Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells

Background: Mitochondrial homeostasis is a highly dynamic process involving continuous fission and fusion cycles and mitophagy to maintain mitochondrial functionality. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, is used to treat various skin malignancies. IMQ also induces apoptotic and autophagic cell death in various cancers through a TLR7-independent pathway. Objective: To investigate whether IMQ-induced ROS production is involved in mitochondrial dysfunction, mitochondrial fragmentation and mitophagy in skin cancer cells. Methods: BCC/KMC-1, B16F10 and A375 skin cancer cells, AGS gastric cancer cells and primary human keratinocytes were treated with 50 mu g/mL IMQ. After 4 h, ROS were detected by CM-H(2)DCFDA, DHE, and MitoSOX Red staining. After 24 h, cell viability and the mitochondrial membrane potential were evaluated by a CCK-8 assay and JC-1 staining, respectively. Oxygen consumption was assessed with an Oroboros instrument. Mitochondrial morphology and mitophagy were evaluated by MitoTracker and LysoTracker staining. Mitochondrial dynamics markers, including MEN-1, DRP-1 and OPAL, and mitophagy markers, including LC3, S65-phosphorylated ubiquitin, PINK1 and T0M20, were detected by immunoblotting. Results: IMQ not only induced severe ROS production but also resulted in increased mitochondrial membrane potential loss, mitochondrial fission and mitophagy and decreased oxygen consumption in skin cancer cells compared with normal keratinocytes. Pretreatment with the antioxidant NAC reduced IMQ-induced ROS production and attenuated IMQ-induced mitochondrial fission and mitophagy in skin cancer cells. Conclusions: IMQ-induced ROS might be associated with mitochondrial dysfunction, mitochondrial fission and mitophagy in cancer cells. Alleviating IMQ-induced ROS production would reduce mitochondrial fission-to-fusion skewing and further reduce IMQ-induced mitophagy. (C) 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.