Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line.
The photokilling activity of a porphyrin-C(60) (P-C(60)) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 microM P-C(60) incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 microM P-C(60) concentration and during long incubation time (24h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10(6)cells was found after 4h of incubation showing practically no change even after 24h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C(60), which inactivated 80% of the cells after 54 J/cm(2) of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitizer is localized, this compound could produce a biological photodamage through either a (1)O(2)-mediated photoreaction process or a free radical mechanism under low oxygen concentration. The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24h with P-C(60) and irradiated with a dose of 54 J/cm(2) showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C(60) induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.
Alvarez
M Gabriela
MG
Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro 3, 5800 Río Cuarto, Argentina.
Prucca
César
C
Milanesio
M Elisa
ME
Durantini
Edgardo N
EN
Rivarola
Viviana
V
D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D002460:Cell Line; D004273:DNA, Neoplasm / Q000378:metabolism; D003624:Darkness; D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007822:Laryngeal Neoplasms / Q000473:pathology*; D008027:Light; D017319:Photosensitizing Agents / Q000737:chemistry* / Q000494:pharmacology*; D011166:Porphyrins / Q000737:chemistry*; D013997:Time Factors
D004273:DNA, Neoplasm; D017319:Photosensitizing Agents; D011166:Porphyrins; C033886:porphyrin c; D053148:Caspase 3