Photodynamic therapy (PDT) is widely used for the treatment
of skin cancer. Mechanistically, in delta-aminolevulinic acid (ALA)-mediated
PDT, the addition of ALA to cells bypasses the negative feedback control of
heme biosynthesis, leading to accumulation of photosensitizing concentrations
of protoporphyrin IX (PPIX). Subsequent activation of cellular PPIX with an
external light source (usually red light, 550-750 nm) leads to generation of
reactive oxygen species (ROS), resulting in cell death. The major side effect
of ALA-PDT treatment is the pain experienced by patients. Management of
treatment-related pain still remains a considerable challenge in patients. Further optimization of the treatment protocol
including light source, dose and duration therefore seems crucial to try and
address this issue. To improve the efficiency of ALA-PDT of skin cells
in the present study three approaches were used: (i) The conventional light
source was changed to UVA (320-400 nm) that is absorbed more efficiently by
PPIX and is 40-fold more potent in killing cultured skin cells than red light;
(ii) ALA treatment was combined with the potent iron chelators, salicylaldehyde
isonicotinoyl hydrazone (SIH), pyridoxal isonicotinoyl hydrazone (PIH) or
desferrioxamine (DFO) to further increase the accumulation of PPIX through the
depletion of iron available for ferrochelatase-mediated bioconversion of PPIX
to heme; (iii) ALA treatment was combined with UVA-activatable caged iron
chelators (CICs) that do not chelate iron unless activated by UVA. The CICs
used were aminocinnamoyl-based SIH derivatives, ‘BY123’ and ‘BY128’. Upon
activation by UVA, these CICs release the active SIH allowing for specific localised release of iron chelator in the cells.
Spontaneously immortalised HaCaT cell line and Met2
cancer line (squamous cell carcinoma) were used as cell models. Cells were pre-treated (or not) for 18 h with SIH, PIH or DFO (20-100 µM),
then subjected to ALA (0.5 mM) for 2 h and irradiated with low doses of UVA
(5-50 kJ/m2). The quantification of intracellular PPIX was carried
out by both HPLC and spectrofluorimetry after treatments of cells with ALA
alone or combined with chelators. Cell death was examined 24 h after UVA
exposure of ALA+/-chelators-treated cells by flow cytometry using Annexin
V-propidium iodide dual staining assay. Pretreatment of HaCaT cells with ALA
caused a substantial increase in the intracellular levels of PPIX which in turn
sensitized the cells to very low non-cytotoxic UVA doses. Pre-treatment with
DFO, PIH and SIH followed by ALA treatment further enhanced the PPIX level in
HaCaT cells and caused an additional level of photosensitization to low UVA
doses. Among the chelators used, SIH combined with ALA provided the most
efficient increase in PPIX and cell killing following UVA irradiation, even at
a lower SIH concentration of 20 µM. Among the CICs used, both UVA-activated
BY123 and BY128 were as effective as SIH in increasing the level of PPIX and
cell killing in ALA-treated cells following exposure to low doses of UVA. UVA-based
ALA-PDT combined with SIH (or its caged-derivatives BY123 and BY128) appears
therefore to be a promising modality for topical PDT. The high lipophilicity of
SIH (and its caged-derivatives) which facilitates skin penetration and their
potent cytotoxicity at low UVA doses should therefore allow the current
modality for topical PDT to be improved, through a reduction of the time of
irradiation and therefore the duration of pain experienced during the
treatment. The use of SIH-based CICs will be a safer
alternative to topical ALA-PDT than ‘naked’ SIH, as application of these
pro-chelators will substantially decrease
the exposure of the surrounding normal skin tissue to strong iron chelators and
their toxic side effects.
Improving the Efficiency of Aminolevulinate-Photodynamic Therapy of Skin Cells by Combining UVA Irradiation and Potent Iron Chelating Agents
Radka, T. (Author). 24 Feb 2014
Student thesis: Doctoral Thesis › PhD