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.