This article is mentioned in:

Introduction

A critical area of obesity research is centered on circulating proteins, the levels of which are altered by changes in the body mass index. Understanding how these proteins affect obesity-associated diseases may provide invaluable clues to constrain the devastating consequences of the current obesity epidemic (1). One such protein, visfatin/eNampt/PBEF, has recently received much attention, but its biological effects and mechanisms of action are not well understood. Obesity is the risk factor of breast (2), endometrial (3) and hepatocellular cancer (4), as well as skin melanoma (5). It has been reported that visfatin augments oxidative stress in differentiated myotubes (6) and endothelial cells (7). Nevertheless, the causal relationship between these facts remains unclear. Visfatin is a novel fat derived adipocytokine, secreted by visceral and subcutaneous fat tissues (8,9), hepatocytes (10), monocytes and macrophages (11–13). Visfatin has both intra- and extracellular forms in mammals (14,15). An extracellular form of this protein has been reported to act as a cytokine known as visfatin/PBEF (16), an insulin-mimetic hormone (17), or an extracellular NAD biosynthetic enzyme, visfatin/eNampt (18). Only few studies have investigated the effect of visfatin on cancer cells in vitro and in vivo. Increased expression of visfatin was confirmed in ovarian cancer (19); malignant astrocytoma (20); the human prostate cancer cell lines, LNCaP (androgen-sensitive) and PC3 (androgen-insensitive); as well as in human prostate cancer (21).

Reactive oxygen metabolites (ROMs), such as superoxide anions (O2*−) hydrogen peroxide (H2O2), and hydroxyl radical (OH*), malondialdehyde (MDA) and nitric oxide (NO) are directly or indirectly involved in the multistage process of carcinogenesis (22). They are mainly involved in DNA damage leading to mutations in tumor suppressor genes. They also act as initiators and/or promoters in carcinogenesis (23). MDA, a by-product of lipid peroxidation is likely involved in DNA adduct formations and DNA damage which are believed to also be responsible for carcinogenesis (24). To counteract ROS-induced DNA damage, cells have developed defensive mechanisms that prevent or repair such damage (25). Antioxidant enzyme systems belong to those regulatory mechanisms whose role is to protect against oxidative stress. Under oxidative stress, ROS are rapidly eliminated by antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) (26).

No studies exploring the effects of adipokines (such as visfatin) on oxidative stress, DNA damage in melanoma cells have been conducted so far. This topic is of utmost interest owing to the potential paracrine interactions between subcutaneous adipose tissue and cancer cells. A better understanding of the role of visfatin in melanoma redox states in vitro may provide sound insight into the association between obesity-related fat adipokines and the antioxidant defense system in vitro in cancer progression.

Materials and methods

Cell culture and reagents

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.

Enzyme assay

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.

GSH-Px activity assay

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).

SOD activity assay

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.

CAT activity

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).

MDA assay

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).

Alkaline comet assay

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.

Assessment of cell proliferation

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.

Statistical analysis

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.

Results

Influence of visfatin on selected antioxidative enzyme activities as well as malondialdehyde level in human malignant melanoma Me45 cells

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 assay

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.

Figure 1 (A) Manganese-dependent (MnSOD) and (B) copper and zinc containing (CuZnSOD) superoxide dismutase activity expressed in NU/mg of protein in the cell supernatants of the human malignant melanoma Me45 cell line upon visfatin stimulation. Data represent the means ± SD; n=12 and were analyzed with the one-way ANOVA test and post hoc Bonferroni correction: #p<0.001 vs. control, *p<0.001 vs. 10 ng/ml visfatin, ^p<0.001 vs. 50 ng/ml visfatin, $p<0.001 vs. 100 ng/ml visfatin, @p<0.001 vs. 250 ng/ml.

CAT assay

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).

Figure 2 (A) Catalase (CAT); (B) Glutathione peroxidase (GSH-Px) enzyme activities; and (C) malondialdehyde (MDA) levels in the cell supernatants of the human malignant melanoma Me45 cell line upon visfatin stimulation. C, control (untreated) group. Data represent the means ± SD; n=12, and were analyzed with the one-way ANOVA test and post hoc Bonferroni correction [(enzymes activity; #p<0.001 vs. C, *p<0.001 vs. 10 ng/ml visfatin, ^p<0.001 vs. 50 ng/ml visfatin, $p<0.001 vs. 100 ng/ml visfatin, @p<0.001 vs. 250 ng/ml visfatin); (MDA levels; #p<0.001 vs. C, *p<0.001 vs. 10 ng/ml visfatin, ^p<0.001 vs. 50 ng/ml, $p<0.001 vs. 100 ng/ml, @p<0.05 vs. 250 ng/ml)].

GSH-Px assay

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).

MDA assay

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).

Evaluation of DNA damage

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).

Figure 3 (A) Tail length and (B) tail moment parameter of DNA damage at different time-points (0–180 min) after hydrogen peroxide (H2O2) removal in the human malignant melanoma Me45 cells pre-incubated with visfatin (100 ng/ml) for 24 h and then treated with H2O2 (100 μM) for 5 min (vertically striped bars) in comparison to the cells exposed solely with hydrogen peroxide (slantwise striped bars) or cells treated with visfatin without adding H2O2 (black bars). The negative control groups were cells not exposed to hydrogen peroxide and/or visfatin (white bar). Results are shown as a median tail moment expressed in arbitrary units (a.u.) or tail length ± first quartile (Q1), third quartile (Q3); n=100; Mann-Whitney U test. For repeated measures a Friedman test with the Schaich and Hamerle post hoc procedure was used (p<0.05, respectively).

Effects of visfatin on the proliferation and viability of Me45 melanoma cells

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).

Figure 4 Effects of various concentrations of visfatin or insulin on [3H]thymidine incorporation and cell number in human malignant melanoma Me45 cells. (A) Cells were exposed to 10–250 ng/ml visfatin or 100 ng/ml insulin for 24 h. Cell proliferation was determined by measuring [3H]thymidine incorporation. Results are expressed as counts per minute. Bars represent means ± SD (n=12). #p<0.001 vs. control (untreated cells). (B) Cells were exposed to 10–250 ng/ml visfatin or 100 ng/ml insulin for 24 h. Cell proliferation was determined by cell counts. Results are expressed as the means ± SD fold increases (n=12) over control values. #p<0.001 vs. control.

Figure 5 Effects of visfatin or insulin in combination with inhibitor of insulin receptor (HNMPA-AM)3 on the proliferation of human malignant melanoma Me45 cells. Cells were incubated with HNMPA-(AM)3 (50 μM) for 3 h prior to treatment with 100 ng/ml visfatin or insulin 100 ng/ml for 24 h. Cell proliferation was determined using [3H]thymidine incorporation. Bars represent the means ± SD (n=12); *p<0.001 vs. untreated cells, #p<0.001 vs. visfatin-treated control, ^p<0.001 vs. insulin-treated control.

Insulin receptor signaling

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).

Discussion

There are no data describing the interactions in subcutaneous microenvironments between adipocytes that secrete various adipokines (such as visfatin) and neighboring melanocytes as well as melanoma cells. This relationship might affect the malignant transformation process of melanocytes. Therefore it seems important to initiate studies that explore the potential paracrine influence of adipokines on melanocytes and melanoma cells.

We studied the role of fat derived-visfatin on antioxidative enzymatic activity and the level of lipid peroxidation process in supernatants of Me45 melanoma cells. In the present study, we observed increased activity of antioxidant enzymes (SOD isoenzymes, catalase and glutathione peroxidase) in cell supernatants upon stimulation with visfatin at the concentration of 10–100 ng/ml. Increased mitochondrial MnSOD isoenzyme activity after visfatin treatment suggests that visfatin increases efficiency of the electron transport chain in the inner mitochondrial membrane and stimulates complex I and IV of the electron transport chain in the mitochondrion (33). Cells subjected to the action of the visfatin at the concentration of 250 ng/ml for 24 h showed higher mitochondrial oxidative stress compared to the other group. In our previous study we also observed a marked increase in MnSOD activity after exposure of AT478 tumor cells to extremely low frequency electromagnetic field (ELF-EMF) and vitamin E. This action of ELF-EMF and vitamin E is expected to increase efficiency of oxidative phosphorylation and adenosine triphosphate production and the high efficiency of ROS elimination by MnSOD itself (34). In this study we also reported increase in CuZnSOD superoxide dismutase isoenzyme activity in Me45 cells subjected for 24 h to different concentrations of visfatin.

SOD isoenzymes catalyze the conversion of O2− to H2O2, which can then be converted to water by catalase (CAT) or glutathione peroxidase (GSH-Px) coupled with glutathione reductase (GR) (26). We observed a marked increase in CAT activity in Me45 melanoma cells treated with all concentrations of visfatin. Increased activity of GSH-Px or CAT in cancer cells can make tumor cells less susceptible to anticancer drugs such as doxorubicin (36,39). Samuels et al(35) demonstrated that the addition of radical scavengers and compounds with peroxide activity can reduce the cytotoxic effect of anticancer drugs in vitro. Increased GSH-Px or CAT activity enables cells to survive with the high level of ROS and maintain cellular viability (36).

In our study, application of visfatin (10–100 ng/ml) also decreased the level of lipid peroxidation in Me45 cells with augmented GSH-Px activity. On the contrary, MDA concentration was increased in melanoma cells stimulated by visfatin in higher concentrations and also decreased GSH-Px activity. ROS are known to cause DNA damage and lipid peroxidation/oxidation in cellular membranes (26). MDA is one of the final products of polyunsaturated fatty acid peroxidation in cells. An increase in free radicals causes overproduction of MDA (37). Glutathione peroxidase is an enzyme that reduces hydrogen peroxide as well as organic superoxides. In such reactions, an organic superoxide (ROOH) becomes reduced to an appropriate alcohol (ROH). In the case of lipid superoxide this means that it cannot become an initiator of lipid peroxidation and, therefore, GSH-Px inhibits the lipid peroxidation process (31,37). The results of our experiments with low concentrations of MDA in the group stimulated with 10–100 ng/ml visfatin is concordant with the high activity of the enzyme inhibiting lipid peroxidation process-glutathione peroxidase (GSH-Px) in this study group when compared to the control group. However, we speculate that the Me45 melanoma cells exposed to visfatin at the concentration of 250 ng/ml may show decreased resistance toward exogenic H2O2 and higher oxidative stress in comparison to the cells treated with lower concentrations of visfatin.

We suppose that visfatin at the concentration of 10–100 ng/ml triggers a redox adaptation response, leading to an upregulation of the antioxidant capacity (SOD isoenzymes, CAT and GSH-Px enzymes).

Moreover, the increased oxidative-stress induces oxidative DNA damage and lipid peroxidation process. We hypothesized that visfatin mediated activation of antioxidant enzymes with decreased levels of MDA may lead to the reduction of DNA damage in Me45 melanoma cells. We pretreated these cells with visfatin at the concentration of 100 ng/ml for 24 h. At every time-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. The results suggested a protective role of visfatin in the Me45 melanoma cells exposed to the genotoxic reagents. Pretreatment with visfatin before adding hydrogen peroxide decreased DNA damage in the Me45 cells. Our observation of increased antioxidant enzyme activity in melanoma cells after visfatin stimulation shows that enzymatic defense mechanisms are used for ROS scavenging and reduce the level of DNA damage in these cells. Moreover, the stationary DNA damage (without adding H2O2) was also decreased in the visfatin-treated group when compared to the untreated group (but only in tail moment parameter of DNA damage). Collectively, we demonstrated for the first time the protective role of visfatin in hydrogen peroxide-induced DNA damage in a human malignant melanoma cell line. However, Wang et al(38) demonstrated that visfatin exerted anti-apoptotic effects under a cell apoptotic state induced by H2O2 in vascular smooth muscle cells (38). Moreover, Adya et al(7) reported that visfatin exerted a protective role in H2O2-induced endothelial cell apoptosis. Visfatin at 100 ng/ml in combination with H2O2 (200 μM) markedly increased the number of viable cells by 73% in comparison to the group treated solely with hydrogen peroxide (only 19% of viable endothelial cells in the culture) (7). This is consistent with our report, which demonstrates that visfatin at 100 ng/ml also increased the level of viable melanoma cells in the group (visfatin + H2O2) along with a decreased level of DNA damage when compared to cells treated solely with H2O2.

We also examined the effect of visfatin on Me45 melanoma cell proliferation. Previous studies reported that visfatin stimulates proliferation of osteoblasts (39), vascular smooth muscle cells (VSMC) (38) and prostate cancer cells (PC-3 line) (21). The present study demonstrated that visfatin (10–100 ng/ml), similar to insulin, stimulates proliferation of Me45 cells in vitro in a dose-dependent manner. Patel et al(21) also reported that visfatin (25–400 ng/ml) increases proliferation of the PC-3 prostate cancer cell line in a dose-dependent manner after 24 h of incubation. The most pronounced proliferation rate increase (approximately 2-fold) was observed in the PC-3 cells subjected to visfatin at the concentration of 400 ng/ml. In the present study, an almost 2-fold increase of the proliferation rate was observed in Me45 cells treated with 100 ng/ml visfatin with increased number of tumor viable cells. The supraphysiological concentration of visfatin in culture medium was similar to the concentrations used in other in vitro studies (6,7,13,17,21,38,39). These observation are concordant with a report which shows that increased GSH-Px or CAT activity enables cells to survive with the high level of ROS and maintain cellular viability (36). By contrast, in this case, high concentration of visfatin (250 ng/ml) led to a significant decrease of [3H]thymidine incorporation of Me45 cells with respect to the untreated cells. The effect on proliferation was demonstrated in a concentration-dependent manner and perhaps more importantly at levels of visfatin similar to those that have been demonstrated in obese patients (40). This provides novel evidence that elevated levels of circulating visfatin may influence the growth of melanoma cancer cells. According to other research studies, visfatin can mimic the effects of insulin on the activation of insulin signaling (17,39). In particular, two studies in monocytes and in osteoblasts have indicated that IR may be involved in visfatin function; both studies demonstrated that visfatin effects could be blocked by IR tyrosine kinase inhibitor, HNMPA-(AM)3(13,39). However, recent studies indicated that visfatin cannot activate IR (41) and proposed that visfatin may act on its own unidentified receptor (42). Therefore, to determine whether visfatin-induced Me45 melanoma cell proliferation is mediated by IR, we used HNMPA-(AM)3 - inhibitor of IR to elucidate this matter, with insulin as a comparison. In our study melanoma cultures pretreated with visfatin and the IR tyrosine kinase inhibitor HNMPA-(AM)3 acted similarly to cells cultured in the absence of the latter. These observations indicated that visfatin-induced melanoma cell proliferation is not mediated by the IR transduction signaling pathway. We also demonstrated that the HNMPA-(AM)3 significantly inhibited insulin-stimulated proliferation of melanoma Me45 cells.

Our study has a few limitations. Its in vitro setting may not fully reflect more complex relationships in organisms as a whole. Additionally, we observed an increased proliferation rate occurring during cell culture. However the magnitude of proliferation was relatively much lower than the effect of visfatin on studied parameters, therefore we considered the influence of this observation as negligible in the study results.

In conclusion, this study provides the first evidence that visfatin can reduce the level of DNA damage of Me45 melanoma cells, probably due to increased levels of antioxidative enzyme activities, and can promote melanoma cell proliferation in a dose-dependent manner.

Acknowledgements

This study was supported by grants from the Silesian Medical University in Katowice, Poland. Grant nos. KNW-1-134/08, KNW-1-078/09 and KNW-1-040/P/1/0.

References