窄頻影像系統未來之可能應用: 氯喹(chloroquine)有效增強吖啶橙(acridine orange)之光毒性阻殺人類膀胱癌細胞
林宜佳1,4、林致凡2、楊尚哲2、鄧雅旻2、蔡德甫1,4、陳宏恩1、仇光宇1,4、黃一勝1,3,4
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
A novel application of narrow band imaging (NBI) system: Chloroquine enhanced Acridine orange inducedphototoxicity against human bladder cancer cells
Yi-Chia Lin1,4, Ji-Fan Lin2, Shan-Che Yang2,Ya-Ming Teng2,Te-Fu Tsai 1,4, Hung-En Chen1,Kuang-Yu Chou1,4, and Thomas I-Sheng Hwang1,2,3,4
Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital1, Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital2, Department of Urology, Taipei Medical University3, Division of Urology, School of Medicine, Fu-Jen Catholic University4, Taipei, Taiwan.
Background: Bladder cancer (BC) is a common noncutaneousmalignancies with high mortality and recurrence rate. Human BC cell lines exhibited a high basal level of autophagic activity demonstrated by accumulating of acridine orange (AO)-stained acidic vesicular organelles (AVOs) in BC cells. When autophagy in BC cells was inhibited using chloroquine (CQ) that block autophagosomes and lysosomes fusion, aincreased number of red AO-positive AVOs and the loss of cell viability were routinely observed. In this study, we aim to investigate the effect of CQ that enhances photodynamic therapy (PDT) with AO on the human BC cell lines.
Materials and methods: Cell viability was determined using (a) WST-1 reagents (immediately after treatment) or (b) time-lapse imaging (for 24 hrs) with multi-mode image-based reader in human immortalized uroepithelial (SV-Huc1) and BC cell lines (5637 and T24) treated with indicated concentration of AO, CQ or blue light exposure alone or in combination. Changes in the morphology of organelles in AO- or CQ-treated BC cells were detected by transmission electron microscopy (TEM). The AO relocalization in treated-BC cells was recorded in real-time using fluorescent microscopy equipped with color CCD camera. The regression of red fluorescent in single cell was calculated.
Results: CQ, AO and blue-light exposure alone did not cause a significant decrease of cell viability in BC cells in one hour post-treatment. However, we found that AO exhibited a dose-dependent increment of cytotoxicity with blue-light exposure toward BC cells, and pretreatment of CQ for 1 hr further decreased the cell viability. In addition, we showed that this treatment caused severe cytotoxicity in human BC cell lines compared to immortalized uroepithelial cells. AO relocation was clearly monitored in fluorescent microscopy with decreased red fluorescent intensity over exposure duration. These data suggest that AO, as a photosensitizer, disrupts acidic organelles in BC cells under blue light irradiation, and treatment with CQ enhances the cytotoxic effect by increasing the AO-targeted organelles in BC cells.
Conclusion: AO has been successfully applied in photodynamic therapy (PDT) in musculoskeletal sarcomas. Because intravesical infusion is routinely performed in BC patients and with the aid of the narrow band imaging system (NBI), blue light could be easily introduced into the bladder. Our results suggest that the combination of AO-PDT with autophagy inhibitors may be an attractive therapeutic strategy against human bladder cancer.