Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross‐Resistance in Vitro in Radiation‐Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin‐Mediated Photodynamic Therapy

The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.