18β-Glycyrrhetinic Acid and Glabridin Prevent Oxidative DNA Fragmentation in UVB-irradiated Human Keratinocyte Cultures

Materials and Methods

Chemicals. GL (Sigma, St. Louis, MO, USA), 18β-GA (Sigma) and GLB (Chromadex Inc, Irvine, CA, USA) were tested (Figure 1). All the substances were dissolved in dimethylsulphoxide (DMSO) (final concentration 0.01%) and the solutions of each compound sterilized by filtration. Final concentrations were obtained in keratinocyte basal medium (KBM, Lonza, Basel, Switzerland).

Cells and cell cultures. Normal human keratinocytes (NHK) were obtained after excision of skin specimens from plastic surgery procedures in accordance with consent procedures approved by the Internal Review Boards of the General Hospital of Modena and Reggio Emilia. NHK cultures were prepared as described by Rheinwald and Green (20). The skin samples were minced and trypsinized (0.25% trypsin, 0.02% EDTA) at 37°C for 30 minutes and the NHK were grown in 75 cm² culture flasks (Costar, Cambridge, MA, USA) with 10 mg/ml mitomycin-treated (Sigma) 3T3 fibroblasts for 2 hours at 37°C. NHK were cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/F12, 3:1) (Seromed-Biochrom KG, Berlin, Germany) containing insulin 5 μg/ml, transferrin 5 μg/ml, triiodothyronine 2 nM, hydrocortisone 0.4 μg/ml, adenine 180 mM, mouse epidermal growth factors (EGF) 10 ng/ml (all from Sigma), 10% foetal calf serum and 100 U/ml penicillin-100 μg/ml streptomycin-amphotericin B (PSA) (Lonza). Sub-confluent primary cultures were transferred to secondary culture in keratinocyte growth medium (KGM; Lonza) containing bovine pituitary extract (BPE; Lonza) until near confluence. Once cell growth was well established, the cells were plated at 10⁴/cm² into disposable 96-multiwell plates (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) for the MTT test and ROS assay and at 1.5×10⁴/cm² in P60 culture dishes (Costar) for the comet and Western blot tests.

Cell viability assay. Cell viability was measured by the 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide test (MTT) according to the method described by Mossman (21). The MTT test was performed in triplicate on 12 and 24 hour cultures of NHK, added with equimolecular increasing concentrations of GL, 18β-GA or GLB (upper concentration: 120 μM). Control cells were maintained in KBM. The reaction was carried out in situ in the multiwell plates and the reaction product was measured colorimetrically at 540 nm using a Bio-Rad 550 microplate reader (Bio-Rad, Monaco, Germany). The results were expressed as the percentage of cell viability with respect to the control (plates without test molecules).

UVB treatment. GL (30 μM), 18β-GA (30 μM) or GLB (15 μM) were added to the KBM and NHK were treated with the compounds for 12 hours. After this period, KBM was removed and replaced with PBS+ (with Ca²⁺ and Mg²⁺; Lonza) (22) added with GL, 18β-GA or GLB. The cells were immediately exposed to UVB radiation (50 mJ/cm², 75 mJ/cm² or 100 mJ/cm²) using a UVB TL-20 W/12 RS lamp. After UVB irradiation, PBS+ was removed and the cells provided with fresh KBM added with GL, 18β-GA or GLB and left for an additional 12 hours. Two controls were prepared: the first was UVB irradiated, but without GL, 18β-GA or GLB; the second was with GL, 18β-GA or GLB, but without UVB irradiation. The effects of GL, 18β-GA and GLB after UVB treatment were evaluated by the comet assay, the measurement of intracellular ROS levels and Western blot analysis.

Comet assay. The comet assay was performed as described by Alapetite et al. (23). The treated cells were trypsinized and 100 μl of the re-suspended pellet, mixed with a 1% low-melting point agarose, were transferred onto a pre-coated microscope slide. The slide with solidified gel was placed into pH10 lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM TRIS, 1% Triton X-100 and 10% DMSO), at 4°C for at least 1 hour. The slides were then incubated in alkaline unwinding electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13) and electrophoresis was performed at 20 V, 300 mA for 30 minutes. The gels were stained with 2 μg/ml ethidium bromide and examined using an Axioskope 40 epifluorescence microscope (Zeiss, Jena, Germany).

Measurement of intracellular ROS levels. The generation of ROS was measured using a ROS-detecting probe, 2’7’-dichlorodihydro-fluorescein diacetate (H2DCFDA, Invitrogen, Carlsbad, CA, USA) (24).

Briefly, after UVB treatment, the cells were loaded with H2DCFDA for 30 minutes (10 μg/ml in PBS+). The production of intracellular ROS was measured spectrofluorometrically. Fluorescence was quantified with a Shimadzu RF5301 PC spectrofluorophotometer (Shimadzu, Duisburg, Germany), set at an excitation of 485 nm and an emission of 535 nm. The test was carried out in triplicate.

Western blotting assay. Control and treated cells were washed in cold PBS then were lysed in 70 μl of lysis buffer pH 7.4 (50 mM Tris-HCl, 150 mM NaCl, 1% Na deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 0.2% NaN₃) with protease inhibitor cocktail tablets (Roche, Penzberg, Germany). Equal quantities of protein were separated by SDS polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred onto a nitrocellulose membrane and blocked with 5% non-fat dry milk in PBS. Red Ponceau was used to verify equal loading of the total protein. The blot was incubated with the following primary antibodies: anti-BCL-2 (Dako, Glostrup, Denmark), anti-poly(ADP-ribose)polymerase (PARP) (Biomol, Hamburg, Germany) and anti-p53 (Calbiochem, Darmstadt, Germany). Subsequently, the blots were incubated with horseradish peroxidase-conjugated goat antimouse IgG (Bio-Rad) at 1:3000 dilution at room temperature. Immunoblotted bands were visualized by an ECL chemiluminescent system (Amersham, Glattbrugg, Switzerland). β-Actin antibody (Sigma) was included as a loading control. Relative density of each band was measured as arbitrary units by MacBiophotonics ImageJ software (MacBiophotonics; Hamilton, ON, Canada).

Statistical analysis. Differences between control and treated samples were determined by Student's t-test. Values of p≤0.01 and p≤0.001 were considered statistically significant. The results are expressed as the mean value±standard deviation (SD).