Blue-light exposure accelerates photo-oxidative disruption of lysosomal membranes and apoptosis in acridine orange-loaded chloroquine-treated human bladder cancer cells
Yi-Chia Lin1,3, Ji-Fan Lin2, Te-Fu Tsai1,3, Hung-En Chen1, Kuang-Yu Chou1,3, and Thomas I-Sheng Hwang1,3
1Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital,
2Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital,
3School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
Introduction: We previously showed that human bladder cancer cells exhibited high basal level of autophagic activity. Administration of chloroquine (CQ) or hydroxychloroquine (HCQ) inhibits autophagy and causes apoptotic cell death in bladder cancer cells. Acridione orange (AO) is commonly used to identify acidic vescular organelles (AVOs) in autophagic cells. The uncharged state of AO in cells was characterized by green fluorescence; while protonated form of AO accumulates in acidic compartments and forms aggregates that characterized by red fluorescence. An incidentally found disruption of the lysosome under treatment of CQ and blue light was observed. An experiment was conducted to verify the phenomenon is meaningful.
Methods: Inhibition of basal autophagy was achieved using CQ inT24 human bladder cancer cell lines by detecting LC3-II formation. Acridine orange relocalization was performed in T24 cells with or without CQ treatment for 2 hours. Immunofluorescence and Western blot were used for detection of cathepsin B and D release from lysosome. Cell viability and induction of cell death were detected using ApoTox-Glo Triplex Assay kit from Promega.
Results: CQ inhibited basal autophagy and decreased cell viability in T24 cells. AO relocalization was detected only in CQ-treated T24 cells. CQ-treated T24 cells that exposed for a short period of time to AO and under ordinary culture condition, accumulate the AO within AVOs, giving rise to a mainly red, granular fluorescence upon excitation with blue light. When AO-loaded CQ-treated cells are irradiated with intense blue light, AO soon starts to leak from lysosomes to nuclear and cytosol diffusely. Severe lysosomal damage that causing necrosis and apoptosis was only detected in AO-loaded CQ-treated T24 cells.This photo-oxidative disruption of lysosome was responsible for triggering cell death in bladder cancer cells.Necrotic and apoptotic death were both detected in cell treated with CQ up to 50%.
Conclusions: Photo-oxidative disruption of lysosomal membrane with AO and CQ may be an effective cancer therapy in human bladder cancer.