The mechanisms of Photodynamic therapy (PDT) include singlet oxygen and reactive oxygen species (ROS) production
that damage tumor cells and vasculature. The resulting effect is a balance between photo-oxidations via primary or
secondary ROS and scavenging activity. Sensitizers distribute in the extra-cellular space before and during cell
sensitization, suggesting that PDT could act directly on cell structures and on extra-cellular compartments, including
sera. In this paper we endeavored to determine whether the application of PDT to culture media could have an effect on
cell survival. Culture media (RPMI supplemented with Fetal Calf Serum (FCS)) was incubated with Rose Bengal (RB)
and irradiated before being added to cells for various times of contact, as a replacement for untreated media. Treatedmedia
reduced cell survival by up to 40% after 30 min of contact for 10 μg/mL of RB and 20 J/cm2. This effect was RB
or light dose-dependent. The survival reduction observed when adding treated-media was more pronounced when cell
doubling time was shorter. Analysis of ROS or peroxide production in treated-media revealed a long-lasting oxidizing
activity. Our findings support the hypothesis of a ROS or peroxide-mediated, PDT-induced, delayed cell toxicity
Type 2 photoreactions are singlet oxygen (1O2) mediated. This singlet oxygen will de activate by reacting with a number
of targets and produce ROS and peroxides that will in turn give Reactive Oxygen Species (ROS). In order to verify
whether sera had the same capability to do so we provoked a photoreaction using Rose Bengal (RB) added to sera of 53
healthy donors and at the end of light delivery 2' -7' dichlorofluorescin (DCFH) had been added. In order to avoid any
optical artefact we analysed the influence of haemolysis on the fluorescent 2' -7' dichlorofluorescin (DCF) induced by
oxidising species. Reactivity to singlet oxygen was slightly different between men and women, varied slightly with age
and platelet number. It was remarkably stable over a large period of time and thus represents a characteristic of each
serum. Studies are in progress to verify whether ROS dependent diseases would impair this reactivity to singlet oxygen.
In this study the effect of PDT on the antitumoral specific immunologic response was evaluated. We compared the specific cytolytic activity (CLA) by a chromium release assay of primed mouse spleen T lymphocytes sensitized against syngeneic mastocytoma P511 cells. P511 cells, or lymphocytes, or both cells were treated or not with photofrin and/or light (514 nm). Photofrin II alone (1 (mu) g/ml, 2 hours) reduced CLA 59% when P511 were treated. Photofrin II (1 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 35%. Photofrin II alone (0.5 (mu) g/ml, 2 hours) reduced CLA 8% when only lymphocytes were treated. And Photofrin II (0.5 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 45%. When both cells were treated with Photofrin II alone or followed by light (25 Joules/sq cm) the CLA was also reduced respectively 19, 41%.
Analysis of sensitizer kinetics is essential for the performance of light irradiation when tumor concentration and tumor-to-normal tissue ratios are optimal. In our study, nude mice were grafted with human adenocarcinoma 15 days before m-THPC IP injection. Fluorescence was recorded through an optic fiber spectrofluorometer at 650, the most intense, and 714 nm, and intensity was proportional to the injected dose. In tumor, skin and muscle, maximum fluorescence was obtained with 1.6 mg/kg 72 h after injection (44 counts per second). Tumor- to-skin and -muscle ratios obtained by HPLC analysis and spectrofluorometric measurements decreased between 12 and 72 h (15 to 1.5), indicating that tumor selectivity decreases with time.
We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.
A new photosensitizer (PS), meso-tetrahydroxyphenyl-chlorin(m-THPC), has been clinically evaluated for photodynamic therapy (PDT) of early squamous cell carcinomas located in the upper aerodigestive tract, the oesophagus and the tracheobronchial tree. The injected doses ranged between 0.1 - 0.3 mg/kg m-THPC and the wavelength of the excitation light was either at 514 nm or 652 nm. The evaluation of the m-THPC induced phototoxicity was carried out on healthy mucosae of the bronchi, the oral cavity and the skone cell population to the other. Appearance of aneuploid populations after PDT suggests that destruction of sensitive cell populations allows the growth of initially non FCM detectable aneuploid clones. MDA assay could thus be a good prognostic tool although larger series of patients are needed. 115
We designed a laser spectrofluorimeter to determine the optimal delay between drug injection and laser irradiation in which both excitation and fluorescence emission are transmitted through a single optic fiber. Preclinical studies were performed in nude mice after ALA administration. The maximum fluorescence (632 nm) in HT 29 tumors was obtained after 90 min (excitation 488 nm, 20 mW, integration time 3 sec, ALA 250 mg/kg), after 180 min in liver. Iron chelators as EDTA (10 or 20 mg/kg) or desferrioxamine (20 or 40 mg/kg) increased the delay for reaching maximum fluorescence to 2 hours as well as fluorescence intensity in tumors but decreased intensity in liver. Desferrioxamine induced the appearance of protoporphyrin IX in the bladder or kidneys, normally absent in urines. These results indicate that drug distribution varies with tissues and also adjuvant therapies that are often administered together with cancer drugs.
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