Hypocrellin B-Acetate-mediated photodynamic activation drives HeLe cells to apoptotic death. Rome ECDO 2012,

Photosensitizing molecules (PSs) are used to activate photodynamic process as an approach for the diagnosis and treatment of several non-oncological and oncological diseases. The PS excitation with light of a suitable wavelength induces preferential dissipation of the absorbed energy through the activation of photochemical processes rather than by fluorescence emission, with the production of reactive chemical species (free radicals, singlet-oxygen, or other oxidizing oxygen species). The very short life-time of these new active species allows them to react only with the closely surrounding molecules, making the PS intracellular localization crucial for the generation of photo-cytotoxic effects, leading to cell death and subsequent destroying the affected tissue. The addition of chemical groups to PS structure increases its ability to enter the cells and makes it more selective in localization in sensitive subcellular sites. In this work, we used Hypocrellin B Acetate (HypB-Ac), a fluorescent perilene-quinonoid pigment modified through the addiction of acetate groups. HeLa cells grown on coverslip were treated for 1 h with HypB-Ac in the 7.5 x 10-7 M to 2.5 x 10-5 M concentration range, irradiated at 480±15 nm (light emitting diode LED, 20 mW/cm2, total dose 1.6 J/cm2) and submitted to 24 h of recovery in PS free medium. Previous experiments with in vivo imaging techniques, allowed to observe a lysosomial preferenzial localization of HypB-Ac, not dependent on its concentration (Croce et al., 2011). In this work the ability of HypB-Ac to induce intracellular phototoxic effects was investigated by means of flow cytometry, electronic microscopy and immunocytochemical techniques. We found that HypB-Ac at the highest concentration induced massive cell necrosis, while the ability to activate sub-cellular damages and apoptotic pathways was detected at the lower doses. In the latter case, the analyses with Annexin V-FITC/Propidium Iodide showed a cell fraction with apoptotic features. This result was supported by the study of ultra-structural morphology, that highlighted apoptotic blebbing cells with condensed chromatine. Immunocytochemical techniques demonstrated the damage of several organelles (mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and cytoskeletal apparatus), driving the cells to apoptotic cell death. However, the flow cytometric analysis of DNA amount revealed that there were no sub-G1 apoptotic peak, indicating that multisite, subcellular damages are detected before the nuclei fragmentation. The targeting of several subcellular sensitive sites, likely inducing early multiorganelle photodamage, has been thus demonstrated for HypB-Ac, leading to apoptotic cell death and providing a suitable model for the study of the induction of different apoptotic pathways. References: Croce AC, Fasani E, Bottone MG, De Simone U, Santin G, Pellicciari C, Bottiroli G. Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization. Photochem Photobiol Sci. 2011;10(11):1783-90.