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Glutathione in human melanoma cells
Journal of Dermatological
Science,
1 (1990) 39-46
Elsevier
39
DESC 00010
Glutathione in human melanoma cells Effects of cysteine, cysteine esters and glutathione
isopropyl
ester
Eszter Karg ‘*, Anders Tunek2, Harald Br6tel13, Anders Hallberg4, Rosengren 5 and Hans Rorsman ’ Departments
of ‘Dermatology
and 5Pharmacology,
University of Lund, Departments of 2Pharmacology,
Evald
3Bioanalysti, and 40rganic
Chemistry Draco AB, Lund, Sweden
(Received 24 May 1989; accepted 24 October
1989)
Key words: Thiols; L-cysteine methyl ester; L-cysteine octyl ester
Abstract Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of r_-cysteine methyl and octyl esters, ofN,S-diacetyl+cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl+cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.
Introduction
Glutathione (GSH), the most prevalent intracellular thiol, acts in cellular protection, metabolism and transport. Its protective functions include those against damage by toxic compounds, reactive oxygen intermediates and irradiation, thus playing an important role in tumour therapy [ l-31. Its intracellular level and the activity of its enzymes have also been reported to be in correlation with different states of melanogenesis [ 4-61. A possible role of GSH in melanin synthesis, the Correspondence to: Hans Rorsman, Department of Dermatology, University of Lund, Lasarettet, S-221 85, Lund, Sweden. * Eszter Karg is on leave from the Department of Dermatology, University Medical School, Pets, Hungary.
0923-181 l/90/$03.50
0 1990 Elsevier Science Publishers
formation of glutathionyldopa and its subsequent metabolism to cysteinyl-dopa, have been extensively investigated [ 7-101. The increasing amount of data seems to be in favor of formation of S-cysteinyldopas by addition of cysteine itself to dopaquinone [lo]. The importance of GSH with regard to pigment production seems therefore to be indirect. The intracellular concentration of glutathione is much higher than that of cysteine and it appears that GSH serves as a means for storage as well as transport of cysteine [ 111. Furthermore, also the protective function of GSH against free radicals and reactive oxygen intermediates may be of special significance with regard to melanin synthesis. In a previous study we have applied N-acetylL-cysteine, an intracellular cysteine delivery pro-
B.V. (Biomedical
Division)
40
drug that proved to be capable of elevating the cellular glutathione level, though also exerted considerable toxic effects on the melanoma cell culture [ 121. Other compounds that might increase the intracellular cysteine level and thus stimulate GSH synthesis have been suggested [ 131. These include, among others, the cysteine esters. Many cells appear to have a high level of esterase that can hydrolyze such compounds and, indeed, both the methyl and ethyl esters of cysteine have been reported to be metabolized to cysteine after in vivo administration to rats [ 14,151. The cellular GSH level can also be elevated by the administration of glutathione derivates. Glutathione itself does not seem to enter most cells to an appreciable extent [ 16,171, however, the monoethyl or methyl esters of glutathione have been reported to lead to a substantial increase in the GSH levels of murine liver and kidney [ 181. The aim of the present work was to examine the potential usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems in the human IGR 1 melanoma cell line in order to lind compounds which by interference with the thiol system would be of value in the study of melanoma cell metabolism and tumour therapy. (The effects of cysteine esters were compared with those of L-cysteine).
Materials and Methods Materials N-acetyl+cysteine (NAC), L-cysteine methyl ester, L-cysteine octyl ester, iV,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester (esterified on the glycyl carboxyl group) were kindly supplied by Draco Co., Lund, Sweden. L-cysteine and glutathione were purchased from Sigma Chemical Co., St Louis, MO, USA. Dithiotreitol (DTT) and monobromobimane were from Merck, Darmstadt, F.R.G. and Behring Diagnostics, La Jolla, CA, USA, respectively. Tissue culture media supplies were obtained from
Flow Laboratories, McLean, VA, USA. All the other chemicals were of high commercial grades. Cell culture Cultures of a pigment producing human melanoma cell line (IGR 1) were obtained from Dr. Christian Aubert, Marseille, France and have been kept since March, 1982 in culture at the Tornblad Institute, University of Lund, using methods previously described [ 191. The medium used was Eagle’s minimal essential medium (MEM) supplemented with 15 % fetal calf serum. About 0.75 x lo6 cells were seeded in 5 ml medium in polystyrene tissue culture flasks, volume 50 ml. Treatment of cell cultures After two days of replating, the medium of the cultures was removed and replaced by fresh medium containing L-cysteine (0.3, 1, 3 mM), L-cysteine methyl ester (0.1,0.2, 1 mM), L-cysteine octyl ester (0.05, 0.1 mM), N,S-diacetyl-L-cysteine methyl ester (0.1, 1, 3 mM) and glutathione isopropyl ester (1, 3 mM). (In a pilot study L-cysteine methyl ester and L-cysteine octyl ester proved to be highly toxic to the cells, thus in the present experiment they were applied in the above indicated lower doses.) The pH values of the media were adjusted to 7.2 with 1 M NaOH solution. Cells and culture media were analysed for thiols 24 h after the administration of the test substances. In all sets of experiments controls were treated in the same way as test samples except for the addition of the drugs to the medium. Analysis of cellular thiols The total cysteine, glutathione and L-NAC content of the cells was determined after a prederivatization reduction with dithiotreitol [ 20,211. After in situ derivatization of DDT reduced thiols with monobromobimane HPLC was used for the quantitation of fluorescent bimane adducts. The separation was performed isocratically on a 150 x 4.6 mm stainless steel column packed
41
with 3 pm Supelcosil LC-18 (Supelco, Bellafonte, PA, USA). With the use of the precolumn the lifetime of the analytical column was substantially increased. The precolumn was a Waters Guard PAKTM, resolve C,, (Waters, Milford, MA, USA). Its insert was changed weekly. The isocratic conditions using an acetonitrile/acetic acid/perchloric acid buffer at pH 3.7 were maintained for 7 min and a step gradient was used to wash out late eluting compounds. Protein The assay was done according to the method of Lowry et al. [22]. Results Effect of L-cysteine on the melanoma cells L-cysteine applied in 0.3, 1 or 3 mM concentrations increased the intracellular cysteine level in a dose dependent manner. The administration of its highest concentration (3 mM) was followed by a considerable decrease in cellular GSH content (Table I.). Besides influencing the thiol level of the cells, L-cysteine also exerted toxic effects resulting in cell loss as determined by protein assay (Table II.). After a 24 h treatment of the two-dayold cultures with 1 or 3 mM L-cysteine, the amount of cells that had adhered to the flasks was found to be markedly lower than that of the controls. This toxic effect was dose-dependent
TABLE
I
Effect of L-cysteine on the total thioi content of melanoma cells. Values are expressed as nmol thiol/mg protein
Control Cysteine
Cont. (mM)
Cysteine
0.3 1 3
2.4 5.8 15.4 46.0
+ f. + +
0.4 (140 1.6 (440 0.9 (1100 5.9 (2900
GSH & 10)” 32.0 + 2.3 f 8) 35.7 f 4.4 k 20) 38.3 f 6.0 i 80) 7.5 f 0.5
a Numbers in parenthesis = concentration of the cysteine in the medium. Values are expressed as nmol cysteine/ml medium. (Mean + SD of 5 samples).
TABLE II Effect of L-cysteine on the amount of cells as determined protein assay Control 0.3 Protein (%)
100 f 6
99 * 3
by
Cysteine (mM) 1 3 74 + 4
Values of test flasks are expressed in percentage trol flasks. Mean _+SD of 5 samples.
30 f 5 of the con-
without apparent cell loss in the case of the 0.3 mM concentration. Effect of L-cysteine methyl and octyl esters and N, S-diacetyl-L-cysteine methyl ester on the melanoma cell culture The r_.-cysteine methyl ester (0.2 mM) increased the intracellular cysteine level, but neither of the lower concentrations (0.1 or 0.2 mM) induced marked changes in the cellular GSH level (Table III.). The octyl ester of L-cysteine (0.05 mM) also elevated the cysteine level of the cells and this was followed by an increase in the intracellular GSH level, too (Table III.). Both esters were found to be toxic to the cultures, 1 mM concentration of the methyl ester and 0.1 mM concentration of the octyl ester was still enough to kill all of the cells in the flasks (Table IV.). Even by further reducing their concentration, there was still a considerable cell loss with the octyl ester being the more toxic compound. The NJ-diacetyl-L-cysteine methyl ester (1, 3 mM) increased the intracellular cysteine level in a dose-dependent manner (Table III.). The application of 1 mM concentration of the compound also resulted in an elevation of the GSH content of the melanoma cells. Besides cysteine and glutathione, a certain amount of another thiol, namely that of N-acetyl-L-cysteine was detected both in the medium and in the cells. The NJ-substituted cysteine ester was less toxic than the L-cysteine methyl ester (Table IV.),
42
TABLE
III
Effects of r_-cysteine esters and glutathione isopropyl ester on the total thiol content of the melanoma cells. Values are expressed as nmol thiol/mg protein
Control t_-cysteine methyl ester L-cysteine octyl ester NJ-diacetyl-L-cysteine
Glutathione
methyl ester
isopropyl ester
il Numbers in parenthesis = concentration Mean f SD of 5 samples.
Cysteine
GSH
0.1 0.2 0.05 0.1
2.1 2.8 4.7 4.0 2.0
30.6 36.2 29.1 46.3 28.8
1
3.4 + 0.6
50.4 k 8.4
3
4.3 * 0.4
26.5 f 2.4
1 3
1.3 f 0.1 1.5 _+0.2
14.0 f 1.0 52.0 _+ 10.1
_+0.4 f 0.5 * 1.1 * 0.5 * 0.3
of L-NAC in the medium. Values are expressed
marked cell loss was induced only by its highest concentration. Effect of glutathione isopropyl ester on the melanoma cells Glutathione isopropyl ester, while slightly decreasing the cellular cysteine level in 1 mM concentration, induced a marked decrease in the TABLE
L-NAC
Cont. (mM)
_+3.6 & 8.3 + 5.5 + 6.6 f 3.8
as nmol L-NAG/ml
1) 3) 8 12)
medium.
GSH level of the cells (Table III.). In contrast to this, the 3 mM concentration of the ester induced a considerable elevation in the cellular glutathione content. A decrease in the amount of adhered cells was also observed in the case of this compound, but this effect was less pronounced than that observed after the application of cysteine or its esters (Table IV.).
IV
Effects of cysteine esters and glutathione isopropyl ester on the amount of cells as detemined by protein assay
Control L-cysteine methyl ester
L-cysteine octyl ester N,S-diactetyl-t_-cysteine methyl ester Glutathione isopropyl ester
Cont. (mM)
Protein (%)
0.1 0.2 1 0.05 0.1 0.1 1 3 1 3
lOOk 9 85& 5 14* 10 all cells died 31* 7 all cells died 102* 9 89k I 44+ 11 86* 3 79* 2
Values of the test flasks are expressed in percentage control flasks. Mean t SD of 5 samples.
of the
Discussion The treatment of the melanoma cells with cysteine resulted in a substantial and dose-dependent increase in the cellular cysteine level. In 3 mM concentration the amino acid considerably decreased the cellular GSH level, though there was no change in the glutathione content with the lower concentrations. The amino acid also exerted toxic effects on the melanoma cells resulting in a pronounced cell loss. Cysteine applied in higher doses causes GSH depletion and other signs of cell damage [23,24] by the hydroxyl and thiyl radicals generated via its autooxidation [25]. This amino acid is toxic at least in part by virtue of its extracellular effects
43
which may include oxidation of membrane thiol groups and lipid peroxidation [ 25,261. The uptake of cysteine is mediated through specific amino acid transport systems [27]. Our results proved its cellular level to be roughly proportional to the cysteine concentration applied in the medium over a wide range, thus also indicating the possibility of passive diffusion, probably via membrane damage. The application of L-cysteine methyl (0.2 mM) and octyl (0.05 mM) esters increased the cellular cysteine level which in the case of the octyl ester was followed by an elevation of cellular GSH content. Esters are usually readily accepted into the cell membrane, however, their transport and also the rate of their enzymatic hydrolysis may vary according to the characteristics of the acyl and alkyl groups. The methyl and ethyl esters of cysteine have been reported to be hydrolysed to cysteine after in vivo application to rats [ 14,151. The elevated cellular cysteine level observed in the melanoma cells indicate an effective transport and hydrolysis for both the methyl and octyl esters. The ester derivates in equimolar doses to r_-cysteine proved to have a much more pronounced toxicity than the parent amino acid as indicated by the cell loss. The higher toxicity of the octyl ester may be explained by its more lipophilic character and even this difference in the degree of cell loss caused by the esters and L-cysteine suggests the involvement of intracellular effects. Furthermore, the various transport and hydrolysis rates with the subsequent differences in the intracellular concentration and time related action of the compounds may also contribute to the differences in their effects on the thiol levels of the melanoma cells. The NJ-diacetyl-L-cysteine methyl ester induced a dose-dependent increase in the intracellular cysteine level and the application of 1 mM concentration resulted also in a pronounced elevation of the cellular glutathione content. Furthermore, after the application of this substance there was both intracellularly and extracellularly quite a high amount of detectable L-NAC. In fact, the intracellular L-NAC concentration after the appli-
cation of this substituted ester derivate was several fold higher than the L-NAC level we could achieve in a previous experiment with the administration of equimolar doses of L-NAC itself [ 121. This phenomenon may probably be explained by the different transport rates of the substituted ester and L-NAC, by the higher deacetylation rate at the S-site compared with that at the N-site, and by a possibly low level of N-acylase activity in the melanoma cells. Though the oxidation rate of r_-NAC is substantially lower compared with L-cysteine, this compound can also deplete cellular GSH by free radical production [23]. Thus the lower efficacy of the higher (3 mM) concentration of N,N-diacetyl-L-cysteine methyl ester compared with the lower (1 mM) one to induce an increase in the glutathione level may also be due to the presence of the high intracellular L-NAC level. The toxicity of this NJ-substituted ester was less pronounced than that of the L-cysteine methyl ester, though this substance also induced a considerable cell loss in its highest (3 mM) concentration. It seems likely that the N- and S-substitutions influence the transport rate of the molecule and also affect the hydrolysis of the ester bond by cellular esterases. The difference in the toxicity may also be due to the originally lacking and via deacetylation gradually liberated free SH-groups. The application of 3 mM glutathione isopropyl ester resulted in an elevation of the cellular GSH level similar to that produced by the administration of the NJ-diacetyl-L-cysteine methyl ester (1 mM). However, in the lower concentration (1 mM) the glutathione ester decreased the cellular cysteine level also inducing a considerable decrease in the cellular GSH concentration. The monoethyl ester of GSH has been reported to be transported into human lymphocytes and fibroblasts and converted to GSH intracellularly [28]. The use of esters of glutathione is attractive because many cells appear to be capable of hydrolysing such compounds. Furthermore, the delivery by esters is not subject to feedback inhibition since the enzymes required for glutathione synthesis are not involved.
44
The elevation of cellular glutathione achieved in our experiment may indicate the transport and efficient hydrolysis of the substance. The extracellular cleavage of the compound and intracellular resynthesis of the GSH from its precursor amino acids cannot be excluded. However, it seems to be less probable. High levels of extracellular glutathione interfere with the transport of y-glutamyl amino acids, i.e. cyst(e)ine [29], and indeed, also in the present experiment cellular cysteine level was found to be decreased after the application of the GSH ester. Furthermore, the cysteine supply of mammalian cells is considered ratelimiting with regard to GSH biosynthesis [30]. The low cellular glutathione level observed with the smaller dose of the isopropyl ester may be the result of the decreased GSH synthesis and the lower transport and hydrolysis rate of the GSH ester in accordance with its lower extracellular concentration. In conclusion, the 24 h application of 1 mM NJ-diacetylL-cysteine methyl ester or 3 mM glutathione isopropyl ester resulted in a similarly marked elevation of cellular glutathione content without apparent, or in the case of the latter substance with slight cell loss, in the human melanoma cell culture. Thus both substances proved to be more effective with regard to increasing the GSH level and less toxic according to the cell loss than L-NAC as found in the previous experiment. The administration of these compounds seems to be suitable for the study of the action of an elevated GSH level on melanogenesis.
funds of the Faculty of Medicine, Lund.
of
References
10
Acknowledgements
This investigation was supported by grants from the Swedish Cancer Society (project 626B89-17XB), the Swedish Medical Research Council, the Walter, Ellen and Lennart Hesselman Foundation for Scientific Research, the Edvard Welander Foundation for Scientific Research, the Thure Carlsson Foundation for Scientific Research, the Crafoord Foundation for Scientific Research, the donation funds of the University Hospital at Lund, and the donation
University
11
12
13
14
Meister A: Selective modification of glutathione metabolism. Science 220: 470-478, 1983. Revesz L, Bergstrand H, Modig H: Intrinsic non-protein sulfhydryl levels and cellular radiosensitivity. Nature 198: 1275-1277, 1963. Vos 0, Roos-Verhey WSD: Radioprotection by glutathione esters and cysteamine in normal and glutathione-depleted mammalian cells. Int J Radiat Biol 53: 273-281, 1988. Halprin KM, Okhawara A: Human pigmentation, the role of glutathione, in Advances in biology of skin. Vol. 8. The pigmentary system, Edited by W Montagna, F Hu. Pergamon Press, 1967, pp 241-251. Seiji M, Yoshida T, Sakurane T, Ogura R: Sulfhydryl content in subcellular fractions of mouse melanomas. J Invest Dermatol 48: 230-234, 1967. Benedetto JP, Ortonne J-P, Volout C, Katchadourian C, Prota G, Thivolet J: Role of thiol compounds in mammalian melanin pigmentation. Part II. Glutathione and related enzyme activities. J Invest Dermato179: 422-424, 1982. Agrup G, Falck B, Kennedy B-M, Rorsman H, Rosengren A-M, Rosengren E: Formation of cysteinyldopa from glutathionyldopa in melanoma. Acta Derm Venereol 55: l-3, 1975. Mojamdar M, Ichihachi M, Mishima Y: Effect of DOPAloading on glutathione-dependent 5-S-cysteinyldopa genesis in melanoma cells in vitro. J Invest Dermato178: 224-226, 1982. Rorsman H, Agrup G, Hansson C, Rosengren E: Biochemical recorders of malignant melanoma, in Pigment ceN. Vol. 6. edited by R.M. McKie. Karger, Basel, 1983, pp 93-l 15. Carstam R, Hansson C, Lindbladh C, Rorsman H, Rosengren E: Dopaquinone addition products in cultured human melanoma cells. Acta Derm Venereol 67: 100-105, 1987. Higashi T, Tateishi N, Naruse A, Sakamoto Y: A novel physiological role of liver glutathione as a reservoir of L-cysteine. J Biochem 82: 117-124, 1977. Karg E, Tunek A, Brotell H, Rosengren E, Rorsman H: Alteration of glutathione level in human melanoma cells. Effect of N-acetyl+cysteine and its analogues. Pigment Cell Res (in press). Williamson JM, Boettcher B, Meister A: Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis. Proc Nat1 Acad Sci USA. 79: 6246-6249, 1982. Wepierre J, Laprand J, Garcet S, Laurre M: Etude de la
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I5 16
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repartition el de l’elimination de fester methylique de la cysttine 35S chez la Souris et le Rat. Therapie 19: 187-200, 1964. Serwin AL, Goulinet S, Renault H: Pharmakokinetics of cysteine ethyl ester in rat. Xenobiotica 18: 839-847, 1988. Dethmers JK, Meister A: Glutathione export by human lymphoid cells: Depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation. Proc Natl Acad Sci USA. 78: 7492-7496, 1982. Jensen GL, Meister A: Radioprotection of human lymphoid cells by exogenously supplied glutathione is mediated by gamma-glutamyl transpeptidase. Proc Nat1 Acad Sci USA. 80: 4714-4717, 1983. Puri RN, Meister A: Transport of glutathione, as gamma-glutamyl cysteinyl glycyl ester, into liver and kidney. Proc Nat1 Acad Sci USA 80: 5258-5260, 1983. Aubert C, Lagrange C, Rorsman H, Rosengren E; Catechols in primary and metastatic malignant melanoma cells in monolayer culture. Eur J Cancer 12: 441-445, 1976. Kosower EM, Pazhenchevsky B, Hershkowitz E: 1,5Diazabicyclo (3.3.0)-octadienediones (9,10,-dioxabimanes). Strongly fluorescent syn-isomers. J Am Chem Sot 100: 6516-6518, 1978. Cotgreave IA, Mold&us P: Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components in biological systems J Biochem Biophys Methods 13: 231-249, 1986.
22 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275, 1951. 23 Estrela JM, SaezGT, Such L, VifiaJ: The effect of cysteine and N-acetyl cysteine on rat liver glutathione (GSH). Biochem Pharmacol 32: 3483-3485, 1983. 24 Viiia J, Saez GT, Wiggins D, Roberts AFC, Hems R, Krebs HA: The effect of cysteine oxidation on isolated hepatocytes. Biochem J 212: 39-44, 1983. 25 Saez G, Thornalley PJ, Hill HAO, Hems R, Bannister JV: The production of free radicals during the autooxidation of cysteine and their effect on isolated rat hepatocytes. Biochim Biophys Acta 719: 24-31. 1982. 26 Kosower NS, Kosower EM: Protection ofmembranes by glutathione, in Glurathione. Edited by L Floht et al. George Thieme Publishers, Stuttgart, 1973, pp 216-227. 27 Bannai Sh: Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta 779: 289-306, 1984. 28 Wellner VP, Anderson ME, Puri RN, Jensen GL, Meister A: Radioprotection by glutathione ester: Transport of glutathione ester into human lymphoid cells and fibroblasts. Proc Nat1 Acad Sci USA 81: 4732-4735, 1984. 29 Griffith OW, Bridges RJ, Meister A: Formation of y-glutamyl-cyst(e)ine in vivo is catalyzed by y-glutamyl transpeptidase. Proc Nat1 Acad Sci USA 78: 2777-2781,198 1. 30 Richman PG, Meister A: Regulation of y-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250: 1422-1426, 1975.
Science,
1 (1990) 39-46
Elsevier
39
DESC 00010
Glutathione in human melanoma cells Effects of cysteine, cysteine esters and glutathione
isopropyl
ester
Eszter Karg ‘*, Anders Tunek2, Harald Br6tel13, Anders Hallberg4, Rosengren 5 and Hans Rorsman ’ Departments
of ‘Dermatology
and 5Pharmacology,
University of Lund, Departments of 2Pharmacology,
Evald
3Bioanalysti, and 40rganic
Chemistry Draco AB, Lund, Sweden
(Received 24 May 1989; accepted 24 October
1989)
Key words: Thiols; L-cysteine methyl ester; L-cysteine octyl ester
Abstract Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of r_-cysteine methyl and octyl esters, ofN,S-diacetyl+cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl+cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.
Introduction
Glutathione (GSH), the most prevalent intracellular thiol, acts in cellular protection, metabolism and transport. Its protective functions include those against damage by toxic compounds, reactive oxygen intermediates and irradiation, thus playing an important role in tumour therapy [ l-31. Its intracellular level and the activity of its enzymes have also been reported to be in correlation with different states of melanogenesis [ 4-61. A possible role of GSH in melanin synthesis, the Correspondence to: Hans Rorsman, Department of Dermatology, University of Lund, Lasarettet, S-221 85, Lund, Sweden. * Eszter Karg is on leave from the Department of Dermatology, University Medical School, Pets, Hungary.
0923-181 l/90/$03.50
0 1990 Elsevier Science Publishers
formation of glutathionyldopa and its subsequent metabolism to cysteinyl-dopa, have been extensively investigated [ 7-101. The increasing amount of data seems to be in favor of formation of S-cysteinyldopas by addition of cysteine itself to dopaquinone [lo]. The importance of GSH with regard to pigment production seems therefore to be indirect. The intracellular concentration of glutathione is much higher than that of cysteine and it appears that GSH serves as a means for storage as well as transport of cysteine [ 111. Furthermore, also the protective function of GSH against free radicals and reactive oxygen intermediates may be of special significance with regard to melanin synthesis. In a previous study we have applied N-acetylL-cysteine, an intracellular cysteine delivery pro-
B.V. (Biomedical
Division)
40
drug that proved to be capable of elevating the cellular glutathione level, though also exerted considerable toxic effects on the melanoma cell culture [ 121. Other compounds that might increase the intracellular cysteine level and thus stimulate GSH synthesis have been suggested [ 131. These include, among others, the cysteine esters. Many cells appear to have a high level of esterase that can hydrolyze such compounds and, indeed, both the methyl and ethyl esters of cysteine have been reported to be metabolized to cysteine after in vivo administration to rats [ 14,151. The cellular GSH level can also be elevated by the administration of glutathione derivates. Glutathione itself does not seem to enter most cells to an appreciable extent [ 16,171, however, the monoethyl or methyl esters of glutathione have been reported to lead to a substantial increase in the GSH levels of murine liver and kidney [ 181. The aim of the present work was to examine the potential usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems in the human IGR 1 melanoma cell line in order to lind compounds which by interference with the thiol system would be of value in the study of melanoma cell metabolism and tumour therapy. (The effects of cysteine esters were compared with those of L-cysteine).
Materials and Methods Materials N-acetyl+cysteine (NAC), L-cysteine methyl ester, L-cysteine octyl ester, iV,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester (esterified on the glycyl carboxyl group) were kindly supplied by Draco Co., Lund, Sweden. L-cysteine and glutathione were purchased from Sigma Chemical Co., St Louis, MO, USA. Dithiotreitol (DTT) and monobromobimane were from Merck, Darmstadt, F.R.G. and Behring Diagnostics, La Jolla, CA, USA, respectively. Tissue culture media supplies were obtained from
Flow Laboratories, McLean, VA, USA. All the other chemicals were of high commercial grades. Cell culture Cultures of a pigment producing human melanoma cell line (IGR 1) were obtained from Dr. Christian Aubert, Marseille, France and have been kept since March, 1982 in culture at the Tornblad Institute, University of Lund, using methods previously described [ 191. The medium used was Eagle’s minimal essential medium (MEM) supplemented with 15 % fetal calf serum. About 0.75 x lo6 cells were seeded in 5 ml medium in polystyrene tissue culture flasks, volume 50 ml. Treatment of cell cultures After two days of replating, the medium of the cultures was removed and replaced by fresh medium containing L-cysteine (0.3, 1, 3 mM), L-cysteine methyl ester (0.1,0.2, 1 mM), L-cysteine octyl ester (0.05, 0.1 mM), N,S-diacetyl-L-cysteine methyl ester (0.1, 1, 3 mM) and glutathione isopropyl ester (1, 3 mM). (In a pilot study L-cysteine methyl ester and L-cysteine octyl ester proved to be highly toxic to the cells, thus in the present experiment they were applied in the above indicated lower doses.) The pH values of the media were adjusted to 7.2 with 1 M NaOH solution. Cells and culture media were analysed for thiols 24 h after the administration of the test substances. In all sets of experiments controls were treated in the same way as test samples except for the addition of the drugs to the medium. Analysis of cellular thiols The total cysteine, glutathione and L-NAC content of the cells was determined after a prederivatization reduction with dithiotreitol [ 20,211. After in situ derivatization of DDT reduced thiols with monobromobimane HPLC was used for the quantitation of fluorescent bimane adducts. The separation was performed isocratically on a 150 x 4.6 mm stainless steel column packed
41
with 3 pm Supelcosil LC-18 (Supelco, Bellafonte, PA, USA). With the use of the precolumn the lifetime of the analytical column was substantially increased. The precolumn was a Waters Guard PAKTM, resolve C,, (Waters, Milford, MA, USA). Its insert was changed weekly. The isocratic conditions using an acetonitrile/acetic acid/perchloric acid buffer at pH 3.7 were maintained for 7 min and a step gradient was used to wash out late eluting compounds. Protein The assay was done according to the method of Lowry et al. [22]. Results Effect of L-cysteine on the melanoma cells L-cysteine applied in 0.3, 1 or 3 mM concentrations increased the intracellular cysteine level in a dose dependent manner. The administration of its highest concentration (3 mM) was followed by a considerable decrease in cellular GSH content (Table I.). Besides influencing the thiol level of the cells, L-cysteine also exerted toxic effects resulting in cell loss as determined by protein assay (Table II.). After a 24 h treatment of the two-dayold cultures with 1 or 3 mM L-cysteine, the amount of cells that had adhered to the flasks was found to be markedly lower than that of the controls. This toxic effect was dose-dependent
TABLE
I
Effect of L-cysteine on the total thioi content of melanoma cells. Values are expressed as nmol thiol/mg protein
Control Cysteine
Cont. (mM)
Cysteine
0.3 1 3
2.4 5.8 15.4 46.0
+ f. + +
0.4 (140 1.6 (440 0.9 (1100 5.9 (2900
GSH & 10)” 32.0 + 2.3 f 8) 35.7 f 4.4 k 20) 38.3 f 6.0 i 80) 7.5 f 0.5
a Numbers in parenthesis = concentration of the cysteine in the medium. Values are expressed as nmol cysteine/ml medium. (Mean + SD of 5 samples).
TABLE II Effect of L-cysteine on the amount of cells as determined protein assay Control 0.3 Protein (%)
100 f 6
99 * 3
by
Cysteine (mM) 1 3 74 + 4
Values of test flasks are expressed in percentage trol flasks. Mean _+SD of 5 samples.
30 f 5 of the con-
without apparent cell loss in the case of the 0.3 mM concentration. Effect of L-cysteine methyl and octyl esters and N, S-diacetyl-L-cysteine methyl ester on the melanoma cell culture The r_.-cysteine methyl ester (0.2 mM) increased the intracellular cysteine level, but neither of the lower concentrations (0.1 or 0.2 mM) induced marked changes in the cellular GSH level (Table III.). The octyl ester of L-cysteine (0.05 mM) also elevated the cysteine level of the cells and this was followed by an increase in the intracellular GSH level, too (Table III.). Both esters were found to be toxic to the cultures, 1 mM concentration of the methyl ester and 0.1 mM concentration of the octyl ester was still enough to kill all of the cells in the flasks (Table IV.). Even by further reducing their concentration, there was still a considerable cell loss with the octyl ester being the more toxic compound. The NJ-diacetyl-L-cysteine methyl ester (1, 3 mM) increased the intracellular cysteine level in a dose-dependent manner (Table III.). The application of 1 mM concentration of the compound also resulted in an elevation of the GSH content of the melanoma cells. Besides cysteine and glutathione, a certain amount of another thiol, namely that of N-acetyl-L-cysteine was detected both in the medium and in the cells. The NJ-substituted cysteine ester was less toxic than the L-cysteine methyl ester (Table IV.),
42
TABLE
III
Effects of r_-cysteine esters and glutathione isopropyl ester on the total thiol content of the melanoma cells. Values are expressed as nmol thiol/mg protein
Control t_-cysteine methyl ester L-cysteine octyl ester NJ-diacetyl-L-cysteine
Glutathione
methyl ester
isopropyl ester
il Numbers in parenthesis = concentration Mean f SD of 5 samples.
Cysteine
GSH
0.1 0.2 0.05 0.1
2.1 2.8 4.7 4.0 2.0
30.6 36.2 29.1 46.3 28.8
1
3.4 + 0.6
50.4 k 8.4
3
4.3 * 0.4
26.5 f 2.4
1 3
1.3 f 0.1 1.5 _+0.2
14.0 f 1.0 52.0 _+ 10.1
_+0.4 f 0.5 * 1.1 * 0.5 * 0.3
of L-NAC in the medium. Values are expressed
marked cell loss was induced only by its highest concentration. Effect of glutathione isopropyl ester on the melanoma cells Glutathione isopropyl ester, while slightly decreasing the cellular cysteine level in 1 mM concentration, induced a marked decrease in the TABLE
L-NAC
Cont. (mM)
_+3.6 & 8.3 + 5.5 + 6.6 f 3.8
as nmol L-NAG/ml
1) 3) 8 12)
medium.
GSH level of the cells (Table III.). In contrast to this, the 3 mM concentration of the ester induced a considerable elevation in the cellular glutathione content. A decrease in the amount of adhered cells was also observed in the case of this compound, but this effect was less pronounced than that observed after the application of cysteine or its esters (Table IV.).
IV
Effects of cysteine esters and glutathione isopropyl ester on the amount of cells as detemined by protein assay
Control L-cysteine methyl ester
L-cysteine octyl ester N,S-diactetyl-t_-cysteine methyl ester Glutathione isopropyl ester
Cont. (mM)
Protein (%)
0.1 0.2 1 0.05 0.1 0.1 1 3 1 3
lOOk 9 85& 5 14* 10 all cells died 31* 7 all cells died 102* 9 89k I 44+ 11 86* 3 79* 2
Values of the test flasks are expressed in percentage control flasks. Mean t SD of 5 samples.
of the
Discussion The treatment of the melanoma cells with cysteine resulted in a substantial and dose-dependent increase in the cellular cysteine level. In 3 mM concentration the amino acid considerably decreased the cellular GSH level, though there was no change in the glutathione content with the lower concentrations. The amino acid also exerted toxic effects on the melanoma cells resulting in a pronounced cell loss. Cysteine applied in higher doses causes GSH depletion and other signs of cell damage [23,24] by the hydroxyl and thiyl radicals generated via its autooxidation [25]. This amino acid is toxic at least in part by virtue of its extracellular effects
43
which may include oxidation of membrane thiol groups and lipid peroxidation [ 25,261. The uptake of cysteine is mediated through specific amino acid transport systems [27]. Our results proved its cellular level to be roughly proportional to the cysteine concentration applied in the medium over a wide range, thus also indicating the possibility of passive diffusion, probably via membrane damage. The application of L-cysteine methyl (0.2 mM) and octyl (0.05 mM) esters increased the cellular cysteine level which in the case of the octyl ester was followed by an elevation of cellular GSH content. Esters are usually readily accepted into the cell membrane, however, their transport and also the rate of their enzymatic hydrolysis may vary according to the characteristics of the acyl and alkyl groups. The methyl and ethyl esters of cysteine have been reported to be hydrolysed to cysteine after in vivo application to rats [ 14,151. The elevated cellular cysteine level observed in the melanoma cells indicate an effective transport and hydrolysis for both the methyl and octyl esters. The ester derivates in equimolar doses to r_-cysteine proved to have a much more pronounced toxicity than the parent amino acid as indicated by the cell loss. The higher toxicity of the octyl ester may be explained by its more lipophilic character and even this difference in the degree of cell loss caused by the esters and L-cysteine suggests the involvement of intracellular effects. Furthermore, the various transport and hydrolysis rates with the subsequent differences in the intracellular concentration and time related action of the compounds may also contribute to the differences in their effects on the thiol levels of the melanoma cells. The NJ-diacetyl-L-cysteine methyl ester induced a dose-dependent increase in the intracellular cysteine level and the application of 1 mM concentration resulted also in a pronounced elevation of the cellular glutathione content. Furthermore, after the application of this substance there was both intracellularly and extracellularly quite a high amount of detectable L-NAC. In fact, the intracellular L-NAC concentration after the appli-
cation of this substituted ester derivate was several fold higher than the L-NAC level we could achieve in a previous experiment with the administration of equimolar doses of L-NAC itself [ 121. This phenomenon may probably be explained by the different transport rates of the substituted ester and L-NAC, by the higher deacetylation rate at the S-site compared with that at the N-site, and by a possibly low level of N-acylase activity in the melanoma cells. Though the oxidation rate of r_-NAC is substantially lower compared with L-cysteine, this compound can also deplete cellular GSH by free radical production [23]. Thus the lower efficacy of the higher (3 mM) concentration of N,N-diacetyl-L-cysteine methyl ester compared with the lower (1 mM) one to induce an increase in the glutathione level may also be due to the presence of the high intracellular L-NAC level. The toxicity of this NJ-substituted ester was less pronounced than that of the L-cysteine methyl ester, though this substance also induced a considerable cell loss in its highest (3 mM) concentration. It seems likely that the N- and S-substitutions influence the transport rate of the molecule and also affect the hydrolysis of the ester bond by cellular esterases. The difference in the toxicity may also be due to the originally lacking and via deacetylation gradually liberated free SH-groups. The application of 3 mM glutathione isopropyl ester resulted in an elevation of the cellular GSH level similar to that produced by the administration of the NJ-diacetyl-L-cysteine methyl ester (1 mM). However, in the lower concentration (1 mM) the glutathione ester decreased the cellular cysteine level also inducing a considerable decrease in the cellular GSH concentration. The monoethyl ester of GSH has been reported to be transported into human lymphocytes and fibroblasts and converted to GSH intracellularly [28]. The use of esters of glutathione is attractive because many cells appear to be capable of hydrolysing such compounds. Furthermore, the delivery by esters is not subject to feedback inhibition since the enzymes required for glutathione synthesis are not involved.
44
The elevation of cellular glutathione achieved in our experiment may indicate the transport and efficient hydrolysis of the substance. The extracellular cleavage of the compound and intracellular resynthesis of the GSH from its precursor amino acids cannot be excluded. However, it seems to be less probable. High levels of extracellular glutathione interfere with the transport of y-glutamyl amino acids, i.e. cyst(e)ine [29], and indeed, also in the present experiment cellular cysteine level was found to be decreased after the application of the GSH ester. Furthermore, the cysteine supply of mammalian cells is considered ratelimiting with regard to GSH biosynthesis [30]. The low cellular glutathione level observed with the smaller dose of the isopropyl ester may be the result of the decreased GSH synthesis and the lower transport and hydrolysis rate of the GSH ester in accordance with its lower extracellular concentration. In conclusion, the 24 h application of 1 mM NJ-diacetylL-cysteine methyl ester or 3 mM glutathione isopropyl ester resulted in a similarly marked elevation of cellular glutathione content without apparent, or in the case of the latter substance with slight cell loss, in the human melanoma cell culture. Thus both substances proved to be more effective with regard to increasing the GSH level and less toxic according to the cell loss than L-NAC as found in the previous experiment. The administration of these compounds seems to be suitable for the study of the action of an elevated GSH level on melanogenesis.
funds of the Faculty of Medicine, Lund.
of
References
10
Acknowledgements
This investigation was supported by grants from the Swedish Cancer Society (project 626B89-17XB), the Swedish Medical Research Council, the Walter, Ellen and Lennart Hesselman Foundation for Scientific Research, the Edvard Welander Foundation for Scientific Research, the Thure Carlsson Foundation for Scientific Research, the Crafoord Foundation for Scientific Research, the donation funds of the University Hospital at Lund, and the donation
University
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14
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