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

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

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica, LXIV Reunión Anual de la Sociedad Argentina de Inmunología, XLVIII Reunión Científica Anual de la Sociedad Argentina de Farmacología Experimental; 2016

SAIC, SAI, SAFE, NANOMEDAR,AACYTAL

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.