Boran Tugce, Zengin Ozge Sultan, Seker Zehra, Akyildiz Aysenur Gunaydin, Oztas Ezgi, Özhan Gül
Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 34116 Istanbul, Turkey; Istanbul University-Cerrahpaşa, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 34500 Istanbul, Turkey.
Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 34116 Istanbul, Turkey; Institute of Graduate Studies in Health Sciences, Istanbul University, 34116 Istanbul, Turkey.
Toxicology. 2023 May 1;489:153489. doi: 10.1016/j.tox.2023.153489. Epub 2023 Mar 16.
Ripretinib is a multikinase inhibitor drug approved in 2020 by the FDA and in 2021 by EMA for use in the treatment of advanced gastrointestinal stromal tumors (GIST) which have not adequately responded to previous treatments with kinase inhibitors. The most common side effects of the drug are myalgia and fatigue, which likely causes interruption of the treatment or reduction of the dose. Skeletal muscle cells highly depend on ATP to perform their functions and mitochondrial damage may play a role in skeletal muscle toxicity induced by kinase inhibitors. However, the molecular mechanism has not been clearly identified in the literature yet. In this study, it has been aimed to elucidate the role of mitochondria in the toxic effect of ripretinib on skeletal muscle using the mouse C2C12 myoblast-derived myotubes. The myotubes were exposed to ripretinib at the range of 1-20 μM concentrations for 24 h. To determine the potential role of mitochondrial impairment in ripretinib-induced skeletal muscle toxicity, intracellular ATP level, mitochondrial membrane potential (MMP), mitochondrial ROS production (mtROS), mitochondrial DNA (mtDNA) copy number, and mitochondrial mass were examined after ripretinib treatment. Furthermore, changes in PGC 1α/NRF 1/NRF 2 expression levels that play a role in mitochondrial biogenesis and mitophagy were investigated. Additionally, the mitochondrial electron transport chain (ETC) enzyme activities were evaluated. Lastly, a molecular docking study was done to see ripretinib's possible interaction with DNA polymerase gamma (POLG) which is important for DNA replication in the mitochondria. According to the findings, ripretinib decreases the ATP level and mtDNA copy number, induces loss of MMP, and reduces mitochondrial mass. The activities of the ETC complexes were inhibited with ripretinib exposure which is in line with the observed ATP depletion and MMP loss. The molecular docking study revealed that ripretinib has inhibitory potential against POLG which supports the observed inhibition of mtDNA. The expression of PGC 1α was reduced in the nuclear fraction indicating that PGC-1α was not activated since the NRF 1 expression was reduced and NRF 2 level did not show significant change. Consequently, mtROS production increased in all treatment groups and mitophagy-related gene expressions and Parkin protein expression level were up-regulated at high doses. In conclusion, mitochondrial damage/loss can be one of the underlying causes of ripretinib-induced skeletal muscle toxicity. However, further studies are needed to confirm the results in vivo.
瑞派替尼是一种多激酶抑制剂药物，2020年获美国食品药品监督管理局（FDA）批准，2021年获欧洲药品管理局（EMA）批准，用于治疗对先前激酶抑制剂治疗反应不佳的晚期胃肠道间质瘤（GIST）。该药物最常见的副作用是肌痛和疲劳，这可能导致治疗中断或剂量减少。骨骼肌细胞高度依赖三磷酸腺苷（ATP）来执行其功能，线粒体损伤可能在激酶抑制剂诱导的骨骼肌毒性中起作用。然而，文献中尚未明确其分子机制。在本研究中，旨在使用小鼠C2C12成肌细胞衍生的肌管阐明线粒体在瑞派替尼对骨骼肌的毒性作用中的作用。将肌管暴露于浓度为1-20μM的瑞派替尼中24小时。为了确定线粒体损伤在瑞派替尼诱导的骨骼肌毒性中的潜在作用，在瑞派替尼处理后检测细胞内ATP水平、线粒体膜电位（MMP）、线粒体活性氧（mtROS）产生、线粒体DNA（mtDNA）拷贝数和线粒体质量。此外，研究了在线粒体生物发生和线粒体自噬中起作用的PGC 1α/NRF 1/NRF 2表达水平的变化。另外，评估了线粒体电子传递链（ETC）酶活性。最后，进行了分子对接研究，以观察瑞派替尼与线粒体DNA复制中重要的DNA聚合酶γ（POLG）的可能相互作用。根据研究结果，瑞派替尼降低了ATP水平和mtDNA拷贝数，诱导了MMP的丧失，并减少了线粒体质量。暴露于瑞派替尼会抑制ETC复合物的活性，这与观察到的ATP消耗和MMP丧失一致。分子对接研究表明瑞派替尼对POLG具有抑制潜力，这支持了观察到的对mtDNA的抑制作用。核部分中PGC 1α的表达降低，表明由于NRF 1表达降低且NRF 2水平未显示出显著变化，PGC-1α未被激活。因此，所有治疗组中的mtROS产生均增加，高剂量时线粒体自噬相关基因表达和帕金蛋白表达水平上调。总之，线粒体损伤/丧失可能是瑞派替尼诱导的骨骼肌毒性的潜在原因之一。然而，需要进一步的研究在体内证实这些结果。
