1新光醫院 外科部 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
Inhibition of autophagy with antimalarial drugs chloroquine or hydrochloroquine induces apoptosis in human bladder cancer cells
Thomas I-Sheng Hwang1,3,4、Te-Fu Tsai 1,4, Shan-Che Yang2, Hung-En Chen1, Yi-Chia Lin1,4, Kuang-Yu Chou1,4, and Ji-Fan Lin2
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, Taipei Medical University,
4School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan.
# These authors contributed equally to this work.
Background: Cancer cells adapt to the stress resulting from accelerated cells growth, lack of nutrients or challenges of anti-cancer drugs by activation of the autophagy pathway. We demonstrated that human bladder cancer cells exhibit high level of basal autophagy in our previous studies. We therefore hypothesize that autophagy inhibitors such as chloroquine (CQ) and hydroxychloroquine (HCQ) that inhibits autophagosome function will result in cancer cell death by induction of apoptosis in human bladder cancer cells.
Materials and methods: Human Immortalized uroepithelial cell, SV-Huc-1; human bladder cancer cells including: transitional cell carcinoma (TCC) grade I, RT-4; TCC grade II, 5637; TCC grade III, T24; human prostate cancer cell line: PC3 and human breast cancer cell line: MCF-7 were used in this study. Inhibition of basal autophagy was achieved using CQ, HCQ and Bafilomycin A1 (Baf A1) in multiple human bladder cancer cell lines. Cell viability was assessed by WST-1 assay. Western blot detection of LC3-II was performed to monitor autophagy, while detection of caspase 3/7 activities and DNA fragmentation were conducted to investigate apoptotic induction in treated cells. The disruption of mitochondria membrane potential (MMP), the generation of reactive oxygen species (ROS) and lysosome permeability were accessed by JC-1, H2DCFDA staining and IF detection of cathepsin D and E, respectively, in CQ- and HCQ-treated cells.
Results: Changes in LC3 flux, monitored by Western blot and IF, indicated inhibition of autophagy at the level of the autophagosome by CQ and HCQ. Both two autophagy inhibitors induced cytotoxicity in multiple human bladder cell lines in time- and dose-dependent manner especially in advanced cancer cell lines. CQ and HCQ also significantly impacted the clonogenic formation of bladder cancer cells. However, the inhibition of cell viability was only observed in bladder cancer cell lines but not in SV-Huc-1, PC3 and MCF-7 cells that reported to be with low basal autophagy activity. Induction of apoptosis was found in cells treated with CQ and HCQ. We cannot detected the disruption of mitochondria membrane potential nor the generation of reactive oxygen species in CQ- or HCQ-treated cells. Translocation of cathepsin B in CQ-treated cells suggesting the change of lysosome permeability that leads to the blockage of autophagy and increasing apoptotic cell death by these agents.
Targeting autophagy with CQ or HCQ may be an effective cancer therapy in human bladder