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Cell Death Discovery Oct 2023Lung ischemia/reperfusion injury (LIRI) is a complex pathophysiological process, with the histopathological hallmark of neutrophils migrating into the lungs. Neutrophil...
Lung ischemia/reperfusion injury (LIRI) is a complex pathophysiological process, with the histopathological hallmark of neutrophils migrating into the lungs. Neutrophil extracellular traps (NETs) have been suggested to exert a critical role in the pathogenesis of inflammation and infection in humans and animals, while the exact functions and underlying mechanisms of NETs in LIRI remain insufficiently elucidated. In this study, we investigated the role of pore-forming protein gasdermin D (GSDMD) on NETs release in LIRI induced by lung ischemia/reperfusion (I/R). We found that disulfiram, a GSDMD inhibitor, dramatically reduced NETs release and pathological injury in lung I/R in vivo and in vitro. Additionally, GSDMD caused mitochondrial DNA (mtDNA) leaking into the neutrophil cytosol, and then the cytoplasmic mtDNA activated the cGAS-STING signaling pathway and stimulated NETs formation in lung I/R. Furthermore, inhibition of cGAS/STING pathway could inhibit cytosol mtDNA mediated NETs formation.
PubMed: 37794018
DOI: 10.1038/s41420-023-01663-z -
Cell Death & Disease Sep 2023Gasdermin D (GSDMD)-mediated pyroptosis has a significant pro-inflammation characteristic due to dramatic secretion of pro-inflammatory substances. However, its role...
Gasdermin D (GSDMD)-mediated pyroptosis has a significant pro-inflammation characteristic due to dramatic secretion of pro-inflammatory substances. However, its role remains unclear in psoriasis as one chronic inflammatory skin disorder with high prevalence. We found that N-terminal GSDMD (N-GSDMD) was aberrantly expressed in epidermis of skin lesion in psoriasis patients and imiquimod-induced psoriasis-like dermatitis (IIPLD) mice. In epidermis of IIPLD mice and M5 (simulating psoriatic inflammatory challenge)-treated keratinocytes cultured in vitro, cleavage products of caspase-1, GSDMD and IL-1β were increased. M5-stimulated keratinocyte presented typical pyroptosis morphology accompanied with PI-staining. Gsdmd keratinocytes could not present pyroptosis morphology while stimulated with M5. Electroporation of recombinant N-GSDMD could make the pyroptosis morphology reappear. In Gsdmd mice or keratinocyte-specific Gsdmd conditional knockout mice, we observed the alleviation of psoriatic inflammation and epidermal aberrant expression of Ki-67 and differentiation markers (loricrin and keratin 5) after imiquimod stimulation. Transplanting skin tissue from control mice to Gsdmd mice can evoke the response to imiquimod stimulation in the background of Gsdmd mice (not limited in transplanting area). In M5-stimulated keratinocytes, disulfiram or GSDMD siRNA transfection can inhibit pyroptosis and eliminate disproportionate increases of Ki-67 and PI. We further validated that topically application of disulfiram (pyroptosis inhibitor) also alleviated IIPLD in mice. These findings indicate a novel mechanism that GSDMD-mediated keratinocyte pyroptosis facilitates hyperproliferation and aberrant differentiation induced by immune microenvironment in psoriatic skin inflammation, which contributes to pathogenesis of psoriasis. Our study provides an innovative insight that targeting pyroptosis can be considered as a therapeutic strategy against psoriasis.
Topics: Animals; Mice; Gasdermins; Disulfiram; Imiquimod; Ki-67 Antigen; Pyroptosis; Keratinocytes; Psoriasis; Dermatitis; Inflammation
PubMed: 37673869
DOI: 10.1038/s41419-023-06094-3 -
Proceedings of the National Academy of... Aug 2023Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and...
Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.
Topics: Animals; Mice; Disulfiram; Toll-Like Receptor 4; Lipopolysaccharides; Signal Transduction; Alcoholism; Cytokines
PubMed: 37487070
DOI: 10.1073/pnas.2306399120 -
Nature Communications Sep 2023The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion,...
The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquire early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogram and reverse the immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells of healthy donors or metastatic female breast cancer patients, induce robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a promising therapy for solid tumors.
Topics: Humans; Female; Animals; Mice; Leukocytes, Mononuclear; Receptors, Chimeric Antigen; Tumor Microenvironment; Breast Neoplasms; Disease Models, Animal; Immunosuppressive Agents; T-Lymphocytes
PubMed: 37714830
DOI: 10.1038/s41467-023-41282-x -
Redox Biology Feb 2024Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the fourth leading cause of cancer-related death globally, which is characterized by...
Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the fourth leading cause of cancer-related death globally, which is characterized by complicated pathophysiology, high recurrence rate, and poor prognosis. Our previous study has demonstrated that disulfiram (DSF)/Cu could be repurposed for the treatment of HCC by inducing ferroptosis. However, the effectiveness of DSF/Cu may be compromised by compensatory mechanisms that weaken its sensitivity. The mechanisms underlying these compensatory responses are currently unknown. Herein, we found DSF/Cu induces endoplasmic reticulum stress with disrupted ER structures, increased Ca level and activated expression of ATF4. Further studies verified that DSF/Cu induces both ferroptosis and cuproptosis, accompanied by the depletion of GSH, elevation of lipid peroxides, and compensatory increase of xCT. Comparing ferroptosis and cuproptosis, it is interesting to note that GSH acts at the crossing point of the regulation network and therefore, we hypothesized that compensatory elevation of xCT may be a key aspect of the therapeutic target. Mechanically, knockdown of ATF4 facilitated the DSF/Cu-induced cell death and exacerbated the generation of lipid peroxides under the challenge of DSF/Cu. However, ATF4 knockdown was unable to block the compensatory elevation of xCT and the GSH reduction. Notably, we found that DSF/Cu induced the accumulation of ubiquitinated proteins, promoted the half-life of xCT protein, and dramatically dampened the ubiquitination-proteasome mediated degradation of xCT. Moreover, both pharmacologically and genetically suppressing xCT exacerbated DSF/Cu-induced cell death. In conclusion, the current work provides an in-depth study of the mechanism of DSF/Cu-induced cell death and describes a framework for the further understanding of the crosstalk between ferroptosis and cuproptosis. Inhibiting the compensatory increase of xCT renders HCC cells more susceptible to DSF/Cu, which may provide a promising synergistic strategy to sensitize tumor therapy and overcome drug resistance, as it activates different programmed cell death.
Topics: Humans; Disulfiram; Copper; Carcinoma, Hepatocellular; Cell Line, Tumor; Ferroptosis; Lipid Peroxides; Liver Neoplasms
PubMed: 38150993
DOI: 10.1016/j.redox.2023.103007 -
The Primary Care Companion For CNS... Nov 2023
Topics: Humans; Disulfiram; Acetic Acid; Ethanol; Alcohol Deterrents; Alcoholism
PubMed: 38055873
DOI: 10.4088/PCC.23cr03537 -
BioRxiv : the Preprint Server For... Oct 2023Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DSF) was recently shown to potently inhibit GsdmD, but the in-vivo...
Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DSF) was recently shown to potently inhibit GsdmD, but the in-vivo efficacy and mechanism of DSF's anti-atherosclerotic activity is yet to be explored. We used human/mouse macrophages and a hyperlipidemic mouse model of atherosclerosis to determine DSF anti-atherosclerotic efficacy and mechanism. DSF-fed hyperlipidemic apoE mice showed significantly reduced IL-1β release upon in-vivo Nlrp3 inflammasome assembly and showed smaller atherosclerotic lesions (∼27% and 29% reduction in males and females, respectively). The necrotic core area was also smaller (∼50% and 46% reduction in DSF-fed males and females, respectively). DSF induced autophagy in macrophages, hepatocytes/liver, and in atherosclerotic plaques. DSF modulated other atheroprotective pathways such as efferocytosis, phagocytosis, and gut microbiota. DSF-treated macrophages showed enhanced phagocytosis/efferocytosis, with a mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic-force microscopy analysis revealed altered biophysical membrane properties of DSF treated macrophages, showing increased ordered-state of the plasma membrane and increased adhesion strength. Furthermore, the 16sRNA sequencing of DSF-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota and a reduction in atherogenic species. Taken together, our data shows that DSF can simultaneously modulate multiple atheroprotective pathways, and thus may serve as novel adjuvant therapeutic to treat atherosclerosis.
PubMed: 37905037
DOI: 10.1101/2023.10.17.562757 -
International Journal of Pharmaceutics Oct 2023The respiratory tract, as the first and most afflicted target of many viruses such as SARS-CoV-2, seems to be the logical choice for delivering antiviral agents against...
The respiratory tract, as the first and most afflicted target of many viruses such as SARS-CoV-2, seems to be the logical choice for delivering antiviral agents against this and other respiratory viruses. A combination of remdesivir and disulfiram, targeting two different steps in the viral replication cycle, has showed synergistic activity against SARS-CoV-2 in-vitro. In this study, we have developed an inhalable dry powder containing a combination of remdesivir and disulfiram utilizing the spray-drying technique, with the final goal of delivering this drug combination to the respiratory tract. The prepared dry powders were spherical, and crystalline. The particle size was between 1 and 5 μm indicating their suitability for inhalation. The spray-dried combinational dry powder containing remdesivir and disulfiram (RD) showed a higher emitted dose (ED) of >88% than single dry powder of remdesivir (R) (∼72%) and disulfiram (D) (∼84%), with a fine particle fraction (FPF) of ∼55%. Addition of L-leucine to RD showed >60% FPF with a similar ED. The in vitro aerosolization was not significantly affected after the stability study conducted at different humidity conditions. Interestingly, the single (R and D) and combined (RD) spray-dried powders showed limited cellular toxicity (CC values from 39.4 to >100 µM), while maintaining their anti-SARS-CoV-2 in vitro (EC values from 4.43 to 6.63 µM). In a summary, a combinational dry powder formulation containing remdesivir and disulfiram suitable for inhalation was developed by spray-drying technique which showed high cell viability in the respiratory cell line (Calu-3 cells) retaining their anti-SARS-CoV-2 property. In the future, in vivo studies will test the ability of these formulations to inhibit SARS-CoV-2 which is essential for clinical translation.
PubMed: 37703955
DOI: 10.1016/j.ijpharm.2023.123411 -
Journal of the American Heart... Apr 2024Pyroptosis executor GsdmD (gasdermin D) promotes atherosclerosis in mice and humans. Disulfiram was recently shown to potently inhibit GsdmD, but the in vivo efficacy...
BACKGROUND
Pyroptosis executor GsdmD (gasdermin D) promotes atherosclerosis in mice and humans. Disulfiram was recently shown to potently inhibit GsdmD, but the in vivo efficacy and mechanism of disulfiram's antiatherosclerotic activity is yet to be explored.
METHODS AND RESULTS
We used human/mouse macrophages, endothelial cells, and smooth muscle cells and a hyperlipidemic mouse model of atherosclerosis to determine disulfiram antiatherosclerotic efficacy and mechanism. The effects of disulfiram on several atheroprotective pathways such as autophagy, efferocytosis, phagocytosis, and gut microbiota were determined. Atomic force microscopy was used to determine the effects of disulfiram on the biophysical properties of the plasma membrane of macrophages. Disulfiram-fed hyperlipidemic apolipoprotein E mice showed significantly reduced interleukin-1β release upon in vivo Nlrp3 (NLR family pyrin domain containing 3) inflammasome activation. Disulfiram-fed mice showed smaller atherosclerotic lesions (~27% and 29% reduction in males and females, respectively) and necrotic core areas (~50% and 46% reduction in males and females, respectively). Disulfiram induced autophagy in macrophages, smooth muscle cells, endothelial cells, hepatocytes/liver, and atherosclerotic plaques. Disulfiram modulated other atheroprotective pathways (eg, efferocytosis, phagocytosis) and gut microbiota. Disulfiram-treated macrophages showed enhanced phagocytosis/efferocytosis, with the mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic force microscopy analysis revealed altered biophysical properties of disulfiram-treated macrophages, showing increased order-state of plasma membrane and increased adhesion strength. Furthermore, 16sRNA sequencing of disulfiram-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota and a reduction in atherogenic species.
CONCLUSIONS
Taken together, our data show that disulfiram can simultaneously modulate several atheroprotective pathways in a GsdmD-dependent as well as GsdmD-independent manner.
Topics: Male; Female; Mice; Humans; Animals; Disulfiram; Efferocytosis; Endothelial Cells; Gastrointestinal Microbiome; Atherosclerosis; Autophagy
PubMed: 38563369
DOI: 10.1161/JAHA.123.033881