title: Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes.
abstract: Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 microM) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.
authors.affiliation: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
authors.forename: Calivarathan
authors.initials: C
authors.lastname: Latchoumycandane
authors.forename: Quee Ming
authors.initials: QM
authors.lastname: Seah
authors.forename: Rachel C H
authors.initials: RC
authors.lastname: Tan
authors.forename: Jetsumon
authors.initials: J
authors.lastname: Sattabongkot
authors.forename: Walter
authors.initials: W
authors.lastname: Beerheide
authors.forename: Urs A
authors.initials: UA
authors.lastname: Boelsterli
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D000814:Aniline Compounds / Q000494:pharmacology*; D000873:Anthracenes / Q000494:pharmacology; D018501:Antirheumatic Agents / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003437:Crotonates; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006885:Hydroxybutyrates / Q000494:pharmacology*; D007555:Isoxazoles / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000077339:Leflunomide; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles; D010766:Phosphorylation; D050050:Protein Carbonylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014052:Toluidines
chemical_list: D000814:Aniline Compounds; D000873:Anthracenes; D018501:Antirheumatic Agents; D003437:Crotonates; D006885:Hydroxybutyrates; D007555:Isoxazoles; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D014052:Toluidines; C527525:teriflunomide; C432165:pyrazolanthrone; D000082:Acetaminophen; D045304:Cytochromes c; D048056:Mitogen-Activated Protein Kinase 9; D048031:JNK Mitogen-Activated Protein Kinases; D048055:Mitogen-Activated Protein Kinase 8; D053148:Caspase 3; D000077339:Leflunomide; D005978:Glutathione