A human malignant melanoma Me45 cell line was obtained from the Silesian University of Technology, as a kind gift from Dr M. Wideł from the Marie Curie Memorial Cancer Centre and Institute of Oncology, Gliwice, Poland. The cells were derived from metastatic lesions (local lymph nodes) of a 35-year-old patient of the Institute of Oncology (27). Me45 cells were plated at the density of 1×10⁶ cells per 25 cm² flask and cultured in the Dulbecco’s modified Eagle’s medium with L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum (Gibco, North Androver, MA, USA), antibiotics: penicillin (10,000 IU/ml), streptomycin (10,000 μg/ml), amphotericin B (2.5 μg/ml) (Sigma-Aldrich) under the atmosphere of 95% air and 5% CO₂ at 37°C. The Me45 cell line was free of mycoplasma, pathogenic viruses and bacteria. Cultures were maintained for no longer than 4 weeks after recovery from the frozen stock.

Visfatin (Alexis Biochemical, Plymouth, PA, USA) was dissolved in phosphate buffered saline (PBS) without Mg²⁺, Ca²⁺ (Sigma-Aldrich). The visfatin solutions were prepared fresh, protected from light, and added to the incubation medium obtaining the following final concentrations: 10, 50, 100 and 250 ng/ml. The purity of visfatin was 96–97% (SDS-PAGE analysis) and contained <0.01 ng/μg LPS as determined by the Limulus amebocyte lysate method.

Hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)₃], and insulin were also obtained from Sigma Chemicals Co.

After exposure of the cultured cells to visfatin for 24 h, antioxidative enzyme activities: MnSOD, Cu/ZnSOD, GSH-Px, CAT, and the level of malondialdehyde (MDA) were measured in cell supernatants. Cells were collected after 24 h of incubation with different doses of visfatin (10–250 ng/ml) and were centrifuged (2,000 rpm, 5 min) and supernatants were kept at −80°C until analysis. All experiments were carried out in duplicate. Sample size in all experiments was 12.

The method of Paglia and Valentine, (28) was used with minor modifications. Briefly, Me45 melanoma cells were pooled to a concentration of 5×10⁶ cells/μl. Following centrifugation, the cell pellet was mixed with cell lysis buffer and then sonicated for 10 sec. The protein concentration was measured using Bio-Rad protein reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal volumes of each sample containing 30 μg of protein were mixed with 2.68 ml of 0.05 M phosphate buffer (pH 7.0) containing 0.005 M ethylenediaminetetraacetic acid (EDTA). The following solutions were then added sequentially: 0.100 ml of 0.0084 M NADPH, 0.010 ml of glutathione reductase (GR), 0.010 ml of 1.125 M sodium nitrate (NaNO₃), and 0.100 ml of 0.15 M reduced glutathione (GSH). The enzymatic reaction was initiated by the addition of 0.100 ml of 0.0022 M H₂O₂. The conversion of NADPH to oxidized NADP⁺ was followed by continuous recording of the change in absorbency at 340 nm between 2 and 4 min after the initiation of the reaction. The control measurements were recorded in a simultaneous assay where the sample was replaced by an equal volume of cell lysis buffer. GSH-Px enzyme activity (1 IU) is defined as 1 mM NADPH converted to NADP⁺ per mg of protein (IU/mg p).

The SOD activity assay was based on a minor modification of the procedure described by Paoletti and Mocali (29). The preparation of cell lysates and the measurement of protein concentration were the same as in the GSH-Px assay. Protein (30 μg) from each sample was mixed with 0.8 ml of 1X triethanolamine-diethanolamine buffer (TDB); (pH 7.4), 40 μl of 7.5 mM NADPH (a reduced form of nicotinamide adenine dinucleotide phosphate), and 25 μl of 100 mM EDTA-MnCl₂. The reaction was initiated by the addition of 0.1 ml of 10 mM mercaptoethanol. The decrease in absorbance at 340 nm was recorded over 20 min at room temperature. The control consisted of a reaction mixture in which the sample was replaced by an equal volume of cell lysis buffer. For the determination of MnSOD activity, CuZnSOD activity was inhibited by incubating samples with 5 mM potassium cyanide (KCN) for 30 min with samples assayed for activity within 2 h of adding cyanide to the sample mixture. Total specific SOD and MnSOD (after CuZnSOD inhibition with KCN) activity levels were measured, and then the CuZnSOD activity was calculated. The enzymatic activity of both SOD isoenzymes was expressed in nitric units (NU) per mg of protein (NU/mg p); 1 NU represents 50% inhibition by SOD of the nitrosol ion formation under these conditions.

Catalase activity was measured spectrophotometrically as described by Aebi (30). Direct disappearance of 10 mM hydrogen peroxide in 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA was measured at 240 nm >30 sec on a Beckman DU-70 spectrophotometer. Enzyme activity was calculated based on the molar extinction coefficient of hydrogen peroxide at 240 nm (ɛ = 39.4 M⁻¹ cm⁻¹) and reported as μmol hydrogen peroxide decomposed per minute, recalculated to kIU per mg of protein (kIU/mg p).

The measurement of MDA, a product of lipid peroxidation, was determined by the thiobarbituric acid (TBA) reaction as described by Placer et al(31). Aliquots of reaction buffer (1.5 ml) that included 50 μg of protein from each sample and 1.4 ml of 0.2 M Tris-0.16 M KCl (pH 7.4) were incubated at 37°C for 30 min. Next, 1.5 ml of TBA reagent were added, and the mixture was heated in a boiling water bath for 10 min. After cooling with ice, 3.0 ml of pyridine:n-butanol (3:1, v/v) and 1.0 ml of 1 M NaOH were added and mixed by shaking, and the absorbance was read at 548 nm. The blank control contained the same reaction mixture but was not incubated. The level of MDA was expressed as μmol MDA per mg of protein (μmol MDA/mg p).

All chemicals were purchased from Sigma. The comet assay was carried out under alkaline conditions, as described by Olive and Banáth (32). Fully frosted slides were covered with 1% normal melting point agarose (NMP agarose). Me45 cells (6×10³), and the same number of cells pretreated with visfatin at the concentration of 100 ng/ml for 24 h were washed twice, trypsinized and resuspended in 2 ml fresh medium. Half of the cells from each group were treated for 5 min with H₂O₂ (100 μM), and the genotoxic factor was removed by centrifugation (2,000 rpm/3 min) with supernatants. Subsequently, cells were resuspended in 2 ml medium and incubated for 0 min (control for determination of the basal DNA damage) or 5, 15, 30, 60, 120 and 180 min (for determination of the residual DNA damage). The resuspended cells (50 μl) were mixed with 100 μl 0.5% low melting agarose at 37°C and transferred (100 μl) onto microscope slides. The cells not treated with H₂O₂ were collected at similar time-points.

The slides were immersed for 1 h in the ice-cold, freshly prepared lysis solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris-HCl, pH 10.0) with 1% Triton X-100 and 1% dimethylsulfoxide added fresh for cell destruction and DNA unfolding. The slides were then randomly placed side by side in the horizontal gel-electrophoresis tank, facing the anode. The unit was filled with freshly prepared electrophoretic buffer (300 mM NaOH, 1 mM Na₂EDTA, pH 13.0) and the slides were set in this alkaline buffer for 15 min to allow DNA to unwind and express alkali-labile sites. Denaturation and electrophoresis were performed at 4°C under dim light. Electrophoresis was carried out for 20 min at 25 V (300 mA). Following electrophoresis, the slides were washed gently 2× at 5-min intervals with the neutralization buffer (0.4 M Tris-HCl, pH 7.5) to remove excess alkali and detergents. Slides were fixed in chilled 70% ethanol and stained with ethidium bromide (20 μg/ml) and coverslipped. Slides were stored at 4°C in humidified sealed containers. To prevent additional DNA damage, handling samples and preparation of the slides for the comet assay were conducted under yellow light or in the dark. To avoid possible position effects during electrophoresis, two parallel replicate slides per sample were prepared. Each replicate was processed in a different electrophoretic run.

The Comet images were visualized after ethidium bromide staining (20 μg/ml) using a fluorescent microscope (Axiophot M1, Zeiss, Jena, Germany) that was fitted with an excitation filter of 515–535 nm and a barrier filter of 590 nm at ×40 magnification. The Comet images were captured using an online charge-coupled device (CCD) camera and analyzed using the Comet Score software (version 5.5) from Andor Technology (Belfast, UK). The tail moments were quantified for a minimum of 100 random cells per sample using the Comet Score software. The tail moment is defined as the ratio of DNA in the comet tail to total DNA.

Me45 cell proliferation was assessed using scintillation counting to measure the incorporation of [³H]thymidine (2 μCi/ml) into trichloroacetic acid (TCA)-insoluble material. Cells were plated at 2×10⁴/well in 24-well plates and treated with 10–250 ng/ml visfatin or insulin (100 ng/ml) for 24 h. To study the effects of insulin receptor (IR) inhibition, HNMPA-(AM)₃ (50 μM) was added 3 h before visfatin or insulin, in the presence of [³H]thymidine. After 24 h, the plates were washed with PBS and 10% TCA was added to the wells. Incorporated [³H]thymidine was released by washing with 0.2 N of NaOH, and radioactivity was measured using β-scintillation counter (Wallac 1410, Pharmacia, Freiburg, Germany). Results were shown as counts per minute. To determine visfatin’s effect on the cell number, melanoma cells were plated into 96-well plates at 2×10³/well and treated with 10–250 ng/ml visfatin or 100 ng/ml insulin for 24 h. Cells were harvested by trypsinization, and the viable ones were counted in an automated cell counter (model no. TC10; Bio-Rad Laboratories) using 0.04% trypan blue.

The normality of distribution was evaluated by means of Shapiro-Wilk’s test. All results are presented as the means ± SD or a median (IQR). Results underwent statistical analysis, applying the one-way ANOVA test with the Bonferroni’s post hoc test or Kruskal-Wallis with Mann-Whitney U test, according to parameter distribution. For repeated measures a Friedman test with Schaich and Hamerle post hoc procedure was used in comet assay data. P<0.05 was considered to indicate statistically significant differences.

Visfatin treatment resulted in an increase in antioxidative enzyme activities SOD: (MnSOD, CuZnSOD isoenzymes) and CAT, GSH-Px compared to the control group.

SOD isoenzyme activities (MnSOD, CuZnSOD) after 24 h of incubation with different concentrations of visfatin are shown in Fig. 1. These experimental groups also showed a statistically significant increase of the activities of both isoenzymes, in relation to the control group. The highest MnSOD isoenzyme activity was observed in the group treated with visfatin at a 100 and 250 ng/ml concentration compared to the control group (6.22±0.55, 6.29±0.34 vs. 3.42±0.34 NU/mg p; p<0.001); (Fig. 1A). The MnSOD activity in the two other groups treated with visfatin remained unaltered. All concentrations of visfatin led to a significant 2-fold increase of copper and zinc containing superoxide dismutase isoenzyme (Cu/ZnSOD) activity when compared to the untreated group; (3.42±0.36 3.43±0.52 3.52±0.75, 4.01±0.90 vs. 1.43±0.49 NU/mg p; p<0.001); (Fig. 1B) The dose-dependency in influencing CuZnSOD by various visfatin concentrations was not observed.

In comparison to controls, a significant increase in catalase (CAT) activity in all experimental groups was found at 24 h. CAT activity was ~2-and 2.5-fold higher in the group stimulated with visfatin at 10 and 50 ng/ml than in the control group (17.96±1.66, 19.12±1.50 vs. 7.55±1.37 kIU/mg p; p<0.001, respectively). Moreover, a 4-fold increase in the CAT activity was observed in the group treated with visfatin at a concentration of 100 and 250 ng/ml with respect to the control group; (32.1±2.83, 35.66±2.51 vs. 7.55±1.37 kIU/mg p; p<0.001; respectively) (Fig. 2A).

GSH-Px activity was ~2-fold higher in the groups treated with visfatin at 50 and 100 ng/ml compared to the control group (190.9±31.6, 197.1±20.3 vs. 93.51±6.1 IU/mg p; ^@p<0.001, respectively). However a significant decrease in GSH-Px activity was observed when cells were treated with visfatin at a 250 ng/ml concentration (55.8±20.03 vs. 93.51±6.1 IU/mg p; p<0.001; respectively) (Fig. 2B).

We also studied the influence of visfatin on the MDA level in the supernatants of Me45 cells. The MDA level was 2-fold lower in the group stimulated with 100 ng visfatin compared to the untreated group (0.79±0.10 vs. 1.49±0.17 μmol MDA/mg p; p<0.001, respectively). On the contrary, the MDA concentration was increased in Me45 cells stimulated by visfatin at a 250 ng/ml concentration (1.69±0.5 vs. 1.49±0.17 μmol MDA/mg p; p<0.001, respectively) (Fig. 2C).

We also examined the effect of visfatin at the concentration of 100 ng/ml on DNA damage in Me45 cells. The DNA damage after an incubation of these cells with the genotoxic factor H₂O₂ was compared with cells pre-incubated with 100 ng/ml of visfatin (24 h) and H₂O₂ at different time-points (0–180 min). Fig. 3 shows the level of DNA damage at different time-points (0–180 min) after visfatin and H₂O₂ treatment, in comparison to the untreated cells and the cells treated separately, only with H₂O₂ or visfatin-control groups. These results are presented as tail length (Fig. 3A) and median tail moment parameter (Fig. 3B) with first and third quartile. In every temporal point after H₂O₂ removal, the DNA damage (expressed as median tail moment or tail length) was decreased in the group treated with visfatin and H₂O₂, compared to the group treated solely with H₂O₂. Me45 melanoma cells treated with both visfatin and hydrogen peroxide exhibited significantly less DNA damage than the cells treated solely with hydrogen peroxide (p<0.05). One hundred and eighty minutes after H₂O₂ treatment, the DNA damage level in cells treated with both visfatin and H₂O₂ was lower than in cells treated solely with H₂O₂ (p<0.05). Addition of visfatin to H₂O₂-treated cells resulted in earlier recovery in DNA damage resulting in similar levels of tail moment and tail length 120 min after removal of H₂O₂. The stationary DNA damage (without adding H₂O₂) was also decreased in the visfatin-treated group when compared to untreated controls (but only in the tail moment parameter of the DNA damage); p<0.05 (Fig. 3).

The measurement of [³H]thymidine incorporation showed that visfatin at 10–100 ng/ml stimulated proliferation of Me45 cells in a dose-dependent manner. Visfatin at the concentration of 250 ng/ml led to a significant decrease in the proliferation rate of these cells (15%) when compared to untreated melanoma cells (p<0.05) (Fig. 4A). Compared to the control group, visfatin significantly increased [³H]thymidine incorporation into melanoma cells by 32.1% (10 ng/ml), 63% (50 ng/ml) and 91.2% (100 ng/ml) (all p<0.05) (Fig. 4A). Moreover, compared to the control group, visfatin at 10, 50 and 100 ng/ml increased the number of viable melanoma cells by 9, 15 and 36.3%, respectively (all p<0.05) (Fig. 4B). On the contrary, visfatin at 250 ng/ml decreased the number of viable melanoma cells by 15% (Fig. 4B) in comparison to the untreated group (p<0.05). Fig. 5 shows that insulin also promoted proliferation of Me45 cells. Insulin at 100 ng/ml markedly increased [³H]thymidine incorporation of Me45 cells by 85.4% with augment number of viable cells (24% rise vs. untreated cells; p<0.05). No statistically significant differences between study groups treated with various concentrations of visfatin were observed at the level of α= 0.05 (excluding visfatin at the concentration of 250 ng/ml).

Melanoma cells pretreated with the IR tyrosine kinase inhibitor HNMPA-(AM)₃ did not show reduced proliferation in visfatin-treated cells (Fig. 5). Additionally, the inhibitor HNMPA-(AM)₃ significantly inhibited insulin-stimulated proliferation of melanoma Me45 cells by 27% (p<0.05) (Fig. 5). Furthermore, the immunoblot analysis revealed that IR protein expression (95 kDa) could be detected in Me45 melanoma lysates (not shown).