Effects of photodynamic therapy on the endocytic pathway

Abstract

In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases.

Fig. 1

Time-course of vacuole formation induced by wortmannin. (A) Control cells. (B) Cultures after a 15 min exposure to 20 nM wortmannin. (C) Cultures after a 30 min exposure to wortmannin. (D,E) Cells incubated with 20 nM wortmannin for 30 min before being washed, placed in fresh medium and incubated with fresh medium for 1 h (D) or 2 h (E). White bar in panel A = 20 μm. These images represent typical fields. Some 1c1c7 cells exhibited a slightly compressed cell morphology (e.g., panel A) but this does not appear to affect the response to wortmannin or PDT. In replicate studies, we found 100% of cells were uniformly highly vacuolated after a 30 min exposure to wortmannin, with <10% of cells showing vacuoles after 2 h in fresh medium.

Fig. 2

Wortmannin vacuoles are not autophagosomes: (A) 1c1c7 cultures were treated with 20 nM wortmannin for 30 min at 37 °C prior to being processed for electron microscopy. White bar = 2 μm. An enlarged portion of the vacuole membrane is shown in the lower image. The wortmannin-induced vacuoles lack the double-membrane structure characteristic of autophagosomes. (B–E) 1c1c7 cells that stably expressed a GFP-LC3 fusion protein were treated with nothing (B,C) or 20 nM wortmannin for 30 min (D,E) before phase (B,D) or fluorescence (C,E) microscopy. White bar in panel B = 20 μm.

Fig. 3

Plasma membrane origin of wortmannin-induced vacuoles. 1c1c7 cells were treated with TDPH for 30 s, the medium replaced, and the cells imaged immediately (A) or 30 min later (B) by phase-contrast (left column) and fluorescence (right column) microscopy. For panel C, cells were treated with TDPH for 30 s, then placed in fresh medium containing 20 nM wortmannin for 30 min prior to imaging. Panel D represents an enlargement of a portion of the cell culture shown in panel C. To examine the effect of PDT on TDPH distribution, cells were given an LD₃₀ PDT dose with NPe6, then exposed to TDPH for 30 s followed by a 30 min chase in fresh medium (Panel E). In other studies, the interval between irradiation and the TDPH treatment was expanded to 4 h (panel F) and 16 h (panel G). A significant partition of TDPH to the cytoplasm only occurred with the 16 h interval. To demonstrate effects of the 4–16 h time frame on wortmannin responsiveness, we treated cells with an LD₃₀ PDT dose followed by exposure to 20 nM wortmannin 4 h (panel H) or 16 h (panel I) later. In only the latter culture were vacuoles detected. White bars in panels A and D = 20 μm.

Fig. 4

PDT-induced lysosomal photodamage and suppression of wortmannin-induced vacuolization in cells photosensitized with NPe6. Each pair of images represents a phase-contrast and a LysoSensor Green fluorescence image, except for panel D that demonstrates the NPe6 labeling pattern. Treatments are as follows: A,B = control cultures; C,D = cultures loaded with 40 μM NPe6; E,F = Cultures treated with 20 nM wortmannin for 30 min. The remaining panels involved cultures that were photosensitized with NPe6, irradiated with different light doses, then incubated with 20 nM wortmannin for 30 min prior to microscopy. The light doses were: G,H = 15 mJ cm⁻²; I,J = 30 mJ cm⁻²; K,L = 45 mJ cm⁻²; M,N = 90 mJ cm⁻²; O,P = 180 mJ cm⁻². In all studies LSG was added for the final 10 min prior to imaging. All exposures for image acquisition were 40 ms except for panel D = 2000 ms. White bar in panel A = 20 μm.

Fig. 5

Released proteases are not responsible for NPe6 PDT suppression of wortmannin-induced vacuolization. 1c1c7 cells were either untreated (left column) or given an LD₃₀ PDT dose using NPe6 (right column). A = control culture; (B) cells imaged 60 min after an LD₃₀ PDT dose using NPe6; (C) cells imaged 30 min after treatment with 20 nM wortmannin; (D) cells given an LD₃₀ PDT dose with NPe6 30 min prior to the addition of wortmannin, then incubated for 30 min. (E) Cells imaged after a 90 min exposure to 50 μM 3-O-MeSM; (F) cells treated with NPe6 + 50 μM 3-O-MeSM for 60 min, irradiated and imaged 30 min later. (G) Cells treated with 50 μM 3-O-MeSM for 1 h prior to addition of 20 nM wortmannin and imaged 30 min later. (H) Cells treated with 50 μM 3-O-MeSM for 60 min prior to irradiation and then treated with wortmannin for 30 min. (I,J) Treatments were similar to those described in panels G and H except that 10 μM E64d was substituted for 3-O-MeSM. White bar in panel A = 20 μm.

Fig. 6

Inhibition of wortmannin-induced vacuole formation as a function of the PDT target. (A) A control culture; (B) effect of a 30 min exposure to 20 nM wortmannin; (C,D) cells given an LD₃₀ PDT dose (C) or an LD₉₀ (D) dose using NPe6 prior to a 30 min exposure to wortmannin. (E,F) Cells treated with an LD₃₀ (E) or an LD₉₀ (F) PDT dose using BPD prior to exposure to 20 nM wortmannin for 30 min. (G,H) Cells were given an LD₃₀ (G) or an LD₉₀ (H) PDT dose with mTHPC before a 30 min exposure to 20 nM wortmannin. (I,J) Cells were exposed to an LD₉₀ PDT dose with BPD (I) or mTHPC (J) and then incubated for 30 min. White bar in panel A = 50 μm.