Imatinib disturbs lysosomal function and morphology and impairs the activity of mTORC1 in human hepatocyte cell lines

The tyrosine kinase inhibitors (TKIs) imatinib and lapatinib are associated with severe hepatotoxicity, whose mechanisms are currently under investigation. As amphiphilic drugs, imatinib and lapatinib enrich in lysosomes. In the present study, we investigated their effects on lysosomal morphology and function in HepG2 and HuH-7 cells and explored possible links between lysosomal dysfunction and hepatotoxicity. Both TKIs increased the lysosomal volume time and concentration-dependently in HepG2 and HuH-7 cells. In HepG2 cells, lapatinib and imatinib raised the lysosomal pH and destabilized the lysosomal membrane, thereby impairing lysosomal proteolytic activity such as cathepsin B processing. Imatinib activated the transcription factor EB (TFEB), a regulator of lysosomal biogenesis and function, as demonstrated by nuclear TFEB accumulation and increased expression of TFEB-target genes. Because of lysosomal dysfunction, imatinib impaired mTORC1 activation, a protein complex activated on the lysosomal surface, which explained TFEB activation. HepG2 cells treated with imatinib showed increased levels of MAP1LC3A/B-II and of ATG13 (S318) phosphorylation, indicating induction of autophagy due to TFEB activation. Finally, imatinib induced apoptosis in HepG2 cells in a time and concentration-dependent manner, explained by lysosomal and mitochondrial toxicity. Our findings provide a new lysosome-centered mechanism for imatinib-induced hepatotoxicity that could be extended to other lysosomotropic drugs

Role of the NRF2-mediated oxidative stress response and lysosomal dysfunction in drug-induced liver injury associated with mitochondrial damage

Drug-induced liver injury is a rare, but potentially severe adverse drug reaction that is caused by various mechanisms including mitochondrial dysfunction and oxidative stress. Highly expressed in the liver, the transcription factor NRF2 stimulates the expression of phase II-detoxifying and antioxidant genes in response to electrophilic and oxidative stress. In unstressed conditions, NRF2 activation is suppressed by the cytosolic redox-sensitive protein KEAP1. As master regulator of the oxidative and electrophilic stress response, NRF2 might protect against drug-induced liver injury caused by mitochondrial damage and oxidative stress. To test this hypothesis, we assessed whether the hepatotoxic drugs benzbromarone and lapatinib, which are both associated with mitochondrial dysfunction and oxidative stress, activate the KEAP1-NRF2 pathway in HepG2 cells, a human hepatoma cell line. Moreover, lapatinib has lysosomotropic properties, which have also been described for the tyrosine kinase inhibitor imatinib. Similar to benzbromarone and lapatinib, imatinib caused severe liver injury in patients. As lipophilic weak bases, lapatinib and imatinib accumulate in acidic cellular compartments such as lysosomes. Thus, we assessed the effects of lapatinib and imatinib on lysosomal functions and related processes such as mammalian target of rapamycin complex 1 activation, lysosomal biogenesis, and autophagy in HepG2 cells. Benzbromarone is a uricosuric drug that was withdrawn from the drug market by its manufacturer due to severe cases of liver toxicity. In our first project, benzbromarone (1-100 μM) lead to accumulation of mitochondrial superoxide radicals and cellular reactive oxygen species in HepG2 cells. The uricosuric drug caused oxidation of glutathione, the most prevalent antioxidant molecule in hepatocytes, to glutathione disulfide. Glutathione disulfide levels increased in entire HepG2 cells and especially in mitochondria. Moreover, benzbromarone increased the level of oxidized mitochondrial antioxidant protein thioredoxin 2. These findings indicate that mainly mitochondria were exposed to benzbromarone-induced oxidative stress and might have been the origin of ROS generation. Furthermore, benzbromarone activated the KEAP1-NRF2 pathway in HepG2 cells, demonstrated by nuclear accumulation of NRF2 and upregulation of several NRF2-regulated antioxidant proteins. Downregulation of KEAP1, which led to NRF2 activation, protected HepG2 cells from benzbromarone-induced ATP depletion and cell membrane permeabilization. Approved for the treatment of human epidermal growth factor receptor 2-positive breast cancer, lapatinib received a black box warning by the U.S. Food and Drug Administration due to severe cases of hepatotoxicity in patients. We observed that lapatinib (2-20 μM) induced the generation of mitochondrial superoxide and cellular hydrogen peroxide in HepG2 cells in our second project. In this cellular system, lapatinib activated the KEAP1-NRF2 pathway at clinically relevant concentrations. Consequently, lapatinib upregulated several prototypical NRF2-regulated antioxidant genes and glutathione biosynthesis. As observed for benzbromarone, lapatinib increased the levels of glutathione disulfide, confirming that lapatinib caused oxidative stress in HepG2 cells. Co-treatment with the antioxidant N-acetylcysteine reduced the accumulation of NRF2 induced by lapatinib, indicating that reactive oxygen species were involved in the activation. N-acetylcysteine co-treatment also reduced the decrease in KEAP1 protein levels caused by lapatinib. Finally, lapatinib upregulated mitochondria-specific antioxidant genes more strongly than their cytosolic counterparts. As lysosomotropic drugs, lapatinib and imatinib increased the lysosomal volume, raised the lysosomal pH, and showed signs of lysosomal membrane permeabilization in HepG2 cells in our third project. Both drugs disturbed the proteolytic activity of lysosomes. Moreover, imatinib reduced the activity of the mammalian target of rapamycin complex 1. This protein complex is activated on the lysosomal surface and regulates essential metabolic pathways such as protein synthesis and autophagy. Consequently, imatinib activated the transcription factor EB. This transcription factor is the master regulator of lysosomal biogenesis as well as autophagy and a substrate of the mammalian target of rapamycin complex 1. In response to imatinib, the transcription factor EB accumulated in the nucleus and upregulated the expression of lysosomal as well as autophagic genes. Inactivation of the mammalian target of rapamycin complex 1 and upregulation of autophagic genes together with the increased expression of autophagic proteins implied that imatinib induced autophagy in HepG2 cells. However, the concomitant lysosomal dysfunction caused by imatinib might impair a complete autophagic flux. In conclusion, the hepatotoxic drugs benzbromarone and lapatinib activated the NRF2 pathway in HepG2 cells as an adaptive stress response. Activation of NRF2 protected against benzbromarone- induced hepatotoxicity. Moreover, oxidative stress was partially involved in the activation of NRF2 by lapatinib. Finally, we revealed that the impairment of lysosomal functions and related pathways might contribute to lapatinib- and imatinib-induced liver toxicity

Lapatinib Activates the Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in HepG2 Cells

The receptor tyrosine kinase inhibitor lapatinib, indicated to treat patients with HER2-positive breast cancer in combination with capecitabine, can cause severe hepatotoxicity. Lapatinib is further associated with mitochondrial toxicity and accumulation of reactive oxygen species. The effect of lapatinib on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, the major cellular defense pathway against oxidative stress, has so far not been studied in detail. In the present study, we show that lapatinib (2-20 µM) activates the Keap1-Nrf2 pathway in HepG2 cells, a hepatocellular carcinoma-derived cell line, in a concentration-dependent manner upon 24 h of treatment. Lapatinib stabilized the transcription factor Nrf2 at concentrations ≥5 µM and caused its nuclear translocation. Well-established Nrf2 regulated genes (; Nqo1; ,; Gsta1; ,; Gclc; , and; Gclm; ) were upregulated at lapatinib concentrations ≥10 µM. Furthermore, cellular and mitochondrial glutathione (GSH) levels increased starting at 10 µM lapatinib. As a marker of oxidative stress, cellular GSSG significantly increased at 10 and 20 µM lapatinib. Furthermore, the gene expression of mitochondrial; Glrx2; and; SOD2; were increased upon lapatinib treatment, which was also observed for the mitochondrial SOD2 protein content. In conclusion, lapatinib treatment for 24 h activated the Keap1-Nrf2 pathway in HepG2 cells starting at 10 μM, which is a clinically relevant concentration. As a consequence, treatment with lapatinib increased the mRNA and protein expression of antioxidative and other cytoprotective genes and induced GSH synthesis, but these measures could not completely block the oxidative stress associated with lapatinib

The uricosuric benzbromarone disturbs the mitochondrial redox homeostasis and activates the NRF2 signaling pathway in HepG2 cells

The uricosuric benzbromarone is a mitochondrial toxicant associated with severe liver injury in patients treated with this drug. Since dysfunctional mitochondria can increase mitochondrial superoxide (O; 2; --; ) production, we investigated the consequences of benzbromarone-induced mitochondrial oxidative stress on the hepatic antioxidative defense system. We exposed HepG2 cells (a human hepatocellular carcinoma cell line) to increasing concentrations of benzbromarone (1-100 μM) for different durations (2-24 h), and investigated markers of antioxidative defense and oxidative damage. At high concentrations (≥50 μM), benzbromarone caused accumulation of mitochondrial superoxide (O; 2; --; ) and cellular reactive oxygen species (ROS). At concentrations >50 μM, benzbromarone increased the mitochondrial and cellular GSSG/GSH ratio and increased the oxidized portion of the mitochondrial thioredoxin 2. Benzbromarone stabilized the transcription factor NRF2 and caused its translocation into the nucleus. Consequently, the expression of the NRF2-regulated antioxidative proteins superoxide dismutase 1 (SOD1) and 2 (SOD2), glutathione peroxidase 1 (GPX1) and 4 (GPX4), as well as thioredoxin 1 (TRX1) and 2 (TRX2) increased. Finally, upregulation of NRF2 by siRNA-mediated knock-down of KEAP1 partially protected HepG2 cells from benzbromarone-induced membrane damage and ATP depletion. In conclusion, benzbromarone increased mitochondrial O; 2; --; accumulation and activates the NRF2 signaling pathway in HepG2 cells, thereby strengthening the cytosolic and mitochondrial antioxidative defense. Impaired antioxidative defense may represent a risk factor for benzbromarone-induced hepatotoxicity

Reactive Metamizole Metabolites Enhance the Toxicity of Hemin on the ATP Pool in HL60 Cells by Inhibition of Glycolysis

Metamizole is an analgesic, whose pharmacological and toxicological properties are attributed to N-methyl-aminoantipyrine (MAA), its major metabolite. In the presence of heme iron, MAA forms reactive metabolites, which are toxic for granulocyte precursors. Since decreased cellular ATP is characteristic for MAA-associated toxicity, we studied the effect of MAA with and without hemin on energy metabolism of HL60 cells, a granulocyte precursor cell line. The combination MAA/hemin depleted the cellular ATP stronger than hemin alone, whereas MAA alone was not toxic. This decrease in cellular ATP was observed before plasma membrane integrity impairment. MAA/hemin and hemin did not affect the proton leak but increased the maximal oxygen consumption by HL60 cells. This effect was reversed by addition of the radical scavenger; N; -acetylcysteine. The mitochondrial copy number was not affected by MAA/hemin or hemin. Hemin increased mitochondrial superoxide generation, which was not accentuated by MAA. MAA decreased cellular ROS accumulation in the presence of hemin. In cells cultured in galactose (favoring mitochondrial ATP generation), MAA/hemin had less effect on the cellular ATP and plasma membrane integrity than in glucose. MAA/hemin impaired glycolysis more than hemin or MAA alone, and; N; -acetylcysteine blunted this effect of MAA/hemin. MAA/hemin decreased protein expression of pyruvate kinase more than hemin or MAA alone. In conclusion, cellular ATP depletion appears to be an important mechanism of MAA/hemin toxicity on HL60 cells. MAA itself is not toxic on HL60 cells up to 100 µM but boosts the inhibitory effect of hemin on glycolysis through the formation of reactive metabolites

PGC-1α plays a pivotal role in simvastatin-induced exercise impairment in mice

Statins decrease cardiovascular complications, but can induce myopathy. Here, we explored the implication of PGC-1α in statin-associated myotoxicity.; We treated PGC-1α knockout (KO), PGC-1α overexpression (OE) and wild-type (WT) mice orally with 5 mg simvastatin kg; -1; day; -1; for 3 weeks and assessed muscle function and metabolism.; In WT and KO mice, but not in OE mice, simvastatin decreased grip strength, maximal running distance and vertical power assessed by ergometry. Post-exercise plasma lactate concentrations were higher in WT and KO compared to OE mice. In glycolytic gastrocnemius, simvastatin decreased mitochondrial respiration, increased mitochondrial ROS production and free radical leak in WT and KO, but not in OE mice. Simvastatin increased mRNA expression of Sod1 and Sod2 in glycolytic and oxidative gastrocnemius of WT, but decreased it in KO mice. OE mice had a higher mitochondrial DNA content in both gastrocnemius than WT or KO mice and simvastatin exhibited a trend to decrease the citrate synthase activity in white and red gastrocnemius in all treatment groups. Simvastatin showed a trend to decrease the mitochondrial volume fraction in both muscle types of all treatment groups. Mitochondria were smaller in WT and KO compared to OE mice and simvastatin further reduced the mitochondrial size in WT and KO mice, but not in OE mice.; Simvastatin impairs skeletal muscle function, muscle oxidative metabolism and mitochondrial morphology preferentially in WT and KO mice, whereas OE mice appear to be protected, suggesting a role of PGC-1α in preventing simvastatin-associated myotoxicity

Imatinib and Dasatinib Provoke Mitochondrial Dysfunction Leading to Oxidative Stress in C2C12 Myotubes and Human RD Cells

Tyrosine kinase inhibitors (TKIs) can cause skeletal muscle toxicity in patients, but the underlying mechanisms are mostly unclear. The goal of the current study was to better characterize the role of mitochondria in TKI-associated myotoxicity. We exposed C2C12 murine myoblasts and myotubes as well as human rhabdomyosarcoma cells (RD cells) for 24 h to imatinib (1-100 µM), erlotinib (1-20 µM), and dasatinib (0.001-100 µM). In C2C12 myoblasts, imatinib was membrane toxic at 50 µM and depleted the cellular ATP pool at 20 µM. In C2C12 myotubes exposed to imatinib, ATP depletion started at 50 µM whereas membrane toxicity was not detectable. In myoblasts and myotubes exposed to dasatinib, membrane toxicity started at 0.5 µM and 2 µM, respectively, and the ATP drop was visible at 0.1 µM and 0.2 µM, respectively. When RD cells were exposed to imatinib, ATP depletion started at 20 µM whereas membrane toxicity was not detectable. Dasatinib was membrane toxic at 20 µM and depleted the cellular ATP pool already at 0.5 µM. Erlotinib was not toxic in both cell models. Imatinib (20 µM) and dasatinib (1 µM) reduced complex I activity in both cell models. Moreover, the mitochondrial membrane potential (; Δψ; m) was dissipated for both TKIs in myotubes. In RD cells, the; Δψ; m was reduced only by dasatinib. Both TKIs increased mitochondrial superoxide accumulation and decreased the mitochondrial copy number in both cell lines. In consequence, they increased protein expression of superoxide dismutase (SOD) 2 and thioredoxin 2 and cleavage of caspase 3, indicating apoptosis in C2C12 myotubes. Moreover, in both cell models, the mRNA expression of; Sod1; and; Sod2; increased when RD cells were exposed to dasatinib. Furthermore, dasatinib increased the mRNA expression of; atrogin-1; and; murf-1; , which are important transcription factors involved in muscle atrophy. The mRNA expression of; atrogin-1; increased also in RD cells exposed to imatinib. In conclusion, imatinib and dasatinib are mitochondrial toxicants in mouse C2C12 myotubes and human RD cells. Mitochondrial superoxide accumulation induced by these two TKIs is due to the inhibition of complex I and is probably related to impaired mitochondrial and myocyte proliferation