Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells

Background: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the sub-sequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused ly-sosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells.Objective: To determine whether lysosomes are involved in IMQ-induced apoptosis.Methods: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluores-cence caspase-8 kit and determined by flow cytometry and confocal microscopy.Results: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo.Conclusions: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.(c) 2022 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.