Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells.

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.