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

  • Shu-Hao Chang
    Affiliations
    Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan
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  • Pei-Ying Lin
    Affiliations
    Center for Cell Therapy and Translation Research, China Medical University Hospital, Taichung, Taiwan
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  • Tsai-Kun Wu
    Affiliations
    Division of Renal Medicine, Tungs’ Taichung MetroHarbor Hospital, Taichung, Taiwan

    Post Baccalaureate Medicine, National Chung Hsing University, Taichung, Taiwan
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  • Chien-Sheng Hsu
    Affiliations
    Frontier Molecular Medical Research Center in Children, Changhua Christian Children Hospital, Changhua County, Taiwan
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  • Shi-Wei Huang
    Affiliations
    Center for Cell Therapy and Translation Research, China Medical University Hospital, Taichung, Taiwan
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  • Zheng-Yi Li
    Affiliations
    Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan
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  • Kuang-Ting Liu
    Affiliations
    Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

    Department of Pathology & Laboratory Medicine, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan
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  • Jun-Kai Kao
    Affiliations
    Post Baccalaureate Medicine, National Chung Hsing University, Taichung, Taiwan

    Frontier Molecular Medical Research Center in Children, Changhua Christian Children Hospital, Changhua County, Taiwan
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  • Yi-Ju Chen
    Affiliations
    Post Baccalaureate Medicine, National Chung Hsing University, Taichung, Taiwan

    Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan
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  • Tak-Wah Wong
    Affiliations
    Department of Dermatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
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  • Chun-Ying Wu
    Correspondence
    Correspondence to: Division of Translational Medicine, Department of Medical Research, Taipei Veterans General Hospital, No. 322, Sec. 2, Shipai Rd, Beitou District, 11217 Taipei, Taiwan.
    Affiliations
    Division of Gastroenterology and Hepatology, Taipei Veterans General Hospital, Taipei, Taiwan
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  • Jeng-Jer Shieh
    Correspondence
    Correspondence to: Institute of Biomedical Sciences, National Chung Hsing University, No. 145 Xingda Rd., South Dist., Taichung City 402, Taiwan.
    Affiliations
    Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

    Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan

    Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan
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      Highlights

      • IMQ causes lysosomal membrane permeabilization and CTSs releasing into cytosol in skin cancer cells.
      • IMQ induces lysosomal cell death which is mediated by CTSD/caspase-8 axis.
      • IMQ-induced ROS generation is crucial for IMQ-induced caspase-8 activation and lysosomal cell death.
      • Combination treatment with lysosome-alkalinizing agents and IMQ enhances anti-tumor response in skin cancer cells.

      Abstract

      Background

      Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent 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 lysosomal 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 fluorescence 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.

      Keywords

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