Abstract
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.
Highlights
Receptor tyrosine kinases (TK) are transmembrane proteins that regulate important cellular pathways such as differentiation, proliferation, and apoptosis by phosphorylation of tyrosine residues (Yarden and Sliwkowski, 2001; Krause and Van Etten, 2005)
We found that lapatinib impairs mitochondrial function in a hepatocellular carcinoma-derived cell line (HepG2), which was accompanied by accumulation of reactive oxygen species (ROS) and release of cytochrome c from mitochondria into the cytosol inducing apoptosis
To confirm the findings of our previous study (Paech et al, 2017), we determined the release of adenylate kinase (AK), a marker for plasma membrane damage, into the surrounding cell culture medium from HepG2 cells treated with lapatinib for 2, 6, or 24 h (Figure 1A)
Summary
Receptor tyrosine kinases (TK) are transmembrane proteins that regulate important cellular pathways such as differentiation, proliferation, and apoptosis by phosphorylation of tyrosine residues (Yarden and Sliwkowski, 2001; Krause and Van Etten, 2005). Lapatinib is a TKI that is used in combination with capecitabine to treat patients with advanced or metastatic HER2-positive breast cancer (Moy et al, 2007). The TK HER2 (human epidermal growth factor receptor 2, known as ErbB2) is overexpressed in up to 25% of invasive or metastatic breast cancers, and is associated with an unfavorable prognosis (Slamon et al, 1987). Lapatinib inhibits the intracellular TK domain of both HER1 ( known as epidermal growth factor receptor [EGFR] or ErbB1) and HER2. We found that lapatinib impairs mitochondrial function in a hepatocellular carcinoma-derived cell line (HepG2), which was accompanied by accumulation of reactive oxygen species (ROS) and release of cytochrome c from mitochondria into the cytosol inducing apoptosis. An effective antioxidative defense is essential, since excess ROS can modify DNA bases, induce lipid peroxidation, and oxidize cellular proteins (Ott et al, 2007)
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