Abstract
Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.
Highlights
Heme biosynthesis occurs partially inside and partially outside of the mitochondria, starting in the cytosol with the condensationNote: Supplementary data for this article are available at Cancer Research Online.I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0510 of two molecules of 5-aminolevulinic acid to porphobilinogen followed by the condensation of four porphobilinogen molecules to uroporphyrinogen III by porphobilinogen deaminase
We found that HAL-based photodynamic therapy (PDT) targeted peripheral benzodiazepine receptor (PBR) and led to an apoptosis-inducing factor (AIF)–dependent pathway of apoptosis in the Reh cells
The intracellular colocalization pattern of HAL-mediated endogenous protoporphyrin IX (PpIX) and a mitochondrial probe, MitoTracker, in the Reh cells was studied by fluorescence microscopy (Fig. 1A)
Summary
Heme biosynthesis occurs partially inside and partially outside of the mitochondria, starting in the cytosol with the condensationNote: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0510 of two molecules of 5-aminolevulinic acid to porphobilinogen followed by the condensation of four porphobilinogen molecules to uroporphyrinogen III by porphobilinogen deaminase. By adding exogenous 5-aminolevulinic acid or its derivatives, the naturally occurring porphyrins, PpIX in particular, may selectively accumulate in some tumors because of a high activity of porphobilinogen deaminase with a concomitant low activity of ferrochelatase [1, 2]. Such selectivity has been exploited in photodynamic therapy (PDT), a modality that involves systemic or topical administration of a tumor-localizing photosensitizer (or prodrug) and its subsequent activation by visible light to result primarily in singlet oxygen– induced photodamage to the tumor [1,2,3]
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