Evidence that photoactivated pheophorbide a causes in human cancer cells a photodynamic effect involving lipid peroxidation

Photodynamic therapy (PDT) is a treatment modality that uses a combination of aphotosensitiser and light to induce a photokilling process in the tumor tissue. Recently we reconsideredpheophorbide a (Pba), a second-generation photosensitiser that has not yet beenthoroughly investigated. Here, we report that Pba irradiated at 14 J/cm2 induces a strong PDTeffect in four tumour cell lines, with IC50 values ranging between 70 and 250 nM. Themechanism of phototoxicity has been investigated in HeLa (IC50= 150 nM) and HepG2 (IC50=95 nM) cells. In both cell lines the primary injury caused by Pba is lipid peroxidation, asindicated by a marked increase of TBARS and oxidized C11 BODIPY581/591. At high doses (>IC50), Pba arrests cell growth completely by activating apoptosis and/or necrosis, while at lowdoses (< IC50), the photosensitizer causes a temporary growth arrest. In the presence of Pbaphotodamage, the cells activate a protective mechanism against oxidative stress mediated by astrong increase of heme oxygenase-1 expression (up to 12-fold in HepG2 and 25-fold inHeLa). Moreover, considering that GSTA1-1 is a response gene to lipid peroxidation, wetreated with Pba a genetically modified HepG2 clone, in which GSTA1-1 was constitutivelysilenced by siRNA, observing a 25% increase of lipid peroxidation as compared to HepG2clone expressing GSTA1-1. These data suggest that combine treatments in which Pba is usedwith gene-silencing molecules against HO-1 and GSTA1-1 should potentiate PDT.