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 cells in the photodynamic therapy of cancers. To elucidate the mechanism of photodamage induced lysosomal disintegration, we investigated the role of losing lysosomal proton translocation in latency loss of photosensitized 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'-dipropylthiadicarbocyanine iodide, loss of Mg-ATP dependent proton translocation and decrease in electrogenicity of the proton pump were observed 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 latency decreased. In the presence of Mg-ATP, the latency loss of incubated lysosomes reduced. Addition of n-ethylmaleimide, a potent inhibitor of lysosomal H+-ATPase, abolished the effect of Mg-ATP on lysosomal latency. It suggests a role of proton translocation in protecting lysosomal integrity. Under the same conditions, Methylene blue photosensitized lysosomes increasingly lost latency of beta-hexosaminidase and beta-galactosidase with light exposure, presumably due to the photodamage induced loss of proton pumping. In contrast, the photosensitization did not decrease lysosomal latency in the absence of Mg-ATP, implying that lysosomal integrity might not be impaired via other photodamage effects under the conditions of this study. These results indicate that lysosomal integrity can be photodestructed via the loss of proton translocation.