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. aDepartment of Pharmacology,Wayne State University School of Medicine, Detroit, MI, 48201, USA bCancer Biology Program, Wayne State University, Detroit, MI, 48201, USA cInstitute of Environmental Health Sciences, Eugene Applebaum Building, Wayne State University, Detroit, MI, 48201, USA Abbreviations BPD Benzoporphyrin derivative E64d 2S,3S-trans-(ethoxycarbonyloxirane-2-carbonyl)- L-leucine-(3-methylbutyl) amide (an inhibitor of calpain and cysteine protease activity) ER Endoplasmic reticulum LDxx PDT dose that reduces viability to xx% of controls LMP Lysosomal membrane permeabilization LSG LysoSensor Green mTHPC meso-Tetrahydroxyphenylporphine NPe6 N-Aspartyl chlorine e63-O-MeSM 3-O-Methyl sphingomyelin PCI Photochemical internalization PDT Photodynamic therapy PI Phosphatidyl inositol TDPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5- Triene