Induction of apoptosis by hexaminolevulinate-mediated photodynamic therapy in human colon carcinoma cell line 320DM.

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.