陳宏恩^1#、林致凡^2#、蔡德甫¹、楊尚哲²、林宜佳^1,4、仇光宇^1,4、黃一勝^1,3,4

¹新光醫院 泌尿科、²新光醫院 中央研究室、³台北醫學大學 醫學院、⁴輔仁大學 醫學院

Hung-En Chen^1#, Ji-Fan Lin^2#, Te-Fu Tsai ^1,4, Yi-Chia Lin^1,4, Kuang-Yu Chou^1,4,
and Thomas I-Sheng Hwang^1,3,4

¹Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, ²Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, ³Department of Urology, Taipei Medical University, ⁴Division of Urology, School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.

# These authors contributed equally to this work.

 

Purpose: Chloroquine (CQ) widely used to treat and prevent malaria, is shown to be an important therapeutic compound to treat diseases such as autoimmune disorders. Recently, CQ is also recognized as a potent adjuvant agent when combined with other chemotherapeutic drugs to treat cancers. However, the effects of a single treatment of CQ on bladder cancer (BC) have not been investigated. In the present study, we aimed to examine the cell death mechanism induced by CQ in BC cells.

Materials and methods: The effect of CQ on the growth and viability of BC cells (5637 and T24) in vitro was examined by cologenic formation assay and WST-1 reagent, respectively. The morphology and numbers of lysosomes in live cells with or without CQ treatment was tracked using a fluorescent dye, LysoTracker. The induction of lysosomal membrane permeability (LMP) was detected by acridine orange translocation, and further investigated by the immunofluorescent detection of cathepsin B and D (CatB and CatD) release. The expression level of bid, caspase-3, and cytosolic cytochrome c in CQ-treated cells was detected by Western blot. Lysosomal protease inhibitors, pepstatin A and E64d were used to attenuate CQ-induced LMP.

Results: CQ exhibited cytotoxicity toward human bladder cancer cell lines. Time-lapse monitoring of lysosomes labeled with LysoTracker showed diminishing of fluorescent in CQ-treated cells, suggesting that CQ targets lysosomal functions. This was further supported by dose-dependent AO translocation detected with increased concentration of CQ, and the releasing of CatB and CatD into cytosol. The increased level of cleavage bid and cytosolic cytochrome c indicated mitochondrial outer membrane permeabilization (MOMP), and subsequently leading to apoptosis induction judged by the increased level of cleaved caspase 3. Furthermore, the reduced cell viability, the release of CatB and CatD, the level of cytosolic cytochrome c and cleaved caspase3 were attenuated by the pretreatment of pepstatinA/E64 which are inhibitors of lysosomal protease suggesting CQ induced LMP is responsible for CQ-induced apoptotic cell death in human BC cells.

Conclusions: CQ exhibits cytotoxicity in human bladder cancer cells by enhancing LMP that amplifies apoptotic signaling and ultimately leading to cell death.