title: Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis.
abstract: The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity.
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Zuquan
authors.initials: Z
authors.lastname: Weng
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Yang
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Greenhaw
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Haibo
authors.initials: H
authors.lastname: Li
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Liming
authors.initials: L
authors.lastname: Xie
authors.affiliation: PharmPoint Consulting, 17014 Hersperger Lane, Poolesville, MD 20837, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Mattes
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA. Electronic address: qiang.shi@fda.hhs.gov.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shi
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D004789:Enzyme Activation; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects*; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000502:physiology; D009336:Necrosis; D010671:Phenylurea Compounds / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D010671:Phenylurea Compounds; D011725:Pyridines; C559147:regorafenib; D055372:AMP-Activated Protein Kinases
keywords: Hepatocyte; Hepatotoxicity; Mitochondrion; Necrosis; Rat; Regorafenib