Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines

Abstract

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse-XF24 analyzer) 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 (measured as adenylate kinase release) and dissipation of adenosine triphosphate 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 reactive oxygen species accumulation 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 showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.

Role of Cytochrome P450 Enzymes in the Metabolic Activation of Tyrosine Kinase Inhibitors

“…Studies by Krahenbuhl and colleagues have focused on defining the mitochondrial mechanisms of hepatocellular toxicity induced by tyrosine kinase inhibitors in hepatic cell lines, including HepG2 and HepaRG cells [65,66]. Paech et al, examined the toxic effects of four tyrosine kinase inhibitors (imatinib, sunitinib, lapatinib, and erlotinib) in HepG2 cells, HepaRG cells, and isolated mouse liver mitochondria [65].…”

“…Mingard et al, examined the toxic effects of six tyrosine kinase inhibitors (crizotinib, dasatinib, pazotinib, ponatinib, regorafenib, and sorafenib) in HepG2 cells [66]. Regorafenib and sorafenib were shown to cause direct mitochondrial toxicity, as evidenced by increased ATP depletion in HepG2 cells in the presence of galactose compared to glucose [66].…”

“…Regorafenib and sorafenib were shown to cause direct mitochondrial toxicity, as evidenced by increased ATP depletion in HepG2 cells in the presence of galactose compared to glucose [66]. Regorafenib and sorafenib (10 µM) decreased mitochondrial membrane potential in HepG2 cells [66]. Studies were also conducted in HepaRG cells to evaluate the impact of CYP-mediated metabolism of tyrosine kinase inhibitors on mitochondrial toxicity.…”

“…Unlike studies with lapatinib, sunitinib, and imatinib in HepaRG cells, pretreatment of HepaRG cells with the CYP3A4 inducer rifampicin (20 µM) did not enhance the toxic effects of crizotinib, dasatinib, pazotinib, ponatinib, regorafenib, and sorafenib on mitochondrial membrane integrity or ATP depletion. Rather, intracellular ATP content modestly increased with rifampicin in cells treated with crizotinib (20 µM), dasatinib (50 µM), regorafenib (5 µM), and sorafenib (5 µM) [66]. Ponatinib, regorafenib, and sorafenib were shown to inhibit the mitochondrial electron transport chain.…”

“…Crizotinib and ponatinib (20 µM) increased superoxide accumulation, and dasatinib and ponatinb (20 µM) caused intracellular GSH depletion. Based on their findings, Mingard et al suggested a mitochondrial mechanism of hepatotoxicity for regorafenib, sorafenib, and potentially ponatinib, and a non-mitochondrial mechanism of hepatotoxicity for crizotinib, dasatinib, and pazopanib [66]. …”