3-(2-4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3h)-quinazolinone and Necrosis

Autoimmune hepatitis (AIH) is a severe necroinflammatory liver disease associated with significant mortality. Although loss of hepatocytes is generally recognised as a key trigger of liver inflammation and liver failure, the regulation of hepatic cell death causing AIH remains poorly understood. The aim of this study was to identify molecular mechanisms that drive hepatocyte cell death in the pathogenesis of acute liver injury.. Acute liver injury was modelled in mice by intravenous administration of concanavalin A (ConA). Liver injury was demonstrated by serum transaminases and histological assessment of liver sections. PGAM5-deficient mice (PGAM5-/-) were used to determine its role in experimental hepatitis. Mdivi-1 was used as an inhibitor of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Mitochondrial fission and the expression of PGAM5 were compared between liver biopsies derived from patients with AIH and control patients.. PGAM5 was highly expressed in hepatocytes of patients with AIH and in mice with ConA-induced experimental hepatitis. Deficiency of PGAM5 protected mice from ConA-induced hepatocellular death and liver injury. PGAM5 regulated ConA-induced mitochondrial fission in hepatocytes. Administration of the Drp1-inhibitor Mdivi-1 blocked mitochondrial fission, diminished hepatocyte cell death and attenuated liver tissue damage induced by ConA.. Our data demonstrate for the first time that PGAM5 plays an indispensable role in the pathogenesis of ConA-induced liver injury. Downstream of PGAM5, Drp1-mediated mitochondrial fission is an obligatory step that drives the execution of hepatic necrosis and tissue damage. Our data highlight the PGAM5-Drp1 axis as a potential therapeutic target for acute immune-mediated liver injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Case-Control Studies; Cell Death; Chemical and Drug Induced Liver Injury; Concanavalin A; Dynamins; Gene Expression; Hepatitis, Autoimmune; Hepatocytes; Humans; Liver; Mice; Mice, Knockout; Mitochondrial Dynamics; Mitochondrial Proteins; Necrosis; Phosphoprotein Phosphatases; Quinazolinones

We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKL(Ser358) and Drp1(Ser616) phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKL(Ser358), and coincidence of phospho-MLKL(ser358) and phospho-Drp1(Ser616) at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.

Topics: Acrylamides; Apoptosis; Carcinoma, Renal Cell; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Kidney Neoplasms; Kidney Tubules; Necrosis; Organ Culture Techniques; Quinazolinones; Receptor-Interacting Protein Serine-Threonine Kinases; Receptors, Tumor Necrosis Factor, Type I; RNA, Messenger; Signal Transduction; Sulfonamides; Tumor Necrosis Factor-alpha; Up-Regulation

Caspase-independent programmed necrotic cell death (necroptosis) has recently been described. Previously described models of necroptosis required 16h or more of induction, which made the interpretation of findings somewhat difficult. In human monocytic leukemia cell line U937 necroptosis could be induced within 6h by combination of TNF and Z-VAD-fmk. Here we show that the reduction in intracellular ATP levels may not be the sole determinant of necroptosis, and that necroptosis is associated with the loss of mitochondrial membrane potential, but not the activation of Bak/Bax or calcineurin.

Topics: Adenosine Triphosphate; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Calcineurin Inhibitors; Caspases; HL-60 Cells; Humans; Imidazoles; Indoles; Jurkat Cells; Membrane Potential, Mitochondrial; Necrosis; Protein Conformation; Quinazolinones; Tumor Necrosis Factor-alpha; U937 Cells

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1(-/-) mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

Topics: Animals; Apoptosis; Cells, Cultured; Dynamins; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Mitophagy; Necrosis; Nicotiana; Protein Kinases; Pulmonary Disease, Chronic Obstructive; Quinazolinones; Smoke; Ubiquitin-Protein Ligases