Cytocidal effects of acridine orange evoked by blue light on human bladder cancer cells

Objectives: Bladder cancer is a major malignancy in urology, and has a high rate of recurrence. Intravesical BCG instillation therapy, despite many adverse effects, is often used to prevent recurrence. Acridine orange (AO), a membrane permeable weakly basic dye, has cytocidal effects when the blue light is illuminated. For this reason, AO was used for the photodynamic therapy (PDT) of musculoskeletal sarcoma. In order to test if AO-PDT can become an effective treatment for bladder cancer, we wanted to determine the cytocidal effects of AO evoked by blue light on the human bladder cancer cell line T-24. Methods: T-24 cells loaded with AO were observed during blue light illumination under a fluorescent microscope, and vesicle disruption and cell death were assessed by trypan blue staining. To determine the cytocidal mechanism, we determined the change of vesicle disruption and cell death using bafilomycin A1, a vacuolar H+-ATPase inhibitor, and antioxidants, and intracellular distribution of cathepsin D, a lysosomal protease, was also investigated. Finally, the efficacy of the evoked AO on normal rat bladder cells was compared to that on T-24 cells. Results: Cells showed the disruption of AO-containing vesicles as a green flash during blue light illumination and died withn 30 minutes. Bafilomycin A1 and antioxidants inhibited vesicle disruption and cell death. Intravesicular cathepsin D increased in the cytoplasm following illumination. Normal bladder cells showed slight vesicular AO accumulation but did not die in the same condition that almost all T-24 cells died. Conclusions: AO evoked by blue light has strong cytotoxicity for the T-24 bladder cancer cells and was caused by the lipid peroxidation-mediated disruption of vesicles, where AO is accumulated by vacuolar H+-ATPase, followed by the subsequent release of cathepsin D into the cytoplasm. PDT with AO may be worthwhile for the treatment of bladder cancer.