Significance of 5-S-Cysteinyldopa as a Marker for Melanoma.

Melanoma is one of the most lethal and malignant cancers and its incidence is increasing worldwide, and Japan is not an exception. Although there are numerous therapeutic options for melanoma, the prognosis is still poor once it has metastasized. The main concern after removal of a primary melanoma is whether it has metastasized, and early detection of metastatic melanoma would be effective in improving the prognosis of patients. Thus, it is very important to identify reliable methods to detect metastases as early as possible. Although many prognostic biomarkers (mainly for metastases) of melanoma have been reported, there are very few effective for an early diagnosis. Serum and urinary biomarkers for melanoma diagnosis have especially received great interest because of the relative ease of sample collection and handling. Several serum and urinary biomarkers appear to have significant potential both as prognostic indicators and as targets for future therapeutic methods, but still there are no efficient serum and urinary biomarkers for early detection, accurate diagnosis and prognosis, efficient monitoring of the disease and reliable prediction of survival and recurrence. Levels of 5-S-cysteinyldopa (5SCD) in the serum or urine as biomarkers of melanoma have been found to be significantly elevated earlier and to reflect melanoma progression better than physical examinations, laboratory tests and imaging techniques, such as scintigraphy and echography. With recent developments in the treatment of melanoma, studies reporting combinations of 5SCD levels and new applications for the treatment of melanoma are gradually increasing. This review summarizes the usefulness of 5SCD, the most widely used and well-known melanoma marker in the serum and urine, compares 5SCD and other useful markers, and finally its application to other fields.

Eumelanins and pheomelanins: characterization by electron spin resonance spectroscopy.

Synthetic dopa melanin and cysteinyldopa melanin have different electron spin resonance spectra. Data are reported for mixtures of these melanins and for dopa-cysteinyldopa copolymers, which are spectroscopically similar. A simple parameterization of the spectra allows estimation of the relative amounts of (i) dopa melanin and cysteinyldopa melanin in mixtures and of (ii) dopa and cysteinyldopa incorporated into copolymers. Several natural eumelanins and pheomelanins have been characterized and shown to be copolymers.

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins.

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6-dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.

Tumorigenicity of human malignant melanocytes in nude mice in relation to their differentiation in vitro.

Of 16 cell lines derived from 12 human melanomas obtained from 11 patients, all were established as permanent cell lines: 7 from primary tumors and 9 from metastatic tumors. Study of the early subcultures and established cell lines showed that melanocytes passed through a phase of dedifferentiation during which they took on a fibroblast-like appearance and were hypodiploid and nontumorigenic in nude (thymus-deficient) mice. Phenotypic modulation in vitro was shown to be dependent on the culture medium. The lines varied considerably in karyologic and phenotypic expression (as assessed by morphologic appearance and 5-S-cysteinyldopa production). Fibroblast-like, epithelioid, nonpigmented, achromic, and pigmented cells were obtained from the same tumor. Heterotransplantation into nude mice revealed wide variations in tumorigenicity: The latency of the tumors, their size, and infrequent metastases bore no relationship to the phenotypic modulation of the melanocytes as expressed in vitro. Melanogenesis is therefore not related to malignancy; they are two independent characteristics.

Ultrastruct and biochemical chantes in cultured human malignant melanoma cells after heterotransplantation into nude mice.

Cells from three lines of cultured human malignant melanomas were heterotransplanted into nude mice and then recultered. The shape of the cells, the aspect of the melanosomes, and the content of 5-S-cyteinyldopa showed pronounced changes induced by the transplantation. Such results indicate that this experimental model should be used with great caution. A relationship was found between the shape of the melanosomes and the content of 5-S-cysteinyldopa in the cells.

Biosynthetic and structural studies on pheomelanin.

13C-NMR spectroscopy of pheomelanin biopolymers, prepared from isotopically enriched precursors, has been developed as a tool for structure elucidation of melanins. By employing large pulse-widths and short cycle time, only the signals originating from labeled carbons are observed in the high-resolution spectra of these polymers.

Metal ions as potential regulatory factors in the biosynthesis of red hair pigments: a new benzothiazole intermediate in the iron or copper assisted oxidation of 5-S-cysteinyldopa.

In the presence of iron or copper ions, the course of the oxidation in air of 5-S-cysteinyldopa (1), the main biosynthetic precursor of pheomelanins and trichochromes, was markedly changed affording two main products. One of these was identified as the oxobenzothiazine 8, previously obtained under nonphysiologically relevant conditions, while the other was characterized as the novel hydroxybenzothiazole 9. Besides 8 and 9, carboxylated and noncarboxylated benzothiazine products were obtained by persulfate oxidation of 1 in the presence of iron or copper ions. The ratio of formation yields of carboxylated/noncarboxylated benzothiazines, determined after reduction of the mixture, was lower than that of the control reaction run in the absence of metal ions, and much lower than that of the oxidation carried out in the presence of zinc ions, in agreement with a recent report. Notably, 8 and 9 were formed in variable yields under different oxidation conditions including tyrosinase/O(2), peroxidase/hydrogen peroxide, and the hydrogen peroxide/or (9E,11Z,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid/Fe(III) systems. Mechanistic routes to 8 and 9 were proposed based on the results of experiments involving in situ generation of labile benzothiazine intermediates. Overall, these results allow to formulate an improved biosynthetic scheme in which metal ions act as critical regulatory factors determining pheomelanin vs. trichochromes formation.

Interaction of radicals from water radiolysis with melanin.

Melanins are considered to be natural photoprotectors in the melanocytes and keratinocytes of the skin. These pigments have also been suggested to play an important role in protection of melanin-containing cells against ionising radiation. Various mechanisms have been proposed to explain the protective role of melanin which invoke the radical scavenging properties of the polymer. In the present work the reactions of melanins with radicals generated in aqueous media by pulse radiolysis have been studied. Time-resolved changes in absorbance of the melanin or the radical species were recorded at selected wavelengths. Experiments were carried out on synthetic dopa- and 5-S-cysteinyldopa-melanins and a natural melanin in phosphate buffer (pH 7.4). Under the conditions employed, melanin reacted predominantly with either oxidising (OH., N3.) or reducing (eaq-, CO2-) species. We were also able to monitor the interaction of melanin with superoxide radical, which was reducing in this case. Detailed analysis of transient changes in melanin absorbance, detected at different wavelengths, was demonstrated to be a convenient method for studying redox processes of this substance, as shown by model experiments using ferricyanide and dithionite as oxidising and reducing agents, respectively. Among the radicals studied, OH. exhibited the strongest reactivity with melanins. Apparent rate constants for the reactions of radicals with autoxidative dopa-melanin (1.5 X 10(9) M-1 X s-1, 2.6 X 10(8) M-1 X s-1, 1.8 X 10(8) M-1 X s-1, 5 X 10(5) M-1 X s-1, 10(6)-10(7) M-1 X s-1 for OH., eaq-, N.3. O2- and CO2-, respectively) are reported. The reactivity of melanins with radicals from water radiolysis and their effect on pigment properties are discussed in terms of the structure and possible biological role of the pigments.

gamma-Glutamyl transpeptidase, tyrosinase, and 5-S-cysteinyldopa production in melanoma cells.

The distribution of gamma-glutamyl transpeptidase (gamma-GTP), tyrosinase, and 5-S-cysteinyldopa (5-S-CD) within melanoma cells has been studied in vitro as well as in vivo. Sodium periodate treatment of intact B-16 melanoma cells has been found to inhibit gamma-GTP present as an ectoenzyme. However, these periodate-treated cells in the presence of 10(-5) M dopa and glutathione have been found to continue to secrete large quantities of 5-S-CD in their medium. The large-granule fraction of Greene's melanotic melanoma contains substantial amounts of both tyrosinase and gamma-GTP. However, further separation of the large-granule fraction into sub-fractions indicates that tyrosinase and gamma-GTP seem to co-exist with premelanosome. It is suggested that glutathione-dependent t-S-CD genesis proceeds within premelanosomes through the formation of glutathione-dopa. The excess of glutathione-dopa and 5-S-CD, unutilized for pheomelanin formation overglow into the cytoplasm.

Characterization of melanogenesis in mouse and guinea pig hair by chemical analysis of melanins and of free and bound dopa and 5-S-cysteinyldopa.

This study examined how various genotypes of coat color in mice and guinea pigs are related to the type and content of melanin and to the levels of free and protein-bound dopa and 5-S-cysteinyldopa in hair. In analysis of black, yellow, and white areas of tortoiseshell guinea pigs, the melanogenesis type was in parallel to the type and content of melanin and was correlated fairly well with the levels of melanin precursors. In mouse hair, substitution of the brown allele (bb) for black (BB) reduced the eumelanin content to 1/2 to 1/3, while it significantly increased the dopa level. The dilution (dd) gene of mice reduced the eumelanin content only slightly, while the gene for pink-eyed dilution (pp) reduced the content of eumelanin and the level of dopa to as much as 1/10. From the eumelanin/pheomelanin ratio, the melanin of brown and dilute brown mice was found to be eumelanic, while the melanin of pink-eyed dilution mice appeared to be a mixed type because of an extremely low content of eumelanin. The levels of bound dopa and 5-S-cysteinyldopa in hair were found to largely reflect the tyrosinase activity.

Exploitation of pigment biosynthesis pathway as a selective chemotherapeutic approach for malignant melanoma.

Human malignant melanoma represents a difficult therapeutic challenge to both medical scientists and practicing physicians. However, the biologic uniqueness of the tumor may provide opportunities for exploitation in therapeutics. This study proposed to undertake a systemic approach to the chemotherapy of malignant melanoma based upon the uniqueness of pigment-cell metabolic pathway pertaining to conversion of tyrosine and dopa with subsequent formation of melanin by tyrosinase and its related enzymes. The sulphur homologue of tyrosine, cysteinylphenol (CP), its amine derivative, cysteaminylphenol (CAP), and their N-acetyl and alpha-methyl derivatives have been synthesized and tested in in vivo and in vitro melanocytotoxicity and antimelanoma effects. These phenolic thioethers (PTEs) and phenolic thioether amine (amides) (PTEAs), which are substrates of tyrosinase, showed significant cytotoxicity that is selective to melanocytes and melanoma cells. Most previous attempts to impair the melanin pathway as a therapeutic strategy have been of limited success because they have been directed to catecholic compounds that are unstable and insufficient in lethality at physiologically tolerable doses. By contrast, our approach relies on phenolic compounds, PTEs and PTEAs, which are more stable than catechols and become toxic only after oxidation by tyrosinase. We found PTEA as the most promising agent for the future development of chemotherapeutic agents. The possible biologic, chemical, and pharmacologic reactions of these synthetic compounds within the melanoma cells are studied and discussed.

Effect of DOPA-loading on glutathione-dependent 5-S-cysteinyldopa genesis in melanoma cells in vitro.

The effect of dopa, cysteine, and glutathione on 5-S-cysteinyldopa (5-S-CD) genesis in melanoma cells cultured in normal and tyrosine- and cysteine-free media has been studied. In normal media only melanotic melanoma cells have been found to secrete 5-S-CD into the medium. In the presence of dopa and cysteine, both, media incubated with and without cells have been found to produce 5-S-CD. In the presence of dopa and glutathione, however, cell-free media did not show the presence of 5-S-CD. In contrast melanoma cell-cultured media has been found to contain large quantities of this amino acid. The optimum condition for maximum production of 5-S-CD via glutathione-dependent pathway has been found to be at the dopa concentration of 10(-5) M when glutathione is present at the concentration of 10(-5) M in the culture medium. Thus dopa concentration with regards to glutathione is 1:1 on the molar basis which is twice the dopa concentration required in in vitro noncellular tyrosinase system. It is suggested that higher dopa requirement in our melanoma cell culture system reflects the co-existence of eu- and pheomelanin synthesis taking place according to their genetically predetermined proportions.

Stimulation of pheomelanogenesis in cultured B16 melanoma cells by 4-tertiary butylcatechol.

Intermediates of pheomelanin in tissue cultured B16 melanoma cells were analyzed by high performance liquid chromatography, and reduced glutathione (GSH), L-dopa, 2-[(L)-S-cysteinyl]-L-dopa (2-SCD) and 5-[(L)-S-cysteinyl]-L-dopa (5-SCD) were quantified. The effects of 4-tertiary butylcatechol (TBC), an antioxidant which causes skin depigmentation, on the levels of the intermediate were then examined. A concentration of 10(-4) M TBC increased the intracellular levels of GSH, 2-SCD and 5-SCD, whereas the L-dopa level was unchanged. The time-course of the increased intermediates corresponded to the elevation of glutathione-metabolizing enzyme activities previously reported by Kawashima et al. [J. invest. Derm. 82, 53 (1984)] in the same cell line exposed to 10(-4) M TBC. The findings establish chemical evidence that TBC stimulates pheomelanogenesis in melanocytes.

Conjugation of dopa and 5-S-cysteinyldopa with cysteine mediated by superoxide radical.

cytotoxicity of catechols has been ascribed to their binding with proteins through sulfhydryl groups. Superoxide radical (O2-) generated in hypoxanthine-xanthine oxidase system at pH 7.4 mediated conjugation of dopa with cysteine to form cysteinyldopas. Similarly, 5-S-cysteinyldopa gave 2,5-S,S-dicysteinyldopa. The rates of oxidation of the catechols by O2- appear to be comparable to that of reduction of nitro-blue tetrazolium by O2-. These results suggest that catechols may exert cytotoxicity in cells where biochemical defence against O2- or the quinone oxidation products is not sufficient.

Comparison of N-acetylcysteine and l-2-oxothiazolidine-4-carboxylate as cysteine deliverers and glutathione precursors in human malignant melanoma transplants in mice.

PURPOSE: Glutathione is an important cellular compound which affects detoxification of electrophiles and may have direct or indirect effects on pigment formation. It is therefore of importance to study interstitial concentrations in melanoma tissue while decreasing its formation with an enzyme inhibitor and increasing its amount with cysteine deliverers. METHOD: Glutathione formation was inhibited by intraperitoneal (i.p.) injection of BSO. N-Acetylcysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) were then given i.p. to subgroups of the animals. Intratumoral microdialysis was performed during BSO treatment, during BSO treatment combined with NAC or OTC and after discontinuation of BSO but ongoing NAC or OTC treatment. RESULTS: Glutathione formation was inhibited during BSO treatment. The dialysate concentrations of both glutathione and cysteine decreased during concomitant treatment with BSO and NAC or OTC. Recovery of the amounts of the two compounds was seen in both groups after discontinuation of BSO treatment. In the NAC group we also observed an acute increase in dialysate concentrations of cysteine after NAC injection. The 5-S-cysteinyldopa concentrations were unaffected by variations in glutathione and cysteine concentrations. CONCLUSIONS: 5-S-Cysteinyldopa in melanoma is not formed from glutathione in vivo to any appreciable extent. The intracellular amount of cysteine is probably not a limiting factor for cysteinyldopa formation. It seems that both NAC and OTC can be used as cysteine deliverers to melanoma cells in vivo to produce recovery of glutathione levels after synthesis inhibition by BSO treatment.

Modulation of 5-S-cysteinyldopa formation by tyrosinase activity and intracellular thiols in human melanoma cells.

The catechol 5-S-cysteinyldopa (5-S-CD) is produced in large amounts in metastatic malignant melanoma. To further understand the mechanism of formation of 5-S-CD, we investigated the effects of thiol modulating agents and melanin precursors on human melanoma cells. Under standard culture conditions (0.1 mM cystine), the cell levels of 5-S-CD were highly correlated with the degree of melanization and the dopa oxidase activity of the four cell lines investigated (Me8, JUSO, GLL19, Swift). Inhibition of glutathione (GSH) biosynthesis with buthionine sulphoximine did not affect 5-S-CD levels in the low melanotic GL 19 cells. In contrast, the highly pigmented Swift cells showed a strong increase in the cell levels of cystine (CysH) and 5-S-CD. When the cystine concentration of the growth medium was increased to 0.2 mM, a similar situation of 5-S-CD synthesis caused by an increase in intracellular CysH levels was observed in the Swift cells. The GLL19 cells showed enhanced 5-S-CD formation in the presence of 0.1 mM L-dopa. This effect was associated with a fourfold increase in dopa oxidase activity. Our data clearly indicate that 5-S-CD is formed in human melanoma cells by a tyrosinase-dependent mechanism involving the addition of CysH to dopaquinone. Based on the enhancing effect of buthionine sulphoximine on 5-S-CD formation, it is proposed that GSH is not directly implicated in 5-S-CD formation, but regulates CysH levels via the enzyme gamma-glutamylcysteine synthetase.

Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants.

Using microdialysis of human melanoma transplants in athymic mice we have shown that interstitial glutathione levels decreased during treatment with buthionine sulphoximine (BSO) and recovered after cessation of treatment. The cysteine concentrations also decreased, while 5-S-cysteinyldopa tended to increase during BSO treatment. Restoration of the glutathione levels was not seen after either N-acetylcysteine (NAC) or L-2-oxothiazolidine-4-carboxylate (OTC) injections, given on the third day of BSO treatment. These results were to be expected since NAC and OTC were given during the BSO treatment, and BSO is a specific and potent inhibitor of glutathione synthesis. Cysteine levels, however, increased after the NAC injection but remained unaltered after the OTC injection, while 5-S-cysteinyldopa remained unaltered after both the NAC and the OTC injections.

Melanin-related biochemistry of IGR 1 human melanoma cells.

Cultured melanoma cells have been of great value in the study of pigment metabolism. IGR 1 human melanoma cells, established by Dr Christian Aubert, produce melanin in large quantities. These cells have been used for isolation of human tyrosinase which enzyme has not previously been obtained in a pure form. IGR 1 cells contain large amounts of 5-S-cysteinyldopa which is the quantitatively most important catecholic amino acid. This review deals with the metabolism of dopa, cysteinyldopa, glutathionyldopa, cysteine and glutathione, compounds of central importance in pigment metabolism. The information available on tyrosinase, catecholic compounds and on thiols in IGR 1 melanoma cells makes these cells most suitable for further investigation of the metabolism of human melanoma cells.

Studies on amelanotic melanoma with the fluorescence method (Falck and Hillarp) and biochemical analysis of 5-S-cysteinyldopa in the tissues.

Histochemical findings of primary and metastatic amelanotic melanomas were shown by the formaldehyde-induced fluorescence method (Falck and Hillarp). All or some of the amelanotic melanoma cells were discovered to emit green specific fluorescence. Results of the determination of 5-S-cysteinyldopa and DOPA in amelanotic melanoma tissues indicated that the specific fluorescence emitted by these cells is primarily due to the presence of 5-S-cysteinyldopa. The values of 5-S-cysteinyldopa in these tissues were lower than those in melanotic melanoma, but were approximately the same as those in pigmented nevus. When unpigmented tumors were histopathologically revealed to be malignant, amelanotic melanoma could be definitely diagnosed by the fluorescence method of Falck and Hillarp and the biochemical analysis of 5-S-cysteinyldopa in the tissues.

Some indolic compounds as markers of the melanocyte activity.

The melanocyte activity was studied by analysis of the urinary excretion of indolic and cysteinyldopa compounds. One eumelanin marker, 5,6-dihydroxy-indole-2-carboxylic acid was identified and quantified in normal urine. However, its low concentration and sensitivity to oxidation made it less suitable for clinical studies. A methylated derivative of this substance, 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI-2-C), was also demonstrated in normal urine. A quantitative method was worked out and the normal urinary concentration of this substance was as high as the concentration of 5-S-cysteinyldopa. The concentrations of the eumelanic marker 6-hydroxy-5-methoxyindole-2-carboxylic acid and the pheomelanic marker 5-S-cysteinyldopa were determined in the urine of psoriasis patients during PUVA treatment and also in the urine of subjects with different skin colour. The melanocyte activity in albinotic patients and in albinotic mice was studied by the same technique. Some in vitro experiments were performed to show that 5-S-glutathionyldopa has the molecular properties of forming a mercapto-substituted indole derivative. The following main conclusions were drawn: 1. 5,6-Dihydroxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid are both present in measurable amounts in normal urine. 2. The urinary concentration of 6-hydroxy-5-methoxyindole-2-carboxylic acid increased during PUVA treatment in a similar way as for 5-S-cysteinyldopa. 3. The eumelanic marker 6-hydroxy-5-methoxyindole-2-carboxylic acid was excreted in larger quantities by people with genetically dark skin, whereas the pheomelanic marker 5-S-cysteinyldopa was not related to pigment type. 4. In the urine of one albino patient and in the urine of albinotic mice a total absence of 6-hydroxy-5-methoxyindole-2-carboxylic acid was found. The urinary concentrations of 5-S-cysteinyldopa in these subjects were measurable but lower than in pigmented subjects. Thus, 6-hydroxy-5-methoxy-indole-2-carboxylic acid seems to be a more specific melanocyte marker than the cysteinyldopas.(ABSTRACT TRUNCATED AT 400 WORDS)