Molecular mechanisms of tyrosine kinase inhibitors-associated hepatotoxicity

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of certain cancers. They are usually well-tolerated, but can cause adverse reaction including liver injury. Currently, mechanisms of hepatotoxicity associated with TKIs are only partially clarified. We therefore aimed at investigating the in vitro mechanisms of hepatotoxicity of 10 TKIs that have been reported to cause liver injury in patients in our first three papers. We treated HepG2 cells, HepaRG cells, and mouse liver mitochondria with TKIs (concentrations 0.5-100 μM) for different periods of time and assessed toxicity.
In the first paper, we investigated erlotinib, imatinib, lapatinib, and sunitinib. In HepG2 cells, all TKIs showed a time- and concentration-dependent cytotoxicity and, except erlotinib, a drop in intracellular ATP. For imatinib, lapatinib, and sunitinib, cytotoxicity increased in HepaRG cells induced with rifampicin, suggesting formation of toxic metabolites. Imatinib, lapatinib, and sunitinib reduced the mitochondrial membrane potential in HepG2 cells and in mouse liver mitochondria. In HepG2 cells, these compounds increased reactive oxygen species (ROS) production, impaired glycolysis, and induced apoptosis. In addition, imatinib and sunitinib impaired oxygen consumption and activities of complex I and/or III of the electron transport chain, and reduced the cellular GSH pool. In conclusion, imatinib and sunitinib are mitochondrial toxicants after acute and long-term exposure and inhibit glycolysis. Lapatinib affects mitochondria only weakly and inhibits glycolysis, whereas the cytotoxicity of erlotinib could not be explained only by a mitochondrial mechanism.
In the second paper, we investigated crizotinib, dasatinib, pazopanib, ponatinib, regorafenib, and sorafenib. Regorafenib and sorafenib strongly inhibited oxidative metabolism and glycolysis, decreased the mitochondrial membrane potential, and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady-state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity and dissipation of ATP stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady-state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial ROS formation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib show no relevant toxicity.
In the third paper, we wanted to better characterize the mechanisms underlying the mitochondrial impairment observed with the multikinase inhibitors ponatinib, regorafenib, and sorafenib in our second paper. The multikinase inhibitors impaired the activity of different complexes of the respiratory chain. As a consequence, they decreased the mitochondrial membrane potential concentration-dependently. They induced mitochondrial fission and mitophagy as well as mitochondrial release of cytochrome c associated with apoptosis and/or necrosis. In conclusion, ponatinib, regorafenib, and sorafenib impair the function of the respiratory chain, which is associated with increased ROS formation and a drop in the mitochondrial membrane potential. Despite mitochondrial fission and mitophagy, some cells are eliminated concentration-dependently by apoptosis or necrosis. Mitochondrial dysfunction may represent a toxicological mechanism of hepatotoxicity associated with certain kinase inhibitors.
In the fourth paper, we switched to the in vivo situation and aimed to investigate the in vivo mechanisms of hepatotoxicity of sunitinib in mice. We treated mice with 7.5 mg/kg sunitinib for 14 days. Sunitinib did not affect nutrient intake or body weight, but was associated with a six-fold increase in plasma ALT. Enzyme activity of the mitochondrial electron transport chain was decreased in liver tissue and significantly for complex III in isolated liver mitochondria. The decreased complex activity was associated with mitochondrial ROS formation and increased SOD2 expression. In addition, the citrate synthase activity and protein expression of PGC-1α were reduced. Caspase 3 cleavage and TUNEL-positive hepatocytes were increased, compatible with hepatocyte apoptosis and increased plasma ALT. In conclusion, mice treated with 7.5 mg/kg sunitinib for 14 days had an impaired mitochondrial function leading to hepatocyte apoptosis with the key regulator of mitochondrial proliferation and function PGC-1α may an important mechanistic factor for these findings.