Photodynamic therapy with a lipophilic Zn(II) phthalocyanine leads to apoptotic cell death through lysosomal permeabilization, ER stress and caspases activation

CHIARANTE N.A; REY O; GARCIA VIOR M.C.; ROGUIN, L. P; MARINO, V. J; AWRUCH J

BUENOS AIRES

Congreso; III International Congress in Translational Medicine; 2016

Facultad de Farmacia y Bioquímica, Facultad de Medicina, Universidad de Buenos Aires

Phthalocyanines (Pcs) have been found to be useful photosensitizers for photodynamic therapy (PDT). In a previous work, we demonstrated the cytotoxic action of a lipophilic Zn(II) phthalocyanine (Pc9) encapsulated into poloxamine polymeric micelles (T1107) in CT26 cells derived from a murine colon carcinoma (IC50=11±1 nM). In order to elucidate the mechanism of phototoxic action, we explored both the subcellular localization of Pc9 as well as the possible induction of an apoptotic response. After staining Pc9-loaded cells with fluorescent dyes for specific organelles, Pc9 was detected in lysosomes and endoplasmic reticulum (ER), but not in mitochondria. A significant increase in the cytosolic levels of the lysosomal enzyme cathepsine D was observed after irradiation of Pc9-treated cells, suggesting the permeabilization of the lysosomal membrane. In addition, an enhancement of cell viability was obtained after incubating Pc9-exposed cells with lysosomal proteases inhibitors, such as Pepstatin (cathepsin D inhibitor), CA-074 Me (cathepsin B inhibitor) or aprotinin (serine proteases inhibitor). A time-dependent increment of the cytosolic amounts of calcium together with the regulation of the expression levels of ER proteins suggested the involvement of ER stress. Consistently, a lower cytotoxic effect was obtained when cells were pretreated with the calcium chelator BAPTA. An apoptotic response was then demonstrated by the increase of caspase-3, -8 and -9 activities, the decrease of anti-apoptotic Bcl-2 family protein levels, the cleavage of PARP and the visualization of apoptotic nuclei in cells stained with Hoechst 33258. Finally, PDT also provoked cell cycle arrest in the S and G2/M phases. In conclusion, our results showed that Pc9 behaves in vitro as a potent photosensitizer capable of inducing an apoptotic cell death through lysosomal permeabilization and ER stress, both contributing to the activation of a mitochondrial caspase cascade.