Carboxyfullerenes Protect Human Keratinocytes from Ultraviolet-B-Induced Apoptosis
2000; Elsevier BV; Volume: 115; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2000.00140.x
ISSN1523-1747
AutoresCristiana Fumelli, Alessandra Marconi, Stefano Salvioli, Elisabetta Straface, Walter Malorni, Anna Maria Offidani, Roberto Pellicciari, Gennaro Schettini, Alberto Giannetti, Daniela Monti, Claudio Franceschi, Carlo Pincelli,
Tópico(s)Selenium in Biological Systems
ResumoCarboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2. Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2. carboxyfullerenes deoxy-D-ribose N-acetyl-L-cysteine poly(ADP-ribose)polymerase Buckminsterfullerenes, or ''Buckyballs'', are spherical carbon molecules (C60 and larger carbon spheres) with a unique cage structure (Figure 1). Since their discovery in 1985 (Kroto et al., 1985Kroto H.W. Heath J.R. O'brien S.C. Curl R.F. Smalley R.E. C60: buckminsterfullerene.Nature. 1985; 318: 162-163Crossref Scopus (12973) Google Scholar) and recent large-scale synthesis (Kratschmer et al., 1990Kratschmer W. Lamb L.D. Fostiropoulos K. Huffman D.R. Solid C60: a new form of carbon.Nature. 1990; 347: 354-358Crossref Scopus (6805) Google Scholar), their physical and chemical properties have been extensively investigated. On the other hand, biologic activities of fullerenes are still largely unknown, mainly because of their poor solubility in water. Recently the availability of water-soluble fullerene derivatives (Yamakoshi et al., 1994Yamakoshi Y.N. Yagami T. Fukuhara K. Sueyoshi S. Miyata N. 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In this study, we investigated the role of CF in UVB-induced damage in human keratinocytes. Normal human keratinocytes were obtained from foreskin and cultured as described previously (Pincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for Bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar). Briefly, keratinocytes were plated on mytomicin-C-treated 3T3 cells (2.4 × 104 per cm2, ATCC, Rockville, MD) and cultivated in Dulbecco's modified Eagle's medium and Ham's F12 media. Subconfluent secondary cultures were trypsinized with 0.05% trypsin/0.02% ethylenediamine tetraacetic acid and replated for the experiments in defined serum-free medium [keratinocyte growth medium (KGM), Clonetics, San Diego, CA]. As previously described (Lamparth and Hirsch, 1994Lamparth I. Hirsch A. Water-soluble malonic acid derivatives of C60 with a defined three dimensional structure.J Chem Soc, Chem Commun. 1994; 14: 1727-1728Crossref Google Scholar;Dugan et al., 1997Dugan L.L. Turetsky D.M. Du C. et al.Carboxyfullerenes as neuroprotective agents.Proc Natl Acad Sci. 1997; 94: 9434-9439Crossref PubMed Scopus (650) Google Scholar), a solution of fullerene-C60 and dry diethyl bromomalonate in toluene was magnetically stirred for 15 h in an argon atmosphere and at room temperature in the presence of dry 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction mixture was then evaporated and the brownish residue was submitted to flash chromatography by means of the Biotage Flash 40 system (Biotage UK, Flash 40 M, KP-SIL cartridges). Elution with toluene:hexane (8:2) to toluene afforded a fraction (A) enriched in the D3 adduct. A following elution with toluene:acetonitrile (99.5:0.5) gave a fraction (B) enriched in the C3 adduct. Fraction A was then submitted to medium pressure chromatography and elution with toluene:hexane (8:2) to afford pure (84.2%) D3 adduct in 19.5% yield. Fraction B was further purified by flash chromatography using toluene:acetonitrile (99.9:0.1), thus obtaining pure (90.1%) C3 adduct in 15.5% yield. Both the tris-adducts showed analytic and spectroscopic data (UV, 13C-NMR) identical with those already reported (Hirsch et al., 1994Hirsch A. Lamparth I. Karfunkel H.R. Fullerene chemistry in three dimensions: isolation of seven regioisomeric bisadducts and chiral trisadduct of C60 and di (ethoxy-carbonyl) methylene.Angew Chem Int Ed Engl. 1994; 33: 437-439Crossref Scopus (439) Google Scholar). Alkaline hydrolysis of the C3 adduct was performed with sodium hydride in toluene as previously reported (Lamparth and Hirsch, 1994Lamparth I. Hirsch A. Water-soluble malonic acid derivatives of C60 with a defined three dimensional structure.J Chem Soc, Chem Commun. 1994; 14: 1727-1728Crossref Google Scholar;Dugan et al., 1997Dugan L.L. Turetsky D.M. Du C. et al.Carboxyfullerenes as neuroprotective agents.Proc Natl Acad Sci. 1997; 94: 9434-9439Crossref PubMed Scopus (650) Google Scholar), with a slightly modified work-up procedure. Accordingly, after stirring for 30 min at room temperature, the reaction mixture was cooled (0°C) and methanol was added. Stirring was continued for 45 min at room temperature, and the red-orange precipitate was collected by centrifugation and washed with toluene and hexane. An equimolar amount of 4 M sulfuric acid was then added to the residue dissolved in water and the resulting solution was concentrated in vacuo and re-dissolved in methanol. Filtration and evaporation of the solvent afforded the pure acid C3 adduct in 70% yield. An analogous sequence was applied to the D3 adduct thus obtaining the free acid in 37% yield. Trypan blue dye exclusion was performed at 24 and 72 h after the addition of either different doses of CF (10, 100 μM) or diluent to subconfluent keratinocyte colonies. Neutral Red was performed in keratinocyte subconfluent cultures, seeded in a 96 well tissue culture plate. At 24 and 72 h after CF (0, 10, 100 μM) addition cells were incubated with the Neutral Red solution for 3 h at 37°C. Cells were fixed with formaldehyde solution and then the extractive solution was added. After 20 min incubation, the sample was analyzed using a scanning multiwell spectrophotometer at 540 nm; the results are expressed as optical density units (OD). The metabolic assay MTT was performed in keratinocyte subconfluent cultures, seeded in a 96 well tissue culture plate. At 24 and 72 h after CF (0, 10, 100 μM) addition cells were incubated with the MTT solution for 4 h at 37°C. After solubilization with dimethylsulfoxide, the formazan dye formation was evaluated using a scanning multiwell spectrophotometer at 540 nm; results are expressed as optical density units (OD). Results are expressed as the mean ± SD of three different experiments. Student's t test was used for comparison of the means. UVB radiation (290–320 nm) was delivered with a battery of lamps (TL 20 W/12 RS UV-B Philips Medical). Normal human keratinocytes were sham or UVB irradiated at subconfluency after the addition of different doses of CF (1, 10, 25, 50, 100 μM) and analyzed 8, 12, 24, 36, and 48 h later. We selected UVB doses of 25, 50, and 100 mJ per cm2, as measured with an International Light Research Radiometer (Newburyport, MA). Normal human keratinocytes (12,000 cells per well) were grown on 96 well plates in KGM. Sham and UVB (25, 50 mJ per cm2) irradiated keratinocytes were pretreated with either 10 μM CF or diluent. 3H-thymidine (1 μCi per well, Amersham-Pharmacia Biotech, Rainham, UK) incorporation was performed 12 h before harvesting of the cells and cells were collected at 24 h after irradiation. The incorporated radioactivity was determined by β counter. Subconfluent keratinocytes were sham or UVB irradiated after the addition of either CF or diluent. Cells were directly stained on chamber slides with the ''In situ cell death detection kit'' (Boehringer), as recommended by the manufacturer and described byPincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for Bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar. Each experiment was repeated from three to seven times. Negative control was obtained by replacing the primary incubation with a nucleotide mixture without TdT. Fluorescent specimens were analyzed with a confocal scanning laser microscope (Leica TCS4D) in conjunction with a conventional optical microscope (Leica DM IRBE). Keratinocytes, cultivated as before, were sham or UVB irradiated after the addition of either CF or diluent. In separate experiments, UVB-irradiated cells were pretreated with the antioxidant compound N-acetyl-L-cysteine (NAC, 10 mM; Calbiochem, LaJolla, CA). After 24 h cells were trypsinized and resuspended (1 × 106 cells) in 400 μl hypotonic fluorochrome solution: propidium iodide (PI), 50 mg per ml, in 0.1% sodium citrate containing 0.1% Tryton X-100 (Sigma). After a 60 min incubation in this solution, cells were analyzed using a FACScan flow cytometer (Becton-Dickinson, San José, CA). Ten thousand events per sample were collected and analyzed with Lysys II software (Becton-Dickinson). The procedure allows the detection of either diploid DNA (normal cells) or hypodiploid DNA content (apoptotic cells). Apoptosis was detected by evaluating the reduced fluorescence of the DNA binding dye PI in the apoptotic nuclei. Cells were washed with phosphate-buffered saline (PBS) and lyzed on ice in RIPA buffer, pH 8.5, as described previously (Pincelli et al., 1997Pincelli C. Haake A.R. Benassi L. et al.Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for Bcl-2.J Invest Dermatol. 1997; 109: 757-764Crossref PubMed Scopus (88) Google Scholar). Thirty micrograms of total protein were analyzed under reducing conditions on 12% and 6% polyacrylamide gels and blotted onto nitrocellulose membranes. To verify equal loading of total proteins in all lanes, the membranes were stained with red ponceau. The blots were blocked for 2 h in blocking buffer (PBS buffer, pH 7.4, with 0.2% Tween 20 and 5% nonfat milk) and incubated overnight at 4°C with 2.5 μg per ml antihuman BCL-2 monoclonal antibody (Dako, Glostrup, Denmark), with a monoclonal antibody against the human enzyme poly(ADP-ribose)polymerase (PARP, 1:1000; Biomol, Plymouth Meeting, PA), and with anti-β-actin monoclonal antibody (1:1000; Sigma) as a control. Then membranes were washed in PBS/Tween 20, incubated with peroxidase-conjugated goat antimouse antibody (1:800; Biorad, Hercules, CA) for 45 min at room temperature, washed, and developed using the ECL chemiluminescent detection system (Amersham). Mitochondrial membrane potential was measured by means of the lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide (JC-1; Molecular Probes, Eugene, OR), as previously described (Cossarizza et al., 1993Cossarizza A. Baccarani-Contri M. Kalashnikova G. Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′6, 6′–tetrachloro-1, 1′3, 3′–tetraethylbenzimidazol carbocyanine iodide (JC-1).Biochem Biophys Res Commun. 1993; 197: 40-45Crossref PubMed Scopus (886) Google Scholar). This molecule, able to selectively enter the mitochondria, exists in a monomeric form that forms aggregates when the mitochondrial membrane is polarized, with a change in color of the dye from green to greenish orange that can be detected by flow cytometry using fluorescence channel 1 (FL1) for the green emission and fluorescence channel 2 (FL2) for the greenish orange emission. Subconfluent keratinocytes were sham or UVB irradiated after the addition of either CF or diluent. At 24 h keratinocytes were trypsinized and the cell suspension (1 × 106 cells in 1 ml KGM) was incubated with JC-1 probe for 10 min at room temperature in the dark. Then cells were washed with PBS, resuspended in a 400 μl total volume, and analyzed by flow cytometry. Subconfluent keratinocytes were treated with a solution of deoxy-D-ribose (dRib; Sigma) at a final concentration of 40 mM after the addition of either 10 μM CF or diluent. At 48 h cells were trypsinized and resuspended (1 × 106 cells) in 400 μl hypotonic fluorochrome solution: 50 mg per ml PI in 0.1% sodium citrate containing 0.1% Triton X-100 (Sigma). After a 60 min incubation in this solution cells were analyzed by flow cytometry. Apoptosis was detected by analyzing the reduced fluorescence of the DNA binding dye PI in the apoptotic nuclei. In order to understand the effects of CF on human keratinocytes, we first evaluated a possible alteration of vital parameters. Increasing doses of CF did not cause keratinocyte cytotoxicity, as shown by Trypan blue dye exclusion, MTT, and Neutral Red (Table 1). Moreover no difference in proliferation rate was detected between CF-pretreated cells and controls (Figure 2).Table ICell viability and cytotoxicity24 h72 hSampleTrypan blue % ± SDNeutral red OD ± SDMTT OD ± SDTrypan blue % ± SDNeutral red OD ± SDMTT OD ± SDControl92.73 ± 6.300.39 ± 0.080.34 ± 0.0396.17 ± 0.900.81 ± 0.140.38 ± 0.04CF 10 μm85.07 ± 2.890.41 ± 0.090.31 ± 0.1095.53 ± 0.610.76 ± 0.090.46 ± 0.12CF 100 μm89.07 ± 2.920.33 ± 0.040.29 ± 0.0990.72 ± 4.570.74 ± 0.070.28 ± 0.11 Open table in a new tab DNA synthesis in cultured keratinocytes was investigated 24 h after irradiation with two different doses of UVB with the addition of either CF or diluent. As expected, UVB radiation induced inhibition of keratinocyte proliferation in a dose-dependent manner. On the other hand, pretreatment with CF significantly prevented this effect. Indeed, proliferation rate was significantly higher in CF-pretreated cells than in cells treated with diluent alone, following UVB irradiation (Figure 2). In order to study the effects of CF in UVB-induced keratinocyte apoptosis, we first tested different doses of CF in keratinocyte cultures before UVB irradiation. We observed a significant and dose-dependent reduction of apoptotic keratinocytes in CF-pretreated cells compared with cells treated with diluent, starting from a dose of 10 μM (Figure 3a). Whereas UVB irradiation induced marked alterations in keratinocytes, which became rounded and detached from the plate, the addition of increasing doses of CF progressively prevented this effect, as shown by the morphology of the cells. Indeed, whereas, after UVB radiation, keratinocytes appear unhealthy and detached from the plate, the addition of increasing doses of CF allows them to progressively recover a morphology that is indistinguishable from that of sham-irradiated cells (Figure 3b). As the minimal protective dose was 10 μM, we selected it for successive experiments. TUNEL staining showed that the percentage of apoptotic cells 24 h after UVB irradiation was significantly lower in CF-pretreated cells than in cells treated with diluent alone (Figure 4a). The protective effect of CF was further confirmed by flow cytometric analysis. A sub-G1 peak, indicating the amount of apoptotic nuclei, was clearly more pronounced in UVB-irradiated keratinocytes than in CF-pretreated cells (Figure 4b,c). Moreover, significant protection was detected up to 72 h in CF-pretreated keratinocytes (Figure 4d). Finally, PARP is a specific substrate for caspases, which play a crucial role in the execution stage of apoptosis. In this study, we showed that CF partially inhibit caspase activation induced by UVB irradiation. Indeed, the 85 kDa fragment, representing the cleaved form of PARP, is clearly less pronounced in CF-pretreated keratinocytes than in UVB-irradiated cells provided with diluent alone, at all time points (Figure 5a,b). Taken together, these results demonstrate that CF exert an effective protection from UVB-induced apoptosis in human keratinocytes.Figure 4CF protect human keratinocytes from UVB-induced apoptosis. (A) Subconfluent keratinocytes were given 10 μM CF or diluent before UVB (100 mJ per cm2) or sham irradiation. At 24 h cells were directly stained with TUNEL. Around 100 cells were counted in randomly selected fields for each point and percentages are expressed as the means ± SD of seven experiments. Student's t test was used for comparison of the means. (B) Cells were treated as in (A), trypsinized at 24 h, stained with PI, and analyzed by flow cytometry. Results are expressed as the mean ± SD of four experiments. Student's t test was used for comparison of the means. (C) Histograms from one representative flow cytometry experiment illustrating sham-irradiated keratinocytes, UVB (100 mJ per cm2) irradiated cells, UVB-irradiated cells after the addition of 10 μM CF. Data from the original histograms were used to generate the mean values expressed in (B). (D) Subconfluent keratinocytes were sham or UVB (50 mJ per cm2) irradiated after the addition of 10 μM CF or diluent. Cells were stained with TUNEL in situ at different time points. About 100 cells were counted in randomly selected fields for each point and percentages are expressed as the means ± SD of three experiments. Student's t test was used for comparison of the means. 10 μM CF + UVB vs UVB at 8 h, not significant; 10 μM CF + UVB vs UVB at 12 h, p < 0.05; 10 μM CF + UVB vs UVB at 24 h, p < 0.01; 10 μM CF + UVB vs UVB at 36 h, p < 0.05; 10 μM CF + UVB vs UVB at 48 h, p < 0.01; 10 μM CF + UVB vs UVB at 72 h, p < 0.05.View Large Image Figure ViewerDownload (PPT)Figure 5CF reduce UVB-induced PARP cleavage. (A) Keratinocytes were cultured in KGM and sham or UVB (50, 100 mJ per cm2) irradiated after the addition of 10 μM CF or diluent. Cells were lyzed at different time points and proteins were analyzed by Western blotting using anti-PARP monoclonal antibody. The 85 kDa fragment represents the cleaved form of PARP. A non-specific band is present immediately below the 85 kDa band. (B) Relative intensity of the band on autoradiograms was quantified by scanning laser densitometry (TN-Image program; Copyright © T. Nelson 1995–97, Version 2.27). Values of 85 kDa bands are expressed as fold variations, compared with UVB 50 irradiated cells at 12 h.View Large Image Figure ViewerDownload (PPT) Bcl-2, the founding member of the large bcl-2 family that protects many cell types from apoptosis (Yang and Korsmeyer, 1996Yang E. Korsmeyer S.J. Molecular thanatopsis: a discourse on the bcl-2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar;Kroemer, 1997aKroemer G. The proto-oncogene bcl-2 and its role in regulating apoptosis.Nat Med. 1997; 3: 614-620Crossref PubMed Scopus (1669) Google Scholar;Green and Reed, 1998Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), is detected in normal human keratinocytes (Polakowska et al., 1994Polakowska R.R. Piacentini M. Bartlett R. Goldsmith L.A. Haake A.R. Apoptosis in human skin development: morphogenesis, periderm, and human stem cells.Dev Dynamics. 1994; 199: 176-188Crossref PubMed Scopus (242) Google Scholar;Rodriguez-Villanova et al., 1995Rodriguez-Villanova J. Colome M.I. Brisbay S. McDonnell T.J. The expression and localization of bcl-2 protein in normal skin and in non-melanoma skin cancers.Pathol Res Pract. 1995; 191: 391-398Crossref PubMed Scopus (53) Google Scholar). To investigate the possible mechanisms of CF interference with UVB-induced apoptosis, Western blot analysis of bcl-2 protein was performed on extracts from UVB-irradiated keratinocytes with or without the addition of CF. As expected, at 24 h bcl-2 protein levels were downregulated by UVB irradiation compared with sham-irradiated keratinocytes (Figure 6a). No difference in bcl-2 protein expression was detected between CF- and diluent-pretreated keratinocytes following UVB irradiation. In a separate experiment, the antioxidant compound NAC also prevented UVB-induced keratinocyte apoptosis (Figure 6e) without affecting bcl-2 levels (Figure 6c). In order to evaluate alterations in mitochondrial membrane potential related to apoptosis (Cossarizza et al., 1995Cossarizza A. Franceschi C. Monti D. et al.Protective effect of N-acetylcysteine in tumor necrosis factor-α-induced apoptosis in U937 cells: the role of mitochondria.Exp Cell Res. 1995; 220: 232-240Crossref PubMed Scopus (266) Google Scholar), flow cytometric analysis using the JC-1 probe was performed. Keratinocytes with depolarized mitochondria are normally represented by events in the lower quadrants with loss of greenish orange fluorescence and increased green fluorescence. We observed an increased number of cells with depolarized mitochondria in UVB-irradi
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