High efficiency of 5-aminolevulinate-photodynamic treatment using UVA irradiation
Darius P. Buchczyk,
Lars-Oliver Klotz,
Kerstin Lang1,,
Clemens Fritsch1, and
Helmut Sies2,
1 Institut für Physiologische Chemie I and Hautklinik der Universität, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany
2 D.P.Buchczyk and L.-O.Klotz contributed equally to this work
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Abstract
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Photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. One concept of delivering the employed photosensitizer directly to target cells is to stimulate cellular synthesis of sensitizers such as porphyrins. ALA (5-aminolevulinate) is applied as a precursor of porphyrins which then serve as endogenous photosensitizers. Upon irradiation, reactive oxygen species, predominantly singlet oxygen, are generated, leading to cell death. ALAPDT using red light (550750 nm) is known to lead to the activation of stress kinases, such as c-Jun-N-terminal kinase and p38. These kinases are also activated by UVA (320400 nm), whose biological effects are mediated in part by singlet oxygen. In the present study, the efficiency of a combination of both treatment strategies, ALAPDT and UVA, in cytotoxicity and activation of stress kinases was investigated taking human skin fibroblasts as a model. Compared with the commonly used ALAPDT with red light (LD50 = 13.5 J/cm2), UVAALAPDT was 40-fold more potent in killing cultured human skin fibroblasts (LD50 = 0.35 J/cm2) and still 10-fold more potent than ALAPDT with green light (LD50 = 4.5 J/cm2). Its toxicity relied on the formation of singlet oxygen, as was shown employing modulators of singlet oxygen lifetime. In line with these data, strong activation of the stress kinase p38 was obtained in ALA-pretreated cells irradiated with UVA at doses two orders of magnitude lower than necessary for a comparable activation of p38 by UVA in control cells. Taken together, these data suggest UVAALAPDT as a potentially interesting new approach in the photodynamic treatment of skin diseases.
Abbreviations: ALA, 5-aminolevulinic acid; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; HBSS, Hank's balanced salt solution; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PDT, photodynamic therapy.
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Introduction
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Photodynamic therapy (PDT) is being used clinically for the treatment of cancers and various skin diseases (1). PDT relies on the application of photosensitizers followed by irradiation. One concept for the delivery of photosensitizers to target cells is the stimulation of cellular synthesis of appropriate compounds such as porphyrins. In 5-aminolevulinic acid (ALA)PDT, ALA, the product of the rate-limiting step in porphyrin biosynthesis, is applied topically to increase intracellular concentrations of protoporphyrin IX, which acts as a photosensitizer. Upon irradiation of cells, reactive oxygen species, predominantly singlet oxygen, are generated in PDT, leading to cell death (2). The accumulation of protoporphyrin IX in cancerous cells is more pronounced than in healthy tissue for several reasons. Higher metabolic activity, a faster uptake of ALA and a lower specific activity of ferrochelatase lead to a higher concentration of protoporphyrin IX and result in a higher sensitivity to irradiation (3).
The wavelengths commonly used for irradiation correspond to the maxima of the absorbance spectrum of protoporphyrin IX (Figure 1
). Red light is used most frequently for ALAPDT since light of longer wavelength allows for better penetration of the skin. Yet therapy with red light is associated with side effects such as burning pain and heating due to the thermal irradiation. ALAPDT using green light is already employed in the therapy of patients with multiple solar keratoses and superficial skin tumours with efficiencies similar to red light ALAPDT but less associated side effects (4).
A wavelength region absorbed more efficiently by protoporphyrin IX than green and red light is UVA (320400 nm; see absorption spectrum in Figure 1
), which is still able to penetrate skin down to the dermal layer with 2030% of the incident intensity (5). Thus, it may be expected that treatment with UVA is most efficient in terms of inducing phototoxicity, if protoporphyrin IX is the main photosensitizer. So far, UVA is used clinically for the treatment of diseases like atopic dermatitis or, in combination with methoxypsoralen, in PUVA therapy (for review see ref. 6 and 7). No clinical use has been made of UVA in combination with ALAPDT yet.
Both ALAPDT and UVA phototherapy exert many of their effects via formation of singlet oxygen (1O2). Singlet oxygen, an electronically excited form of normal ground-state oxygen, which is generated by the irradiation of photosensitizers such as protoporphyrin IX, is one of the major species responsible for the cytotoxicity inflicted by ALAPDT (8) and UVA (9). It is also capable of modulating intracellular signalling pathways (10): singlet oxygen, generated chemically or photochemically and by UVA irradiation, activates the stress-inducible MAP kinases p38 and JNK but not ERK MAP kinases (11). A similar pattern of MAP kinase activation is elicited by ALAPDT using red light (12).
In the present study, we sought to combine the effects of ALAPDT and UVA and investigated the efficiency of a combination of UVA irradiation and ALA treatment by comparing toxicities inflicted by UVA, green and red light on human skin fibroblasts pretreated with ALA. Using 1O2 quenching compounds (sodium azide and imidazole) and an enhancer of 1O2 lifetime (deuterium oxide), we tested whether singlet oxygen is involved in toxicity mediated by UVAALAPDT in fibroblasts and we compared the activation of the stress kinase p38 by UVAALAPDT with that elicited by ALAPDT with green and red light.
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Materials and methods
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Materials and reagents
Dulbecco's modified Eagle's medium (DMEM), Hank's balanced salt solution (HBSS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), protoporphyrin IX, NaN3, imidazole and D2O were purchased from SigmaAldrich (Deisenhofen, Germany). Polyclonal anti-phospho-p38 and anti total p38 antibodies were from New England Biolabs (Schwalbach, Germany). PVDF membranes were purchased from Amersham (Buckinghamshire, UK), dithiothreitol (DTT) from Boehringer (Mannheim, Germany). 5-Aminolevulinic acid and other chemicals and solvents were purchased from Merck (Darmstadt, Gemany).
Cell culture and irradiations
Human skin fibroblasts for determinations of porphyrin accumulation were isolated from foreskin biopsies. The fibroblasts used in all other experiments were also derived from foreskin but were purchased from BioWhittaker Europe (Taufkirchen, Germany). Cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.02 g/l streptomycin and penicillin (20 000 IU/l) and kept at 37°C in a humidified atmosphere of 5% CO2.
The cells were grown to confluence in 35 mm dishes and incubated with 1 mM ALA in culture medium for 24 h. They were then washed once with PBS and covered with 1 ml HBSS for UVA irradiations (320400 nm) which were performed using a UVA700 illuminator from Waldmann Lichttechnik (Villingen, Germany). Irradiation intensities were 44 (without ALA) and 10 mW/cm2 (with ALA). Irradiations with red light (570700 nm) were performed with a PDT1200 illuminator (Waldmann Lichttechnik) in fresh DMEM at an intensity of 50 mW/cm2, and green light irradiation (545 ± 3 nm) was performed with a `PDT Green Light' illuminating device from Saalmann (Herford, Germany) at intensities of 1020 mW/cm2. Different doses were achieved by varying irradiation times between 10 s and 30 min. For irradiation in the presence of sodium azide (20 mM in HBSS) or PBS with 90% deuterium oxide, the cells were pre-incubated with the respective solution for 10 min at 37°C. Imidazole (40 mM) was added to the growth medium 30 min before treatment and was present also during irradiation.
Porphyrin assay and spectra
Porphyrins were determined according to Piomelli (13) with minor modifications. Directly after treatment, cells were homogenized and extracted in 1 N perchloric acid/methanol (1:1, v/v). After a 10 min centrifugation, porphyrins were measured in the supernatants by fluorescence (excitation, 405 nm; emission, 598 nm) using uroporphyrin as a calibration standard. The spectrum of an aqueous solution of protoporphyrin IX (SigmaAldrich) was taken with a Perkin Elmer Lambda 2 Spectrometer.
Viability
Cell viability was determined using the reduction of MTT to the corresponding blue formazan. Skin fibroblasts were treated in 35 mm dishes and incubated with 800 µl 0.5 mg/ml MTT in medium for 34 h. The reaction was stopped by addition of 800 µl 10% (w/v) SDS/0.01 M HCl and the formazan released from cells by incubation at 37°C overnight. Absorbance of the supernatant was then measured at 570 nm against a background at 700 nm.
Western blotting
After irradiation, cells were incubated for 30 min, washed with PBS (2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4, pH 7.4), lysed in 2x SDSPAGE lysis buffer (125 mM Tris, 4% SDS, 20% glycerol, 100 mM DTT, 0.2% bromophenol blue, pH 6.8), heated for 5 min at 95°C and applied to SDS-polyacrylamide gels [10% (w/v) acrylamide]. The gels were blotted onto polyvinylidene difluoride membranes and immunodetection was performed with polyclonal anti-phospho-p38 antibodies. Polyclonal anti total p38 antibodies served as gel loading and protein control. Blots were also routinely controlled for equal loading by testing for the presence of GAPDH (not shown).
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Results
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Toxicity of UVA, green or red light on ALA-treated human skin fibroblasts
In order to evaluate the efficiency of ALAPDT with UVA irradiation, human skin fibroblasts were treated with ALA and irradiated with varying doses of UVA (320400 nm), green (545 ± 3 nm) or red (570750 nm) light. Viabilities of treated cells were determined 24 h after irradiation. Pre-incubations with 1 mM ALA were for 24 h; cellular porphyrins were shown to reach maximum levels at that time point (12) and to be increased 8-fold over control (Table I
). Figure 1A
compares viabilities of ALA-treated fibroblasts after irradiation with UVA (triangles), green (circles) or red light (squares). ALAPDT with UVA turned out to be the most efficient treatment in terms of reducing cell viability: the doses needed to decrease cell viabilities to 50% (LD50) were >10-fold higher for green light and ~40-fold higher for red light than in the case of UVA (Table I
). With UVAALAPDT, a nearly complete loss of cell viability was achieved at about 1 J/cm2, a dose that was not at all toxic with green- or red light-ALAPDT (Figure 1A
). Further, cell viabilities were reduced about 100-fold in ALA-pretreated cultures as compared with the control cells treated with UVA without ALA pretreatment (Figure 1B
). These results are in accordance with the absorption spectrum of protoporphyrin IX, which has its strongest absorbance in the UVA region (Figure 1C
).
For green and red light no toxicity could be observed upon application of the highest doses tested, 25 and 45 J/cm2, respectively (Table I
).
In summary, UVAALAPDT turned out to be the most efficient among the tested treatments, and it had to be tested if singlet oxygen contributes to this effect.
Role of singlet oxygen in the toxicity of UVAALAPDT
Singlet oxygen is known to be a major mediator of cytotoxicity in PDT (2,10). To test for its involvement in UVAALAPDT, fibroblasts were irradiated in the presence the 1O2-quenching compounds, sodium azide (20 mM) or imidazole (40 mM), and in the presence of deuterium oxide [90% (v/v)], an enhancer of 1O2-lifetime. As shown in Figure 2
, cell viability was increased 3- and 4-fold in the presence of azide and imidazole, respectively, while it was strongly decreased in the presence of D2O. This points to the generation of 1O2 during UVAALAPDT and suggests that it plays a crucial role in mediating toxicity.
Activation of p38 by ALAPDT
In order to test whether UVAALAPDT not only is the most effective way of performing ALAPDT with regard to gross toxicity but also to the activation of stress-activated signalling pathways, we analysed the dual phosphorylation (and thus, the activation) of the stress kinase p38. Upon activation, p38 phosphorylates and activates a variety of transcription factors, such as ATF-2, Elk-1 and others (for review see ref. 14), leading to the modulated expression of genes. P38 is known to be activated by 1O2 (11), which is generated during UVAALAPDT (see above and Figure 2
). 1O2 has also been shown to be the mediator of UVA-induced activation of p38 (11).
Cells were either irradiated or irradiated after ALA pre-incubation and analysed for p38-activation 30 min after treatment. A strong activation of p38 was obtained in cells treated with UVA (Figure 3A
) at doses corresponding to an ~80% toxicity (as measured 24 h after treatment). A comparable activation and toxicity was obtained with much lower doses if the cells were pretreated with ALA (Figure 3A
). At those lower doses, however, no activation of p38 could be seen if UVA was applied without prior ALA treatment. Activation of p38 was also seen at doses corresponding to a toxicity of about 20%.

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Fig. 3. Phosphorylation of p38 in ALA-treated cells upon irradiation with green or red light: Human skin fibroblasts were irradiated with UVA (A), green (B) or red light (C) as in Figure 1 . Cells were postincubated for 30 min, lysed and analysed by Western blotting using polyclonal anti-phospho-p38 and anti total p38 antibodies as described in Materials and methods.
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The doses needed to kill some 20% of the cell population were much lower in UVAALAPDT than in ALAPDT with red or green light (Figure 1
); similarly, the doses that had to be applied in green and red light-ALAPDT to achieve p38 activations comparable with those seen after UVA irradiation were much higher (3 and 10 J/cm2, respectively, versus 0.1 J/cm2; Figure 3B and C
). Nevertheless, not only ALAPDT with UVA, but also with green and red light led to an activation of p38.
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Discussion
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ALAPDT is a form of photodynamic therapy that relies on the topical generation of porphyrins as photosensitizers and has been successfully applied clinically in the treatment of skin diseases such as acne vulgaris (15), basal cell carcinoma and squamous cell carcinoma (16,17). Ultraviolet A is also used clinically in the treatment of atopic dermatitis (18,19) and, with methoxypsoralen as photosensitizer, in PUVA therapy, which is employed in treatment of psoriasis (7).
Both PDT and UVA therapy have been shown to rely on the formation of 1O2, leading to apoptotic and necrotic cell death (20), preceded by the activation of stress kinases (11,12), the expression of proapoptotic proteins, such as FasL (19) or bax (21), and the activation of caspases (22,23).
We proposed that a combination of both, ALAPDT and UVA phototherapy, might be superior to either treatment. ALAPDT relies mainly on the de novo generation of protoporphyrin IX whose extinction coefficient is higher in the UVA region (Soret band) than in the red or green region (Figure 1C
). Therefore, more energy is absorbed by protoporphyrin IX and used for the formation of reactive species such as singlet oxygen, leading to stronger cytotoxic effects. Thus, using UVA, lower doses might be needed for equal therapeutic efficiency as penetration into the skin is still sufficient for the treatment of pathological processes in the epidermis and upper subepidermal layers (5).
Here we show that, compared with the commonly used ALAPDT with red light, ALAPDT with UVA is the most efficient with regard to the toxicity inflicted in ALA-treated cultured cells. UVAALAPDT turned out to be 40-fold more potent in killing fibroblasts and still 10-fold more potent than ALAPDT with green light (Figure 1A
).
1O2 was shown to induce stress kinases (11,24). In line with an involvement of 1O2 in UVAALAPDT (Figure 2
), UVA irradiation of ALA-treated cells activated p38 at doses as low as 0.1 J/cm2 (Figure 3A
). Doses of 3 and 10 J/cm2 were necessary for a comparable activation using green or red light, respectively (Figure 3B and C
). Activation of p38 has been shown to mediate apoptotic death of cells after certain forms of PDT (25). Yet, though in the present study p38 activation largely parallels gross toxicity, it is likely that the cell death initiated in our system is not solely dependent on the activation of p38, because treatment with an inhibitor of p38 (PD169316) so far failed to protect against UVAALAPDT mediated toxicity (data not shown). Other factors such as direct damage of membranes, proteins or nucleic acids might be involved (25,26).
In summary, use of UVA is superior to visible light in inducing toxicity and activation of p38 in ALA-treated cells, with only 10 and 2% of the doses necessary when using green or red light, respectively. The dramatic rise in cytotoxicity of UVA that accompanies a mere 8-fold increase in porphyrin levels implies that a further increase in porphyrin content may lead to even higher susceptibility to UVA light. Taken together, ALAPDT in the UVA-region might be preferable to red or green light ALAPDT for treatment of readily accessible tissue because of its higher efficacy.
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Notes
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2 To whom correspondence should be addressed Email: helmut.sies{at}uni-duesseldorf.de 
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Acknowledgments
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We thank Klaus Bolsen for help with porphyrin determinations. This work was supported by Deutsche Forschungsgemeinschaft (Bonn, Germany; SFB 503/B1) and the National Foundation of Cancer Research (Bethesda, MD, USA).
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Received October 10, 2000;
revised January 31, 2001;
accepted February 20, 2001.