title: Differential cytotoxic response in keloid fibroblasts exposed to photodynamic therapy is dependent on photosensitiser precursor, fluence and location of fibroblasts within the lesion.
abstract: Treatment of keloid disease (KD) is ill-defined and remains challenging. We previously reported successful clinical application of photodynamic therapy (PDT) in KD. The aim here was to evaluate cytotoxic effect of PDT using methyl aminolevulinate (M-ALA) and 5-aminolevulinic acid (5-ALA) on keloid fibroblasts (KF) (n = 8) from different lesional sites (top, middle and margin) as compared to normal skin fibroblasts (n = 3). The effect of protoporphyrin IX (PpIX) precursors was evaluated by fluorescence emission, LDH and WST-1 assay, reactive oxygen species (ROS) generation and qRT-PCR analysis. Apoptosis/necrosis differentiation and senescence were studied by fluorometric staining with Hoechst 33258/propidium iodide and β-galactosidase activity, respectively. Three hours post incubation with 4 mM precursors of photosensitisers, PpIX accumulation was site specific and higher with M-ALA. Cytotoxicity was also site specific (higher in fibroblasts from middle of the keloid as compared to cells from other sites) and increased proportionately to fluence rates post-PDT. Additionally, cytoproliferation was significantly decreased post-PDT depending on the light energy. Fluorescence analysis revealed that M-ALA instigated higher KF cytotoxicity at lower fluence (≤20 J/cm(2)) while 5-ALA instigated higher KF cytotoxicity at higher fluence, except in cells derived from middle of the keloid. ROS-mediated cytotoxicity was light energy dependent. Senescence was not observed at higher light energies (>10 J/cm(2)). Compared to other sites, fibroblasts from the middle were more prone to cell death post 5-ALA treatment. We conclude that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site, precursor of intracellular photosensitiser and fluence. Thus, PDT may be used for site-targeted therapy of KD.
authors.affiliation: Plastic and Reconstructive Surgery Research, School of Translational Medicine, Manchester Institute of Biotechnology (MIB), The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jengmg@hotmail.com
authors.forename: Jenifer
authors.initials: J
authors.lastname: Mendoza
authors.forename: Anil
authors.initials: A
authors.lastname: Sebastian
authors.forename: Ernest
authors.initials: E
authors.lastname: Allan
authors.forename: Donald
authors.initials: D
authors.lastname: Allan
authors.forename: Parthasarathi
authors.initials: P
authors.lastname: Mandal
authors.forename: Teresa
authors.initials: T
authors.lastname: Alonso-Rasgado
authors.forename: Ardeshir
authors.initials: A
authors.lastname: Bayat
mesh_terms: D000328:Adult; D000622:Aminolevulinic Acid / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D007627:Keloid / Q000188:drug therapy* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010778:Photochemotherapy; D011524:Protoporphyrins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012867:Skin / Q000473:pathology
chemical_list: D011524:Protoporphyrins; D017382:Reactive Oxygen Species; C475457:methyl 5-aminolevulinate; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX