A comparative study on the enhancement efficacy of specific and non-specific iron chelators for protoporphyrin IX production and photosensitization in HaCat cells

Abstract

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC), and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.

Keywords

aminolaevulinic acid / desferrioxamine / ethylenediaminetetraacetic acid / keratinocytes / photodynamic therapy / protoporphyrin IX

References

[1] KrammerB., PlaetzerK.. ALA and its clinical impact, from bench to bedside. Photochem Photobiol Sci, 2008, 7(3): 283-289

[2] ZhuT.C., FinlayJ.C.. The role of photodynamic therapy (PDT) physics. Med Phys, 2008, 35(7): 3 127-3 136

[3] GodalA., NilsenN.O., KlavenessJ., et al.. New derivatives of 5-aminolevulinic acid for photodynamic therapy: chemical synthesis and porphyrin production in in vitro and in vivo biological systems. J Environ Pathol Toxicol Oncol, 2006, 25(1–2): 109-126

[4] OhgariY., NakayasuY., KitajimaS., et al.. Mechanisms involved in delta-aminolevulinic acid (ALA)-induced photosensitivity of tumor cells: relation of ferrochelatase and uptake of ALA to the accumulation of protoporphyrin. Biochem Pharmacol, 2005, 71(1–2): 42-49

[5] StarW.M., van’t VeenA.J., RobinsonD.J., et al.. Topical 5-aminolevulinic acid mediated photodynamic therapy of superficial basal cell carcinoma using two light fractions with a two-hour interval: long-term follow-up. Acta Derm Venereol, 2006, 86(5): 412-417

[6] BraathenL.R., SzeimiesR.M., Basset-SeguinN., et al.. Guidelines on the use of photodynamic therapy for nonmelanoma skin cancer: an international consensus. International Society for Photodynamic Therapy in Dermatology, 2005. J Am Acad Dermatol, 2007, 56(1): 125-143

[7] SmuclerR., VlkM.. Combination of Er:YAG laser and photodynamic therapy in the treatment of nodular basal cell carcinoma. Lasers Surg Med, 2008, 40(2): 153-158

[8] MarmurE.S., SchmultsC.D., GoldbergD.J.. A review of laser and photodynamic therapy for the treatment of nonmelanoma skin cancer. Dermatol Surg, 2004, 30(2): 264-271

[9] ChenZ., SunQ., XiY., et al.. Speciation of metal-EDTA complexes by flow injection analysis with electrospray ionization mass spectrometry and ion chromatography with inductively coupled plasma mass spectrometry. J Sep Sci, 2008, 31(21): 3796-3802

[10] PyeA., CurnowA.. Direct comparison of delta-aminolevulinic acid and methyl-aminolevulinate- derived protoporphyrin IX accumulations potentiated by desferrioxamine or the novel hydroxypyridinone iron chelator CP94 in cultured human cells. Photochem Photobiol, 2007, 83(3): 766-773

[11] BlanusaM., VarnaiV.M., PiasekM., et al.. Chelators as antidotes of metal toxicity: therapeutic and experimental aspects. Curr Med Chem, 2005, 2(23): 2771-2794

[12] LiuH.F., XuS.Z., ZhangC.R.. Influence of CaNa2 EDTA on topical 5-aminolaevulinic acid photodynamic therapy. Chin Med J, 2004, 117(6): 922-926

[13] AyalaF., GrimaldiE., PerfettoB., et al.. 5-aminolaevulinic acid and photodynamic therapy reduce HSV-1 replication in HaCat cells through an apoptosis-independent mechanism. Photodermatol Photoimmunol Photomed, 2008, 24(5): 237-243

[14] TunstallR.G., BarnettA.A., SchofieldJ., et al.. Porphyrin accumulation induced by 5-aminolaevulinic acid esters in tumour cells growing in vitro and in vivo. Br J Cancer, 2002, 87(2): 246-250

[15] YoungblomJ.H., YoungblomJ.J., WilkinsonJ.. Telepre-sence confocal laser scanning microscopy. Microsc Microanal, 2001, 7(3): 241-248

[16] TrepteO., RokahrI., Andersson-EngelsS., et al.. Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope. J Microsc, 1994, 176(3): 238-244

[17] SaczkoJ., KulbackaJ., ChwiłkowskaA., et al.. The influence of photodynamic therapy on apoptosis in human melanoma cell line. Folia Histochem Cytobiol, 2005, 43(3): 129-132

[18] ChenS., ChengA.C., WangM.S., et al.. Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling. World J Gastroenterol, 2008, 14(14): 2174-2178

[19] HuS.S., ChengH.B., ZhengY.R., et al.. Effects of photodynamic therapy on the ultrastructure of glioma cells. Biomed Environ Sci, 2007, 20(4): 269-273

[20] TsaiJ.C., WuC.L., ChienH.F., et al.. Reorganization of cytoskeleton induced by 5-aminolevulinic acid-mediated photodynamic therapy and its correlation with mitochondrial dysfunction. Lasers Surg Med, 2005, 36(5): 398-408

[21] KuzelováK., GrebenováD., PluskalováM., et al.. Early apoptotic features of K562 cell death induced by 5-aminolaevulinic acid-based photodynamic therapy. J Photochem Photobiol B, 2004, 73(1–2): 67-78

[22] FedorenkoG.M., UzdenskyA.B.. Dynamics of ultrastructural changes in the isolated crayfish mechanoreceptor neuron under photodynamic impact. J Neurosci Res, 2008, 86(6): 1409-1416

[23] CurnowA., PyeA.. Biochemical manipulation via iron chelation to enhance porphyrin production from porphyrin precursors. J Environ Pathol Toxicol Oncol, 2007, 26(2): 89-103

[24] UehlingerP., BalliniJ.P., van den BerghH., et al.. On the role of iron and one of its chelating agents in the production of protoporphyrin IX generated by 5-aminolevulinic acid and its hexyl ester derivative tested on an epidermal equivalent of human skin. Photochem Photobiol, 2006, 82(4): 1069-1076

[25] JuzenasaP., JuzenieneA., MoanJ.. Deferoxamine photosensitizes cancer cells in vitro. Biochem Biophys Res Commun, 2005, 332(2): 388-391