C60 Carboxyfullerene Exerts a Protective Activity against Oxidative Stress-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

Biochemical and Biophysical Research Communications 277, 711–717 (2000) doi:10.1006/bbrc.2000.3715, available online at http://www.idealibrary.com on

C60 Carboxyfullerene Exerts a Protective Activity against Oxidative Stress-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells Daniela Monti,* ,1 Laura Moretti,† Stefano Salvioli,‡ Elisabetta Straface,§ Walter Malorni,§ Roberto Pellicciari, ¶ Gennaro Schettini,储 Michela Bisaglia,储 Carlo Pincelli,** Cristiana Fumelli,** Massimiliano Bonafe`,‡ and Claudio Franceschi‡ ,†† *Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy; †Department of Biomedical Sciences, Section of General Pathology, University of Modena and Reggio Emilia, Modena, Italy; ‡Department of Experimental Pathology, University of Bologna, Bologna, Italy; §Department of Ultrastructures, Istituto Superiore di Sanita`, Rome, Italy; ¶Institute of Chemistry and Technology of Drugs, University of Perugia, Perugia, Italy; 储Unit of Pharmacology and Neuroscience, National Cancer Institute and Biotechnology, Department of Oncology, University of Genova, Genoa, Italy; **Department of Neuropsychosensorial Pathology, Division of Dermatology, University of Modena and Reggio Emilia, Modena, Italy; and ††Department of Gerontological Research, INRCA, Ancona, Italy

Received September 27, 2000

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a freeradical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-D-ribose (dRib) or TNF-␣ plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-D-ribose or TNF-␣ plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria. © 2000 Academic Press Key Words: carboxyfullerenes; lymphocytes; apoptosis; mitochondria.

Buckminsterfullerenes have attracted considerable attention in the last few years either for their particular chemical structure or for their biological properties (1–3). These compounds, thanks to their peculiar structure, are capable of “adding” multiple radicals per mol1 To whom correspondence should be addressed at Department of Experimental Pathology and Oncology, University of Florence, via Morgagni 50, Florence, Italy. Fax: ⫹39 055 416908. E-mail: [email protected].

ecule, leading Krusic et al. to characterise fullerenes since 1991 as “radical sponge” and to hypothesise their potential activity as free-radical scavengers (4). Indeed, owing to the availability of different suitable chemical derivatives, studies on the biological effects exerted by carboxyfullerenes have been performed. In particular, water-soluble fullerene derivatives have revealed some interesting properties on specific biological functions, such as cell ionic homeostasis (5) and viral replication (6, 7). Recently, the capability of carboxyfullerenes to act as free radical scavengers has been shown to be involved in the inhibition of apoptosis in different cell types such as neuronal cells (8, 9), hepatoma (10), or epithelial cells (11). As apoptosis is of critical importance for a variety of physiological (thymic negative selection, down regulation of the immune response, NK- and T cell-mediated cytotoxicity) or pathological phenomena (infections, autoimmunity, lymphoid malignancies), we thought it worthwhile to investigate the possible protective role of the watersoluble carboxyfullerene derivative, C 3-fullero-trismethanodicarboxylic acid (C 3-F-tris-MDC), in human peripheral blood mononuclear cells (PBMCs). Two models of apoptosis, both interfering with cell redox status, have been used. In the former PBMCs are exposed to 2-deoxy-D-ribose (dRib), a reducing sugar capable to induce oxidative stress and GSH depletion while in the latter PBMCs are exposed to Tumour Necrosis Factor-␣ plus cycloheximide. These models of apoptosis have been useful to study the protective effect of antioxidant compounds, such as N-acetyl-

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (100 ␮g/mL) and 10% heat inactivated foetal calf serum (Gibco, Paisley, Scotland, UK). Cell suspensions were seeded into culture plates in presence or absence of dRib (final concentration 10 mM) or TNF-␣ plus cycloheximide (CHX), 50 U/mL and 16 ␮M, respectively and then incubated for 24, 48, and 72 h at 37°C in humidified atmosphere of 5% CO 2 in air. For studies on the effect of carboxyfullerene, PBMCs were incubated with the abovementioned agents plus two concentrations of carboxyfullerene (2 ␮M and 10 ␮M) for 24, 48, and 72 h. Finally, cells were collected and analysed by different assays.

Carboxyfullerene Preparation

FIG. 1. Detection of apoptosis of PBMCs assessed by TUNEL staining and fluorescence microscopy. Sample were collected after 48 h of incubation. (A) Control, untreated PBMCs; (B) PBMCs treated with 10 mM dRib; (C) PBMCs treated with 50 UI/ml TNF-␣ plus CHX.

As previously described (8, 17), 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 submitted to flash chromatography by means of the Biotage Flash 40 system (Biotage UK, Ltd., Flash 40M, KP-SIL cartridges). Elution with toluene-hexane (8:2) to toluene afforded a fraction (A) enriched in the D 3 -adduct. Following elution with tolueneacetonitrile (99.5:0.5) gave a fraction (B) enriched in the C 3 adduct. Fraction A was then submitted to medium pressure chromatography and elution with toluene-hexane (8:2) to afford pure (84.2%) D 3 -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%) C 3 -adduct in 15.5% yield. Both the Trisadducts showed analytical and spectroscopic data (UV, 13 C-NMR) identical with those already reported. Alkaline hydrolysis of the C 3 -adduct was performed with sodium hydride in toluene with a work-up procedure slightly modified. 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, the red-orange precipitate was collected by centrifugation and washed with toluene and hexane. An equimolar amount of 4M sulphuric acid was then added to the residue dissolved in water and the resulting solution was concentrated in vacuum and redissolved in methanol. Filtration and evaporation of the solvent afforded the pure acid C 3 -adduct in 70% yield. An analogous sequence was applied to the D 3 -adduct thus obtaining the free acid in 37% yield.

Apoptosis Assessment cysteine (12, 13), and to reveal different susceptibility to oxidative stress-induced apoptosis in physiological (aging and longevity) (14) and pathological conditions (Fanconi’s Anemia and Werner Syndrome) (15–16). Here we report that the fullerene derivative is able to inhibit dRib- and TNF-␣-induced apoptosis, likely acting as an antioxidising drug and partially preventing the depolarisation of mitochondrial membrane potential. MATERIALS AND METHODS Cell Culture and Induction of Apoptosis Peripheral blood mononuclear cells (PBMCs) from 13 young healthy subjects (25 ⫾ 3 years) were separated by Ficoll-Hypaque sedimentation (Nycomed, Pharma AS, Oslo, Norway) and cultured at a density of 10 6 cells/mL in RPMI-1640 culture medium, containing 2 mM L-glutamine, penicillin (100 units/mL) streptomycin

Morphological analysis. PBMCs were harvested and resuspended at 1 ⫻ 10 5 cells/100 ␮L. One hundred microliters of each cell suspension was loaded into separate cytocentrifuge chambers and spun down for 2–3 min at 500 rpm. Slides were removed and airdried at room temperature and then stained with May-Gru¨nwald/ Giemsa solution. TdT-mediated dUTP nick end labelling (TUNEL). DNA fragmentation associated with apoptosis was detected by TUNEL technique using an in situ cell death detection kit (Boehringer Mannheim, Germany) as described (15). Control and treated PBMCs were harvested and fixed in freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) on slides. After permeabilisation (with 0.1% triton X-100, 0.1% sodium citrate), slides were incubated with TUNEL mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP. Finally, slides were analysed by fluorescence microscopy (Axioscope, Zeiss, Germany). Only the apoptotic cells appeared fluorescent, while the nonapoptotic cells showed no labelled nuclei. DNA content. Apoptosis was quantified by flow cytometry as reduced fluorescence of the propidium iodide (PI) in the apoptotic nuclei, as previously described (12). Briefly, the 200g centrifuged cell

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FIG. 2. Morphological and cytofluorimetrical detection of apoptosis in PBMCs after 48 h of treatment with dRib (see text for further details). (A) Control, untreated cells; (B) PBMCs treated with 10 mM dRib; (C) PBMCs treated with 10 mM dRib and 10 ␮M carboxyfullerene. Pictures were taken with a 630⫻ enlargement. Arrows indicate typical apoptotic cells.

pellet (10 6 cells) was gently resuspended in 500 ␮L of hypotonic fluorochrome solution (PI 50 ␮g/mL in 0.1% sodium citrate plus 0.1% Triton X-100 in bidistilled water; Sigma Chemical Co., St. Louis, MO). Cells were analysed by flow cytometry after a minimum of 20 min of incubation in this solution. Mitochondrial Membrane Potential (MMP). Mitochondrial membrane potential was measured by flow cytometric technique making use of the lipophilic cationic probe 5,5⬘,6,6⬘-tetrachloro-1,1⬘,3,3⬘tetraethylbenzimidazolcarbocyanine iodide (JC-1, Molecular Probes, Eugene, OR). Cell suspension was adjusted to a density of 0.5 ⫻ 10 6

cells/mL and incubated in complete medium for 10 min at room temperature in the dark with 2.5 ␮g/mL JC-1. At the end of the incubation period cells were washed twice in cold phosphate buffer saline (PBS), resuspended in a total volume of 400 ␮L and analysed (18). Samples were analysed using a FACScan flow cytometer (BD, San Jose, CA). A minimum of 10,000 cells per sample was acquired in list mode. Statistical Analysis. Statistical analysis was performed by paired Student’s t test. A P value lower than 0.05 was considered statistically significant.

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FIG. 3. Morphological and cytofluorimetrical detection of apoptosis in PBMCs after 48 h of treatment with TNF-␣ (see text for further details). (A) Control, untreated cells; (B) PBMCs treated with 50 IU/ml TNF-␣ plus 16 ␮M CHX; (C) PBMCs treated with 50 IU/ml TNF-␣ plus 16 ␮M CHX and 10 ␮M carboxyfullerene. Pictures were taken with a 630⫻ enlargement. Arrows indicate typical apoptotic cells.

RESULTS Carboxyfullerene Protects Quiescent PBMCs Undergoing Oxidative Stress-Induced Apoptosis The effects of carboxyfullerene in two models of apoptosis, i.e., dRib- or TNF-␣ plus CHX-induced apoptosis, was analysed in human PBMCs. The presence of carboxyfullerene alone did not modify the percent of

cells with hypodiploid DNA content during “spontaneous” apoptosis. (data not shown). Both stimuli were able to induce apoptosis in resting PBMCs, and the presence of carboxyfullerene significantly reduced the phenomenon, as assessed by TUNEL technique (Fig. 1). Classic alterations of apoptotic cells, i.e., chromatin condensation and clumping and DNA breakage, were evident when PBMCs were treated with the apoptotic

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stimuli. The addition of carboxyfullerene decreased this effect, which was further confirmed by MayGru¨nwald/Giemsa staining and by flow cytometry analysis (Figs. 2 and 3). The morphology of dRibtreated cells and the percentage of apoptotic nuclei in absence and in presence of carboxyfullerene are shown in Fig. 2. The percentage of cells with morphological aspects of apoptosis and with sub-G1 hypodiploid nuclei clearly decreased in dRib-treated cells when carboxyfullerene was present. The same results were obtained when the PBMCs were treated with TNF-␣ plus CHX (Fig. 3). The kinetics of the protective effect exerted by two doses of carboxyfullerene (2 ␮M and 10 ␮M) in PBMCs from 13 healthy donors indicated a significant decrease in the percentage of apoptotic cells at all exposure time considered (24, 48, and 72 h, Fig. 4). This phenomenon was remarkable, about 50%, at 24 and 48 h of drug exposure, becoming less evident at 72 h of culture. As expected, a time-dependent increase of the percentage of PBMCs with decreased mitochondrial membrane potential (MMP) was found after treatment with dRib or TNF-␣ (Fig. 5). Again, the presence of carboxyfullerene alone did not modify the percentage of cells with decreased MMP in untreated cells (data not shown). By contrast, carboxyfullerene was able to significantly reduce the percentage of cells with depolarised mitochondria in PBMCs after dRibinduced apoptosis. In particular, the lower dose (2 ␮M) was effective at 48 and 72 h, while the higher dose (10 ␮M) exerted a significant activity only at 24 h. When PBMCs were exposed to TNF-␣, both concentrations of carboxyfullerene exerted a protective effect on MMP, particularly at 24 and 48 h of culture. DISCUSSION The present study is the first indication of an antiapoptotic activity of the new class of chemical compounds called buckminsterfullerenes in human PBMCs. In particular, we show that carboxyfullerene C60 protects quiescent PBMCs from apoptosis induced by two different agents, such as dRib and TNF-␣ plus CHX. Both stimuli induce oxidative imbalance by interfering with the redox status of the cell (12, 19). In particular, dRib, a highly reducing sugar, has already been found to inhibit cell proliferation and to induce apoptosis in a variety of cell types, including human PBMCs (12, 13, 20, 21). The cell death induced by dRib provokes a depletion of intracellular levels of reduced glutathione (GSH) and can be fully prevented by the antioxidant N-acetylcysteine (13). TNF-␣ exposure is also known to induce an oxidative imbalance during the signal transduction pathway (19, 22). Thus, in both apoptotic stimuli considered here, a key role is played by radical oxygen species (ROS). Hence, on the basis of the above-mentioned studies, we can hypothesise that the protective effect exerted by carboxyfullerene on

FIG. 4. Percentage of apoptotic cells in presence or absence of two concentrations of carboxyfullerene at different hours of culture. (Upper panel) PBMCs treated with dRib; (lower panel) PBMCs treated with TNF-␣ ⫹ CHX. Data are expressed as mean ⫾ SE of 13 experiments.

dRib- and TNF-␣-induced apoptosis is related to their known scavenger activity towards ROS (4, 8). However, it is interesting to note that although at different extent, carboxyfullerene derivative was capable of significantly inhibiting apoptosis in both the experimental conditions considered, i.e., after dRib or after TNF-␣. In the same vein, the antioxidant also decreased the percentage of cells with depolarised mitochondria. Indeed, our data indicate that carboxyfullerene is slightly more effective in preventing mitochondrial membrane depolarisation induced by TNF-␣ than by dRib, being active at both doses employed and at all exposure time. This is in accord with the hypothesis that TNF-␣ plus CHX could induce apoptosis acting as a pro-oxidant cytokine, stimulating superoxide radical production in mitochondria and in-

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REFERENCES

FIG. 5. Percentage of cells with depolarised mitochondria in presence or absence of two concentrations of carboxyfullerene at different hours of culture. (Upper panel) PBMCs treated with dRib; (lower panel) PBMCs treated with TNF-␣ ⫹ CHX. Data are expressed as mean ⫾ SE of 13 experiments.

ducing Ca 2⫹ release and GSH extrusion from theses organelles (23). Moreover, the protective effect of carboxyfullerene on mitochondrial depolarisation appears to be less effective than that exerted on the percentage of apoptotic cells, as assessed by morphological and flow cytometrical analysis. This could mean that the lowering of apoptotic rate or the delaying of apoptosis by carboxyfullerene could also be due to other activities of the compound located “downstream” to mitochondria in the apoptotic cascade. In fact, it has been recently demonstrated that the carboxyfullerene derivatives exert a considerable potential as enzyme inhibitors (24) and therefore they could inhibit the protease cascade. Further studies are in progress to identify possible effects of carboxyfullerene derivatives on the caspase cascade.

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