Inhibition of endocytic processes by photodynamic therapy

Abstract

Background and objectives: Recent studies have demonstrated an effect of photodamage on the endocytic pathway involved in recycling of membrane components. Using a series of agents with known sub-cellular targets, we explored the determinants of photodynamic inhibition of endocytic processes in three cell lines: A murine leukemia, a murine hepatoma, and a non-malignant epithelial cell line of human origin.

Study design/materials and methods: The PI-3 kinase antagonist wortmannin blocks endosomal processing pathway dependent on this enzyme, providing an indication of the "flux" of endocytosis. Microscopic observations were used to assess the effect of photodamage on this pathway. Photosensitizing agents specific for mitochondrial, endoplasmic reticulum (ER), lysosomal, and endosomal photodamage were employed.

Results: Sub-lethal photodamage directed against endosomes or lysosomes interrupted early steps in this endocytic process in the hepatoma cell line. A mechanism for these effects is proposed. Mitochondrial photodamage could interrupt endocytosis, but at levels that also induced apoptosis. ER photodamage did not affect endocytosis even at lethal levels. Somewhat similar results were obtained with other cell lines, but there were sufficient differences to indicate that the cell phenotype is, in part, a determinant of the endocytic response to PDT.

Conclusions: PDT is therefore seen to have an effect on endocytic processes. Further work will be needed to delineate the role of these endocytic effects in the array of responses to photodynamic therapy.

Fig. 1

Wortmannin-induced vacuole formation in 1c1c7 hepatoma cells. A, controls; B–F, images acquired at various times after addition of 20 nM wortmannin. B, 1 hr; C, 2 hr; D, 3 hr, E, 6 hr; F, 8hr.

Fig. 2

Effect of prior photodamage on wortmannin-induced vacuole formation in 1c1c7 hepatoma, P388 leukemia and MCF10A epithelial cells. From top: control = untreated controls; Wm = 90 min after addition of 20 nM wortmannin; NPe6, BPD & mTHPC = cells given an LD₂₀ PDT dose with the specified photosensitizer, then treated with wortmannin for 90 min.

Fig. 3

Phase contrast and fluorescence microscopy showing localization of AlPcS2a in 1c1c7 hepatoma cells. A,B = after a 16 hr incubation with 5 μM AlPcS2a, then washed for 1 hr in fresh medium. C,D = Cells treated as indicated above, then given an LD₂₀ light dose and treated with 20 nM wortmannin for 90 min. E,F = cells labeled with AlPcS2a, then exposed to 20 nM wortmannin for 90 min in the dark.

Fig. 4

Phase-contrast and fluorescence images of 1c1c7 cells showing TDPH labeling patterns. A,E: cell surface labeling by a 30-sec pulse of TDPH. B,F: partition of TDPH into the cell interior when the 30-sec pulse is followed by a 30 min incubation in fresh medium. C,G: effect of the presence of 20 nM wortmannin during the 30 min ‘chase’. D,H: effect of an LD₂₀ PDT dose to cells photosensitized with AlPcS2a showing inhibition of the subsequent cytoplasmic migration of a TDPH pulse.

Fig. 5

Proposed model for effects of endosomal photodamage on endocytosis in 1c1c7 cells. A = A normal membrane recycling pattern. B = Formation of swollen late endosomes when further processing of late endosomes is blocked by wortmannin. C = AlPcS2a labeling pattern. D = Proposed effect of an LD₂₀ light dose on photosensitized endosomes resulting in imperviousness to ‘swollen endosome’ formation. E= Endosomal fragmentation and release of encapsulated AlPcS2a into the cytoplasm after a greater light dose. F = postulated effect of low-dose PDT on cells photosensitized with NPe6. This is based on studies indicating that AlPcS2a targets both lysosomes and late endosomes [14]. Accumulation of photosensitizer is indicated by shading of the organelles;