A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy.
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The mechanism of cell death induction by dimethyl tetrahydroxyhelianthrone (DTHe), a new second-generation photodynamic sensitizer, is analysed in human leukaemic cell lines in comparison with the structurally related hypericin. DTHe has a broad range of light spectrum absorption that enables effective utilization of polychromatic light. Photosensitization of HL-60 cells with low doses of DTHe (0.65 microM DTHe and 7.2 J cm(-2) light energy) induced rapid apoptosis of > or =90% of the cells. At doses > or =2 microM, dying cells assumed morphological necrosis with perinucleolar condensation of chromatin in HL-60 and K-562 cell lines. Although nuclear fragmentation that is characteristic to apoptosis was prevented, DNA digestion to oligonucleosomes proceeded unhindered. Such incomplete apoptosis was more prevalent with the related analogue hypericin throughout most doses of photosensitization. Despite hypericin being a stronger photosensitizer, DTHe exhibited advantageous phototoxic properties to tumour cells, initiating apoptosis at concentrations about threefold lower than hypericin. Photosensitization of the cells induced dissociation of the nuclear envelope, releasing lamins into the cytosol. DTHe also differed from hypericin in effects exerted on the nuclear lamina, causing release of an 86-kDa lamin protein into the cytosol that was unique to DTHe. Within the nucleus, nuclear envelope lamin B underwent covalent polymerization, which did not affect apoptotic nuclear fragmentation at low doses of DTHe. At higher doses, polymerization may have been extensive enough to prevent nuclear collapse. Hut-78, CD4+ cells were resistant to the photodynamically activated apoptotic pathway. Beyond the tolerated levels of photodynamic damage, these cells died exclusively via necrosis. Hut-78 cells overexpress Bcl-X(L) as well as a truncated Bcl-X(L)tr isoform that could contribute to the observed resistance to apoptosis. (+info)
High efficiency of benzoporphyrin derivative in the photodynamic therapy of pigmented malignant melanoma.
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Benzoporphyrin derivative monoacid ring A (verteporfin, BPD-MA) when intravenously injected (5.5 micromol kg(-1)) to C57/BL6 mice bearing a subcutaneously transplanted B1 melanoma gave a maximal accumulation in the tumour within 1-3 h with recoveries of 1.84-1.96 micromol kg(-1). Irradiation of BPD-MA-loaded melanoma with 690-nm light from a dye laser at 3 h and 9 h post injection induced tumour necrosis and delay of tumour growth of 28 and 14 days respectively. The response of the tumour to BPD-MA photosensitization was enhanced by pretreatment with 1064-nm light from a pulse-operated Nd:YAG laser, which caused a selective breakdown of melanosomes. (+info)
Timing of illumination is essential for effective and safe photodynamic therapy: a study in the normal rat oesophagus.
(3/1300)
5-Aminolaevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. This study aimed to optimize the time of illumination after ALA administration. Six groups of eight rats received 200 mg kg(-1) ALA orally, eight rats served as controls. Illumination was performed at 1, 2, 3, 4, 6 or 12 h after ALA administration with a 1-cm cylindrical diffuser placed in a balloon catheter (laser parameters: 633 nm, 25 J radiant energy, power output 100 mW). During illumination, fluorescence measurements and light dosimetry were performed. Animals were sacrificed at 48 h (n = 4) or 28 days (n = 4) after PDT. At day 28, an oesophagogram was performed. Largest PpIX fluorescence was found at 3 h after ALA administration. In vivo fluence rate was three times higher than the calculated incident fluence rate. At 48 h after PDT, major epithelial damage was found in all animals illuminated at 2 h, whereas less epithelial damage was found at 3-6 h and none at 1 and 12 h. In animals illuminated at 4, 6 and 12 h, but not at 2 h, oesophagograms showed severe dilatations and histology showed loss of Schwann cells. These results demonstrate that the choice of time interval between ALA administration and illumination is critical for achieving epithelial damage without oesophageal functional impairment. A short interval of 2-3 h seems to be most appropriate. (+info)
Clinical spectral characterisation of colonic mucosal lesions using autofluorescence and delta aminolevulinic acid sensitisation.
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BACKGROUND AND AIMS: Laser induced fluorescence (LIF) from colonic mucosa was measured in vivo with and without delta aminolevulinic acid (ALA) in an attempt to differentiate between neoplasia and non-neoplasia in real time during colonoscopy. METHODS: Spectra from 32 adenomas, 68 normal sites, and 14 hyperplastic polyps in 41 patients were obtained with a point monitoring system. Twenty one of the patients had been given a low dose of ALA as a photosensitiser before the examination. Light of 337, 405, or 436 nm wavelength was used as excitation. Stepwise multivariate linear regression analysis was performed. RESULTS: With 337 nm excitation, 100% sensitivity and 96% specificity was obtained between normal mucosa and adenomas. Seventy seven per cent of the hyperplastic polyps were classified as non-neoplastic. When exciting with 405 and 436 nm, the possibility of distinguishing different types of tissue was considerably better in the ALA patients than in the non-ALA patients. CONCLUSIONS: The in vivo point measurements imply that a good discrimination between normal tissue and adenomatous polyps can be obtained using the LIF technique. Excitation at 337 nm and at 405 nm or 436 nm using ALA gives good results. LIF also shows potential for distinguishing adenomatous from hyperplastic polyps. The number of detection wavelengths could be reduced if chosen properly. (+info)
Ultrastructural changes in PAM cells after photodynamic treatment with delta-aminolevulinic acid-induced porphyrins or photosan.
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Photodynamic therapy (PDT) is the combination of a photosensitizing drug (Ps) with light in the presence of oxygen leading to the generation of reactive molecular species and destruction of cancer cells. In this study we compared PDT with two Ps, the hematoporphyrin derivative Photosan (Ph) and delta-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX, with respect to mitochondrial function and ultrastructural alterations. The effects of PDT were investigated in PAM 212 cells after different Ps incubation times, light doses, and post-treatment periods. Both Ps induced a light dose-dependent impairment of the mitochondrial function with the dose-response curve being steep for ALA and flat for Ph. The prolongation of the incubation time from 4 to 20 h resulted in an increased reduction of mitochondrial activity after ALA PDT but not after Ph PDT. Treatment with an irradiation dose that decreased mitochondrial activity by 50% (IC50) led to early and profound changes of mitochondrial morphology in ALA photosensitized cells, whereas photosensitization with Ph resulted in more pronounced alterations of lysosomes. We conclude that at bioequivalent sublethal PDT exposures of PAM 212 cells, ALA-induced damage is primarily restricted to mitochondria, whereas Ph-induced cytotoxicity is mediated by damage of the lysosomal system. (+info)
Oxidized low-density lipoprotein as a delivery system for photosensitizers: implications for photodynamic therapy of atherosclerosis.
(6/1300)
Photodynamic therapy is a promising new strategy in the treatment of cardiovascular diseases. Photodynamic therapy for vascular diseases may be improved by the specific delivery of photosensitizers to the atherosclerotic lesion. In this study, we studied whether oxidatively modified low-density lipoprotein (OxLDL) could be used as a specific carrier for photosensitizers, thereby using the scavenger receptor expressed on macrophages as a target. The photosensitizer aluminum phthalocyanine chloride (AlPc) was incorporated into OxLDL, and its photodynamic effects were studied. Macrophages (RAW 264.7) were incubated with various concentrations of OxLDL-AlPc for different periods. After illumination of the cells with red light, cytotoxicity was observed that was dependent on the time of illumination and incubation. Macrophages incubated with OxLDL-AlPc that were not illuminated revealed no cytotoxicity. The uptake of the OxLDL-AlPc complexes was mediated by scavenger receptors expressed on macrophages. In the presence of the polyanion polyinosinic acid, a specific ligand for scavenger receptors, no cytotoxicity could be observed. Serum incubations of the OxLDL-AlPc complexes revealed that these complexes stay intact after incubation. No redistribution of AlPc to other plasma (lipo-) proteins could be detected, and 80-90% of the AlPc remained associated with the OxLDL particle. These results indicate that OxLDL may function as a specific delivery system for photosensitizers to the scavenger receptors expressed on the macrophages in the atherosclerotic lesion, increasing the beneficial effects of photodynamic therapy for cardiovascular diseases. (+info)
Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.
(7/1300)
The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment. (+info)
Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells.
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PURPOSE: The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS. METHODS: BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody. RESULTS: Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively. CONCLUSIONS: The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes. (+info)