Optimization of differential photodynamic effectiveness between normal and tumor urothelial cells using 5-aminolevulinic acid-induced protoporphyrin IX as sensitizer

Photodynamic therapy using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX is a promising tool in bladder-cancer therapy. However, little is known about the cellular mechanisms of phototoxicity. Our aim was to characterize the cellular damage and to optimize differential photodynamic effectiveness between tumor and normal urothelial cells. RT4 tumor and UROtsa normal urothelial cells were used to simulate a papillary bladder tumor in contrast to normal urothelium. Photodynamically induced damage in plasma membrane and mitochondria was monitored by flow cytometry with propidium iodide exclusion and analysis of aggregate formation of the dye JC-I. Cell morphology was investigated by phase-contrast and fluorescence microscopy following acridine orange staining. Long incubation times (3 hr) led to complete RT4 tumor cell kill accompanied by a marked fraction of damaged normal UROtsa cells. Shorter incubation intervals ( 1 hr) also resulted in complete RT4 tumor cell kill; however, most UROtsa cells retained their cell properties, including intact plasma membrane and active mitochondria as well as intact cellular morphology. Phototoxicity depends not only on cellular sensitizer accumulation but also on intracellular localization. Analysis of phototoxic mechanisms is an important step for planning combination therapy regimens with, e.g., DNA-damaging agents. Further, data indicate that differential phototoxicity in normal and tumorous urothelium can be enhanced using differences in cellular protoporphyrin IX distribution following short 5-ALA incubation times. These data are encouraging for the in vivo situation since short incubation times are a more practical approach for local photodynamic therapy of early tumor stages not only in the bladder but also, e.g., in the gastro-intestinal tract or bronchial mucosa.