,China)to Wenfu Ma。
文摘Via an insufficient coat protein complex I(COPI)retrieval signal,the majority of SARSCo V-2 spike(S)is resident in host early secretory organelles and a tiny amount is leaked out in cell surface.Only surface-exposed S can be recognized by B cell receptor(BCR)or anti-S therapeutic monoclonal antibodies(m Abs)that is the trigger step for B cell activation after S m RNA vaccination or infected cell clearance by S m Abs.Now,a drug strategy to promote S host surface exposure is absent.Here,we first combined structural and biochemical analysis to characterize S COPI sorting signals.A potent S COPI sorting inhibitor was then invented,evidently capable of promoting S surface exposure and facilitating infected cell clearance by S antibody-dependent cellular cytotoxicity(ADCC).Importantly,with the inhibitor as a probe,we revealed Omicron BA.1 S is less cell surface exposed than prototypes because of a constellation of S folding mutations,possibly corresponding to its ER chaperone association.Our findings not only suggest COPI is a druggable target against COVID-19,but also highlight SARS-Co V-2 evolution mechanism driven by S folding and trafficking mutations.
