LOSS OF LYSOSOMAL INTEGRITY CAUSED BY THE DECREASE OF PROTON TRANSLOCATION IN METHYLENE BLUE-MEDIATED PHOTOSENSITIZATION

Loss of lysosomal integrity is a critical event for killing tumor cell s in the photodynamic therapy of cancers. To elucidate the mechanism o f photodamage induced lysosomal disintegration, we investigated the ro le of losing lysosomal proton translocation in latency loss of photose nsitized lysosomes. Isolated rat liver lysosomes were light exposed in the presence of Methylene blue. Through monitoring lysosomal Delta pH with Acridine orange and measuring its membrane potential with 3,3'-d ipropylthiadicarbocyanine iodide, loss of Mg-ATP dependent proton tran slocation and decrease in electrogenicity of the proton pump were obse rved after lysosomes were photosensitized. When normal lysosomes were incubated for 60 min in K+ contained medium, percentage free activity of lysosomal enzyme beta-galactosidase increased, i.e. lysosomal laten cy decreased. In the presence of Mg-ATP, the latency loss of incubated lysosomes reduced. Addition of n-ethylmaleimide, a potent inhibitor o f lysosomal H+-ATPase, abolished the effect of Mg-ATP on lysosomal lat ency. It suggests a role of proton translocation in protecting lysosom al integrity. Under the same conditions, Methylene blue photosensitize d lysosomes increasingly lost latency of beta-hexosaminidase and beta- galactosidase with light exposure, presumably due to the photodamage i nduced loss of proton pumping. In contrast, the photosensitization did not decrease lysosomal latency in the absence of Mg-ATP, implying tha t lysosomal integrity might not be impaired via other photodamage effe cts under the conditions of this study. These results indicate that ly sosomal integrity can be photodestructed via the loss of proton transl ocation.