Signaling From Lysosomes Enhances Mitochondria-Mediated Photodynamic Therapy In Cancer Cells

Abstract

In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4 derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.

Fig. 1. Pc 4-PDT-mediated mitochondrial events

Pc 4-PDT induces rapid mitochondrial ROS formation assessed by DCF fluorescence. Green fluorescence is DCF fluorescence, and red fluorescence is TMRM fluorescence. Orange-yellow fluorescence indicates polarized mitochondria, who have increased ROS formation. Green fluorescence indicates depolarized mitochondria with increased ROS production. Inner membrane permeabilization is monitored with calcein fluorescence. Punctate mitochondrial calcein fluorescence changes to diffuse calcein fluorescence as a response of the leakage of calcein from mitochondria due to mitochondrial inner membrane permeabilization. This event is followed by mitochondrial depolarization and swelling, as assessed by MitoTracker Red.

Fig. 2

Photosensitizer structure determines sub-cellular localization and killing efficacy

Fig. 3. Bafilomycin enhances Pc 4-PDT-mediated cell death

Left Panel: A431 cells were incubated with Pc 4 (50 nM) for 18 h followed by incubation with bafilomycin (50 nM) for 1 h. Subsequently, cells were exposed to 670-nm light (198 mJ/cm²). Viability was assessed by PI exclusion using fluorometry. Right panel: Cells were treated and irradiated as in the left panel. Subsequently, cells were trypsinized and plated on Petri dishes. After 14 days, colonies were stained with crystal violet. Results are expressed as mean ± S.E.M. from at least 3 independent experiments for each treatment group.

Fig. 4. Bafilomycin enhances Pc 4-PDT-mediated mitochondrial depolarization

A431 cells were cultured on glass-bottomed Petri dishes, incubated with Pc 4 (50 nM) for 18 h and subsequently incubated with bafilomycin (50 nM) for 1 h before light exposure. Cells were loaded with 100 nM TMRM and Petri dishes were placed on a microscope stage at 37°C. After collecting a baseline image, cells were irradiated at 198 mJ/cm² and subsequently images were collected over time.

Fig. 5. Bafilomycin enhances Pc 4-PDT-mediated caspase-3 activation

A431 cells were incubated with Pc 4 (50 nM) for 18 h and subsequently incubated with bafilomycin (50 nM) for 1 h before light exposure. Cells were irradiated at 198 mJ/cm² at 37°C. After 2 h of post-PDT, whole cell lysates were prepared and caspase-3 activity was measured as described in Methods. Results are expressed as mean ± S.E.M. from three independent experiments.