No abstract

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 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.

While many of the determinants of photodynamic tumor eradication have been identified, the story is not yet complete. Fluorescent probes for reactive oxygen species (ROS) are seldom specific, and the role of different ROS in apoptosis vs. autophagy are not fully delineated. Moreover, the conflicting roles of autophagy as both a death and a survival pathway remain to be explained. Most tissue-culture studies are carried out in 20% oxygen although this is far in excess of the environment of malignant cells in vivo. And while apoptotic and/or autophagic death appears to account for the lethal effects of PDT, an effect on membrane recycling has now been identified. In this report, we summarize some recent experiments designed to examine the specificity of fluorescent ROS probes. We also demonstrate the ability of hydrogen peroxide to accelerate the autophagic response to PDT in an adhering cell line, the 1c1c7 murine hepatoma. In this cell line, autophagy appears to be a pro-survival mechanism since a sub-line (KD) depleted in a critical autophagy protein (atg7) was more responsive to PDT than wild-type (WT) cells. There are clearly multiple determinants of direct tumor cell kill by PDT that depend on the PDT target, the ROS produced and phenotypic variations.