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

Abstract

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.

Singlet oxygen luminescence as an in vivo photodynamic therapy dose metric: validation in normal mouse skin with topical amino-levulinic acid

“…In addition, the 1 O 2 luminescence signal was undoubtedly heterogeneous in origin in that, for example, the photosensitiser was likely concentrated in specific tissue and/or cellular compartments (e.g. mitochondrial membrane; Wilson et al, 1997b) and the oxygenation is higher in membranes than in cytosol. However, the proportion of radiative vs nonradiative 1 O 2 was consistent between experiments, since we used the same photosensitiser and application conditions in all cases.…”

Photodynamic therapy of cancer — Challenges of multidrug resistance

“…The presence of competitive binding agents can also in-°u ence the subcellular localization and/or binding of PSs. 7 As one can expect, the presence of prooxidant factors in cancer cells might scavenge PDT-induced oxygen species and therefore have a negative e®ect on PDT e±cacy. 8 Great variations in PS uptake can be found between individual cell lines, resulting in even more pronounced di®erences in photocytotoxicity.…”

“…30,31 Early studies also suggested that the intra and extracellular PpIX accumulation mediated by ALA were not subjected to the level of P-gp expression, 32 but the rapid e®lux of PpIX had also been demonstrated in DT-resistant variants. 7 Savitskiy et al showed that the speci¯c cellular protein P-gp 170 did not appear to alter the intracellular accumulation of chlorins. 33 Later Saczko et al showed that P-gp appeared to play a role in the intracellular accumulation of Photofrin but not hypericin in doxorubicin-resistant human colon cancer cell lines (LoVo cells).…”

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: Role of alkyl chain length in determining cellular uptake and localization

“…NAO has also been used to reveal properties of the binding of photosensitizers in cultured cells. Wilson et al (Wilson et al, 1997) found that NAO competitively inhibited the uptake of Photofrin® into mitochondria, indicating that some photosensitizers might bind to CL of the inner mitochondrial membrane. Pc 4, a phthalocyanine photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals Case Medical Center, was reported to be localized near CL based on fluorescence resonance energy transfer (FRET) from NAO to Pc 4 (Morris et al, 2003).…”