Apoptotic potential of a new amphiphilic chlorine as photosensitizer for photodynamic therapy

Photodynamic therapy (PDT) is a technique used in the treatment of various types of tumors in that light stimulates a photosensitizer (PS) to generate reactive oxygen species (ROS) that lead to cell death by apoptosis or necrosis. From a chlorine (CHL), whose nomenclature is methylfeoforbidio, hoping to make it over the amphiphilic the TRISMA group (CHL-T) has been added. Several parameters (type of PS, concentration of PS, incubation time and light dose) can trigger different apoptotic pathways. Few studies aiming the type of cell death induced by chlorines as a PS have been done. Thus, the objective of this project was to determine the cytotoxicity of this new chlorine, as well as identify the type of cell death involved in PDT as well as to elucidate the involvement of apoptotic protein p53 in this process comparing with CHL and HY. The main hypothesis of this study is that the modified chlorine has greater efficiency in the induction of apoptosis. To test this hypothesis, it was evaluated the induction of apoptosis by fluorescence microscopy and cytometry and the cytotoxicity by the MTT assay of three PSs: chlorine (CHL), the modified chlorine (CHL-T) and hypericin (HY).The chlorins accumulation was higher for the two cell lines compared with hypericin. Phototoxicity presented by the chlorin was about 10 to 20 times greater than by the chlorine source (CHL) for the two cell lines as demonstrated by the MTT assay. Tests conducted by fluorescence microscopy showed the percentage of cell death increased with increasing concentrations distinguishing higher apoptosis caused by PDT with CHL-T in HEp-2 cells and higher necrosis in HeLa cells. Analysis of apoptosis by flow cytometry also showed a superior response of CHL-T comparing to the others two studied PSs for initial apoptosis (80.35%) for concentration of 0.52 mM, incubation time of 2h and light dose 6 J/\'CM POT.2\'. However the detection of ROS by flow cytometry showed no statistically significant values for PDT in these conditions. A slight increase in the activation of p53 for CHL-T and irradiation was observed, but without being statistically significant. Thus, the results suggest that the induction of cell death in PDT does not depend on this protein and that CHL-T has excellent performance as a PS in PDT. (AU)