IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Induction of an apoptotic response after irradiation of colon carcinoma cells loaded with a lipophilic Zn(II) phthalocyanine incorporated into poloxamine micelles
Autor/es:
CHIARANTE N.A; JULIETA MARINO; GARCIA VIOR M.C.; AWRUCH J; LEONOR ROGUIN
Lugar:
Aveiro
Reunión:
Congreso; 16th Congress of the European Society for Photobiology; 2015
Institución organizadora:
European Society for Photobiology
Resumen:
Phthalocyanines (Pcs) have been found to be useful photosensitizers for photodynamic therapy (PDT). The tendency of Pcs to form aggregates has promoted the use of nanocarriers that improve their solubility. The aim of this work was to study the phototoxic action of 2,9(10),16(17),23(24)-tetrakis[(2?dimethylamino)ethylsulfanyl] phthalocyaninatozinc(II) (Pc9) encapsulated into poloxamine polymeric micelles (T1107) in a murine colon carcinoma cell line (CT26). Pc9-T1107 was found to be cytotoxic after irradiation at 2.8 J.cm-2, being the IC50 value of 11 ± 1 nM. When CT26 cells were incubated with Pc9 micellar preparation, the maximum cellular uptake was found after 4 hours. Studies of the subcellular localization of Pc9-T1107 by confocal microscopy with fluorescent dyes for specific organelles revealed that Pc9 mainly localized in lysosomal vesicles. After cell irradiation, a reduction of the fluorescence emission of the lysosomal probe acridine orange was evident, suggesting the permeabilization of the lysosomal membrane. It was also demonstrated that after light exposure of Pc9-loaded cells, the production of reactive oxygen species (ROS) increased in a concentration-dependent manner. In addition, a lower cytotoxic effect was obtained when cells were pretreated with the antioxidant TROLOX. We also showed a significant increase in the cytosolic amount of the lysosomal enzyme cathepsin D in a time-dependent manner after PDT. The inhibitory activity induced by Pc9-T1107 in cell growth was partially reverted in the presence of pepstatin A, a cathepsin D inhibitor. In order to evaluate mitochondrial membrane damage, we used the fluorescent probe DiOC6. By flow citometry we determined that the percentage of cells with depolarized mitochondrial membrane was increased from 1,1 ± 0,4% to 58,4 ± 0,4% (p< 0,0001) after irradiation of Pc9-treated cells. Western blot analyses (WB) of Bcl-2 family protein expression revealed a reduction of Bid, Bcl-2 and Bcl-XL levels (p< 0,005). Morphological analyses of Pc9 irradiated cells showed an increase of the number of apoptotic nuclei stained with Höechst. In addition, it was detected a decrease in procaspase-3 level by WB and a significant increment of caspase-3, -8 and -9 enzymatic activities. In conclusion, our results showed that Pc9 is a potent cytotoxic agent for PDT, being cell death mediated by the generation of ROS, the release of lysosomal proteases, depolarization of mitochondrial membrane, changes in the expression level of Bcl-2 proteins and caspases activation. These results encourage us to assess Pc9 in vivo phototoxic efficacy.