AbstractNon-melanoma skin cancers are the most common types of cancer in Caucasian populations, with solar UV light as the predominant risk factor. Metastatic squamous cell carcinoma is the most lethal type of skin cancer, with up to 72% of lesions developing from neoplastic actinic keratosis (AK) lesions. Fundamentally, UVA has been shown to promote labile iron release from ferritin and mitochondria, generating free radicals through the redox cycling potential of labile iron. 5-Aminolevulinic acid-based photodynamic therapy (ALA-PDT) is a treatment modality indicated in the management of AK. 5-Aminolevulinic acid (ALA) is a precursor of heme, which when exogenously added results in the selective accumulation of photoactivable protoporphyrin IX (PpIX) in cancer cells. The conversion of PpIX to heme relies on the insertion of iron in the tetrapyrrole core by ferrochelatase, dependent upon iron availability. Despite satisfactory safety profiles, efficiency and cosmetic outcomes, widespread adoption of ALA-PDT has been limited by the burning, stinging, and prickling sensation experienced by the patients upon irradiation, with up to 54% of patients discontinuing the treatment. The pain occurs in relation to reactive oxygen species-mediated covalent modification of TRPA1 and TRPV1 receptor channels present on peripheral nerve endings. In this way, the photochemical reactions responsible for treatment efficiency sustain the covalent alterations of TRPA1 and TRPV1 receptor channels, colloquially described by Morton as “No pain, no gain”.
However, we hypothesized that the redox potential of labile iron could be harnessed with the development of a new treatment modality, referred to as “UVA-based light fractionation of ALA-PDT”. Light fractionation (ALA-fPDT) is an alternative approach ALA-PDT, consisting of two split light doses separated by a single dark interval, promoting higher clearance rate, based on dark interval PpIX re-synthesis, oxygen supply, and PpIX subcellular location. This study evaluated the impact of a series of sub-lethal dose combinations of UVA, designed to promote labile iron release and exacerbate the intracellular oxidative damage following a second UVA light dose. UVA light matches the Soret peak of the PpIX absorption spectrum, exhibiting high quantum yield and reasonable tissue penetration, with up to 50% of UVA light reaching the deepest layers of the epidermis. This work aimed to define the optimal treatment parameters, referring to maximal photo-damage and photo–killing following the lowest UVA light dose, using the immortalized HaCaT keratinocytes as a skin cancer cell model. The biological impact would then differ throughout the depth of cutaneous tissue, which upon design would issue maximal damage to superficial layers and negligible damage to deeper epidermal layers, where nerve endings are located.
This work first demonstrated the optimal parameters for ALA-PDT following a single UVA light dose, defining the intricate relationship of PpIX and cytosolic labile iron levels, via PpIX extraction or cellular porphyrin and calcein fluorescence measurements with a spectrofluorometer. The short-term and long-term biological effect of UVA light doses in ALA-treated cells were investigated using MTT and colony forming ability assays. This determined the treatment parameters upon which comparative studies were later undertaken. The split irradiation scheme consisted of a double UVA dose of 1 kJ/m² and 2.5 kJ/m², as well as the combination of UVA doses of 1 kJ/m² and 2.5 kJ/m². Photokilling of ALA-treated cells was not significantly different throughout a 15-, 30-, 45- and 60 min dark interval. However, the short- and long-term biological impacts of UVA light fractionation were significantly different to a single dose, with the colony forming ability of a double UVA dose of 1 kJ/m² ca 3-fold lower than a single 2.5 kJ/m² dose. This could be beneficial to patient compliance, as treatment would induce negligible pain and adverse effects and significant tumor regression. In both conditions, UVA-mediated cytosolic labile iron release and membrane damage was observed, as monitored by the level of fluorescent calcein dye retention. This effect was confirmed with similar UVA doses at dark intervals of 30 and 60 min, confirming the notion that PpIX levels are the sole critical factor here.
To evaluate the role of labile iron in modulating the efficiency of topical ALA-PDT with UVA-based light fractionation, a series of experiments were undertaken that involved either iron loading of HaCaT cells with iron citrate and hemin prior to ALA-treatment or iron chelation using desferrioxamine mesylate (DFO), salicylaldehyde isonicotinoyl hydrazine (SIH) and deferiprone (DFP) iron chelators during a 60-120 min dark interval, between the two UVA irradiations in ALA-treated cells. Among all the conditions used, only DFO treatment over a 120 min dark interval displayed significant cellular viability reduction in ALA-treated and UVA irradiated cells when compared to DFO-untreated counterparts.
Additionally, our light fractionation scheme was applied to natural sunlight, investigating the biological impact of the solar component of UVA and visible light components together or visible light alone, using the organic UVB-specific filter octocrylene and the commercially available UVB+UVA filter Anthelios™ SPF 50 +, respectively. The synergistic effect of the solar emission bandwidth was significantly higher in the photokilling of ALA-treated cells, and proved to be beneficial in shortening irradiation periods, without compromising treatment efficiency. The latter provided the first proof of concept study for a novel UVA-based daylight PDT with light fractionation involving two short pulses of daylight exposure with a 60-120 min dark interval.
Finally, this study proceeded with the investigation of a series of ALA and 3-hydroxy-4-pyridinone (HPO) iron chelator combination pro-drugs with improved lipophilicity compared to those published so far. Among the 16 prodrugs studied, compound III.B, displayed the desired characteristics notably the lack of dark toxicity, and efficient photokilling with UVA comparable to equimolar concentrations of ALA and the HPO iron chelator, DFP. This compound also exhibited higher cytosolic labile iron depletion and subsequently, higher porphyrin accumulation, when compared to equimolar concentrations of ALA and DFP.
The UVA-based light fractionation of ALA-PDT could be indicated as a treatment modality in the management of superficial AK, adapted for both indoor and outdoor purposes. The treatment would promote tumor regression, while minimizing treatment-induced pain and other adverse skin reactions. Future work must encompass the application of treatment parameter to isogenic skin cancer cells at different stages of carcinogenesis, while evaluating the change in labile iron dynamics and their impact upon irradiation in the cytosol, mitochondria, and nucleus. Additionally, the treatment parameters should be investigated in the context of daylight light fractionation of ALA-PDT, before moving to pre-clinical and clinical studies.
|Date of Award||17 Nov 2021|
|Supervisor||Charareh Pourzand (Supervisor) & Ian Eggleston (Supervisor)|