Abstract
5-Aminolaevulinic acid (ALA)-induced porphyrin biosynthesis and phototoxicity in vitro was investigated in five malignant and two normal cell lines. Intracellular protoporphyrin IX (PpIX) content was quantified by extraction and fluorescence spectroscopy. Cellular PpIX content did not always correlate with cell proliferation rate as measured by the doubling times of cell lines. Cellular efflux of PpIX was also investigated. In a bladder carcinoma cell line, the observed rapid efflux was not blocked by verapamil, an inhibitor of the P-glycoprotein efflux pump. These data support the view that cellular PpIX accumulation is a dynamic process that is determined by both the efflux of PpIX from the cells and enzyme activities in the haem biosynthesis pathway. Desferrioxamine (desferal), a modulator of PpIX biosynthesis, enhanced ALA-induced cellular PpIX content significantly in all carcinoma cell lines but not in non-malignant cell lines. The enhanced PpIX cellular accumulation is attributed to inhibition of ferrochelatase activity, the enzyme responsible for the conversion of PpIX to haem. PpIX-mediated cellular photodestruction following irradiation with an argon ion laser at 514.5 nm was determined by the 'MTT assay'. There appeared to be a 'threshold' effect of cellular PpIX content; cells that synthesised less than 140 ng/mg-1 protein exhibited very little phototoxic damage, while cell lines having greater than 140 ng PpIX/mg-1 protein [corrected] exhibited a consistent phototoxic response. Among the cell lines which did undergo phototoxic damage, there was not a strict correlation between PpIX cellular content and ALA-induced phototoxicity. Desferal enhanced the PpIX content and phototoxic effect in the responsive cells. Fluorescence microscopy of the ALA-treated cells revealed marked accumulation of PpIX in mitochondria (rhodamine 123 co-staining). That the primary site of phototoxic damage is also the mitochondria was confirmed by electron micrographs of cells photosensitised with ALA-induced PpIX, which showed swelling of mitochondria within minutes after irradiation while other suborganelles appeared to be unaffected. The repair or further destruction of the mitochondria was fluence and cell-type dependent. The data from this study suggest that the basis of increased ALA-induced PpIX accumulation in tumours is a combination of various aspects of the metabolic process and pharmacokinetics and that the efficacy of photodestruction of malignancy will be determined not only by the rate of PpIX synthesis but also by specific cellular and tissue characteristics.
Highlights
Counterparts, leading to an increased accumulation of protoporphyrin IX (PpIX) in these cells
FHs738BL derived from human fetal normal bladder was grown in DMEM with 4,500 mg 1- glucose and L-glUtanUin supplemnted with 10% fetal calf serum (FCS)
In order to esimate PpIX loss even while cells were being incubated with Aminolaevulinic acid (ALA), PpIX content in the medium was measured for NBT-ll cells 1, 2, 4, 6, 8, 12 and 24h after start of ALA incubation by extraction and fluorescence spectroscopy
Summary
NBT-II (rat bladder carcinoma cell line) was grown in Eagle's minimal essential medium (MEM) with L-glutamine (Gibco, Grand Island, NY, USA) supplemented with 10%. EJ derived from human transitional cell bladder carcinoma was grown in McCoy's 5A modified medium supplemented with 5% FCS. COPRO, URO and PpIX were dissolved in methanol-water (v/v 9:1) and the ultraviolet-visible absorbance spectra were recorded on a Model 8451A Hewlett-Packard spectrophotometer. Cell suspension absorbance spectra were obtained with NBT-H and PAM cells. Cellular content of PpIX was detmined by fluorescence spectroscopy of the cell extracts and comparison with standard solutions. In order to esimate PpIX loss even while cells were being incubated with ALA, PpIX content in the medium was measured for NBT-ll cells 1, 2, 4, 6, 8, 12 and 24h after start of ALA incubation by extraction and fluorescence spectroscopy. At the end of the incubation period, the medium was diluted 3-fold in methanol-water in a clear four-sided cuvette and fluorescence was measured.
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