Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: first report of a potential combination therapy.
BACKGROUND
We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts.


OBJECTIVE
In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts.


METHODS
The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed.


RESULTS
PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05).


CONCLUSIONS
The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.
Plastic & Reconstructive Surgery Research, School of Translational Medicine, Manchester Interdisciplinary Biocentre (MIB), The University of Manchester, UK.
Anil
A
Sebastian
Ernest
E
Allan
Donald
D
Allan
James
J
Colthurst
Ardeshir
A
Bayat
D000328:Adult; D000368:Aged; D000622:Aminolevulinic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D016022:Case-Control Studies; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D024061:Collagen Type III / Q000378:metabolism; D003131:Combined Modality Therapy; D004599:Electric Stimulation Therapy; D004789:Enzyme Activation; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007627:Keloid / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008875:Middle Aged; D009336:Necrosis; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011524:Protoporphyrins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D024042:Collagen Type I; D024061:Collagen Type III; D017319:Photosensitizing Agents; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; C475457:methyl 5-aminolevulinate; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX; D007770:L-Lactate Dehydrogenase; C505407:CASP3 protein, human; D053148:Caspase 3; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2