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1 From the Department of Ophthalmology, University of L’Aquila; the 2 San Gallicano Dermatological Institute, Rome, Italy; and 3 Medical Genetics, University "La Sapienza," Rome, Italy.
Abstract
PURPOSE. To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes.
METHODS. The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer.
RESULTS. Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P < 0.001) and lower SOD activity. Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022). In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P < 0.005).
CONCLUSIONS. These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution. However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects. Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression.
Despite diagnostic accuracy and the introduction of new conservative treatment modalities over the past decades, the survival rate in patients affected by uveal melanoma has not been substantially modified.1 Furthermore, the identification of risk factors for the development of this tumor has proved to be unsuccessful so far. Although clinical, epidemiologic, and biological observations support the hypothesis that UV light is an etiologic agent for cutaneous melanoma in white populations,2 no irrefutable evidence has appeared in favor of a role for sunlight in uveal melanoma. However, in both cutaneous and uveal melanomas, triggering factors related to the generation of free radical species, and subsequent oxidative stress, can be considered. Oxidative stress results from an imbalance between prooxidant agents and antioxidant systems. Wavelengths in the UVA range (320–400 nm; i.e., not directly absorbed by DNA but capable of generating reactive oxygen species; ROS) are considered to be effective in inducing cutaneous melanoma, at least in animal models.2 On the other hand, uveal vascular changes may significantly increase the amount of ROS generated. Therefore, the evaluation of the cellular antioxidant system could be crucial in understanding the pathway of tumorigenesis.
Recently, we have shown the presence of an antioxidant system imbalance in cultured cutaneous melanoma cells compared with normal cutaneous melanocytes, and we suggested that this alteration could be related to the disease status and progression.3 4 A similar alteration was detected in melanocyte cultures from the apparently normal skin of patients with melanoma. In the present study we investigated the antioxidant status of normal uveal melanocytes from patients with uveal melanoma and cultured uveal melanoma cells, to contribute to the characterization of the biochemical properties of these cells. Therefore, we studied the activity of the intracellular scavenger enzymes, superoxide dismutase (SOD) and catalase, the intracellular levels of vitamin E, and the pattern of fatty acids of cell membranes, and we have compared the results with those previously obtained in normal cutaneous melanocytes.3 4
Methods
Xanthine, xanthine oxidase, and nitro blue of tetrazolium (NBT) were from Sigma (St. Louis, MO). Ham’s F-10 medium, fetal calf serum (FCS), and antibiotics were provided by GIBCO (Paisley, Scotland, UK). Butylated hydroxytoluene, tricosanoic acid (C23:0), N,O-bis(trimethylsilyl)trifluoro-acetamide and trimethyl chorsilane, sodium methoxide, and other reagents and solvents were from Merck AG (Darmastadt, Germany) and were of the highest purity grade.
Cell Cultures
Normal uveal melanocytes from uveal melanoma patients (UM,
n = 10) were collected from eyes, enucleated for large
choroidal and ciliary body melanomas, at the site diametrically opposed
to the tumor, and uveal melanoma cells (UMC, n = 10)
were taken from the same eyes. Uveal melanocytes and uveal melanoma
cells were isolated after mechanical and enzymatic dissection and
cultured as previously described for cutaneous
melanocytes.3
4
Cells were cultured in Ham’s F-10 medium,
with 5% FCS and 5000 UI/ml penicillin and 5000 ng/ml streptomycin, and
in melanocyte cultures, bovine pituitary gland extract (50 µg/ml) was
added. Subconfluent cultures at the fourth or fifth passage were
studied. The batch of FCS was the same for all the experimental periods
and was analyzed for fatty acid pattern and vitamin E level.
Antioxidant Enzyme Assays
Cells (4 x 106) were collected
and sonicated in phosphate-buffered saline (pH 7.4; GIBCO; 1 ml) and
centrifuged at 10,000g for 10 minutes at 4°C. Enzymatic
activities were evaluated by a spectrophotometer (Beckman DU 70) on
cell supernatants. Catalase activity was determined by the
disappearance of hydrogen peroxide,3
4
and SOD activity
was determined by evaluating the inhibition of reduction of NBT by
superoxide produced by xanthine–xanthine oxidase
system.3
4
One unit of catalase was defined as the amount
that degrades 1 µmol of
H2O2, and 1 unit of SOD was
defined as the amount of enzyme that produces 50% inhibition of NBT
reduction. The activities of cell supernatants were compared with the
known purified enzymes and then calculated per
106 cells. At least two determinations were
performed on each supernatant, and experiments were repeated twice. The
results are reported as the mean of different determinations and
expressed as unit/106 cells. For each group of
cultures data are reported as mean ± SD.
Vitamin E Analysis
Cells (4 x 106) were extracted three
times in hexane–ethanol (3:1) with 1% sodium dodecyl sulfate in the
presence of 25 ng of 
and 
tocopherols as internal standards.
Tocopherols were derivatized with
N,O-bis-(trimethylsilyl)-trifluoroacetamide with 1% trimethyl
chlorsilane as catalyst and were analyzed by gas chromatography mass
spectrometry on SPB1 column (30 m x 0.20 µm ID, 0.25 mm,
Supelchem) by a selected ion(s) monitoring technique.3
4
Analyses were repeated twice in each extract, and a SD less than 1%
was found. Results are expressed as nanograms per
106 cells.
Polyunsaturated Fatty Acid Analysis
Cell pellets were extracted twice in chloroform:methanol (1:1) in
the presence of butylated hydroxy toluene (50 µg) as antioxidant and
25 µg tricosanoic acid ethyl ester as internal standard. The fatty
acids of phospholipid fraction were trans-methylated with
sodium methoxide in methanol and analyzed by gas chromatography mass
spectrometry on capillary column (FFA-P, 60 m x 0.32 µm x
0.25 mm; Hewlett–Packard). The results were obtained after
time integration of the chromatogram and final processing of the peak
areas. The identity of each fatty acid was determined by comparing the
mass spectrum of the peaks with those obtained using reference
standards.3
4
Statistical Analysis
Student’s t-test was used to determine the statistical
significance. Statistical significance was accepted as
P < 0.05. The correlation existing was studied by
linear regression analysis and r values were calculated by
the Pearson test.
Morphologic Analysis
Cell cultures of normal uveal melanocytes and uveal melanoma cells
were seeded into 2% gelatin–coated tissue Transwell
andcultured in complete melanocyte medium. Seventy-two
hours afterward cells were washed three times in PBS and incubated for
60 minutes at 4°C in fixative buffer (5% milk, 0.01% Tween-20, 0.3
M NaCl, and 2% glutaraldehyde). After fixative treatment, samples were
postfixed in 1% osmium tetroxide in Veronal acetate buffer (pH 7.4)
for 2 hours at 4°C, stained with uranyl acetate (5 mg/ml), dehydrated
in acetone, and embedded in Epon 812. Thin sections were examined both
unstained or poststained with uranyl acetate and lead hydroxide.
Results
The clinical and histologic features of the lesions of the studied cases are reported in Table 1 . The cultures obtained were studied at subconfluence, at the fourth or fifth passage grown in medium containing the same batch of fetal calf serum, so that the amounts of vitamin E, external fatty acids, and essential elements were the same in each culture. The levels of antioxidants were expressed as units per 106 cells or nanograms per 106 cells, to minimize differences among results due to cell size and shape and to compare the values with those previously obtained in normal cutaneous melanocytes.
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Antioxidant Pattern
In uveal melanocytes mean catalase activity was 1.06 ± 0.81
U/106 cells, a value not significantly different
from that reported in cutaneous melanocytes (CM) (Table 2)
,3
4
but the elevated SD suggested a wide range of
variability in this population. A similar situation was reported in a
previous work of ours, in cultures of cutaneous melanocytes from the
apparently normal skin of subjects with melanoma.3
4
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Levels of vitamin E were 1.15 ± 0.15 ng/106 cells in UM-A and 1.04 ± 0.73 ng/106 cells in UM-B. Both values were significantly lower than those previously observed in normal cutaneous melanocytes (Table 2) .
In UMC, SOD activity was 0.34 ± 0.11 U/106 cells, significantly lower than that observed in the UM-A and UM-B groups, respectively (P < 0.005). In the same cells, catalase activity was 0.42 ± 0.18 U/106 cells, a value significantly lower than that observed in UM-A but not dissimilar from that found in UM-B (Table 2) .
In UMC mean vitamin E concentration was 3.09 ± 2.68 ng/106 cells, higher but not statistically different from that observed in both UM-A and UM-B even if with a wider range of variability (Table 2) . A relationship was found between lipophilic and enzymatic antioxidants, with a significant direct correlation between SOD/catalase ratio and vitamin E level (R = 0.73, P = 0.015; Fig. 1 ).
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Our study demonstrates that cultured uveal melanocytes from patients with melanoma, compared with cultured normal cutaneous melanocytes,3 4 have a higher level of enzymatic antioxidant activities. In particular, SOD activity appears to be significantly higher, possibly related to the high O2 tension found in the choroidal district. SOD, in fact, is important in clinical situations such as in reperfusion after ischemia, where huge amounts of superoxide anion (O2-·) are generated and need to be dismutated by this enzymatic activity.
More interestingly, as previously described in cutaneous melanocytes from melanoma patients,3 4 two subgroups of uveal melanocytes were identified on the basis of catalase activity: one with values comparable to those observed in cutaneous melanocytes (UM-A) and the other with significantly lower values (UM-B). Consequently, in the B group, the SOD/catalase ratio, which is correlated with the susceptibility of the cells to a peroxidative stress,5 was significantly increased compared with that of group A. The SOD/catalase ratio imbalance represents an alteration of the scavenger system. Antioxidants, in fact, interact in a complex fashion, so that changes in the concentration or activity in one component can affect the whole system. SOD dismutates superoxide anion radicals, generating hydrogen peroxide and oxygen. Catalase and glutathione peroxidase (GSH–Px) are the main enzymes involved in removing H2O2.6 If the production of H2O2 overwhelms the activities of these latter enzymes, in the presence of transitional metals (Fe2+, Cu+), H2O2 becomes a substrate of the Fenton reaction, giving rise to extremely toxic and mutagenic hydroxyl radicals (HO·).6 Moreover, the imbalance of the SOD/catalase ratio was associated with an increased PUFA percentage in the cell membranes of UM-B, suggesting that these cells are more susceptible to the deleterious effects of prooxidants. In vitro, cutaneous melanocytes with alteration of the SOD/catalase ratio, in fact, undergo a significant proliferation after treatment with a low concentration of cumene hydroperoxide.4 Moreover, cell cultures deficient in catalase activity showed an increased DNA alteration after exposure to peroxidizing agents.7 Therefore, the imbalance of the intracellular antioxidants has been considered as a possible additional risk factor for the development of melanoma.3 4
In uveal melanoma cells, instead, a significant decrease of SOD activity, compared with that of uveal melanocytes, was detected, whereas catalase activity, even if lower, was not significantly modified. An increased level of the polyunsaturated component of cell membranes and vitamin E concentration was observed. The higher vitamin E level is likely to be a compensatory mechanism adopted by the cells, at least in vitro, to reduce the intracellular oxidative events. In fact, in UMC a significant direct correlation was observed between the SOD/catalase ratio and the vitamin E level. These results are in agreement with previous data that demonstrated the correlation among the differentiation status, antioxidant systems, and percentage of PUFAs in cultured cells: the higher the proliferation rate, the less differentiated the cells; the lower the total antioxidant protection system, the higher the PUFA percentage.8 However, no correlation was found between alteration of the antioxidant pattern and the melanoma cell type.
The source of ROS for the development of cutaneous melanoma could be UV exposure,2 but uveal melanocytes, especially those embedded in the ciliary body, are reached by low amounts of UV radiation; therefore, different free radical sources should be considered. Uveal melanocytes are tightly connected with the vascular bed and the oxidative insults might be related to hemodynamic changes. Alterations in blood flow can induce free radical release, and free radical–mediated injury has been involved in the pathophysiological alterations observed during ischemia and reperfusion.9
Episodes of choroidal ischemia–reperfusion may take place many times during a lifetime without causing clinical manifestations, because of the reservoir in choroidal blood flow.10
In conclusion these data demonstrate that an alteration of the antioxidant pattern can be detected in uveal melanoma cells, as well as in cutaneous ones, possibly related to the disease status and progression. Moreover, data obtained from apparently normal uveal melanocytes suggest that in some subjects a constitutional imbalance of the antioxidant system, detectable by a decrease of catalase activity, can exist. This could be the basis for increased susceptibility to free radical–mediated damage and possibly to the development of uveal melanoma. As for skin melanoma, we can suggest that an individual predisposition together with environmental and general risk factors could play an important role in tumor onset and that the occurrence of the uveal melanoma might be the expression of acute repeated damages.2
Footnotes
Submitted for publication November 13, 1998; revised June 10, 1999; accepted July 7, 1999.
Commercial relationships policy: N.
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1998.
Corresponding author: Maria Antonietta Blasi, Department of Ophthalmology, Via Vetoio, Blocco 11, University of L’Aquila, 67100 L’Aquila, Italy. E-mail: ma.blasi{at}flashnet.it
References
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