Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells.

Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤ 1 µM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥ 10 µM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 µM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions.

Effects of endosomal photodamage on membrane recycling and endocytosis.

The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically substituted porphine or a phthalocyanine also prevents wortmannin-induced endosomal swelling, even when the photodynamic therapy (PDT) dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers.