L-tyrosine induces synthesis of melanogenesis related proteins

In cultured amelanotic hamster melanoma cells L-tyrosine induces melanogenesis. This induction involves an increase in intracellular concentration of proteins precipitated by polyclonal anti-tyrosinase antibodies, and stimulation of the Vmax of tyrosinase activity. Therefore it is suggested that in hamster melanoma cells L-tyrosine induces synthesis of tyrosinase and melanogenesis related proteins.     

Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

MSH binding in bomirski amelanotic hamster melanoma cells is stimulated by L-tyrosine

Bomirski Ab amelanotic melanoma cells have recently been shown to undergo striking phenotypic changes when precursors of the melanogenic pathway, L-tyrosine and L-dopa, are added to the culture medium. The changes include increased tyrosinase activity andde novo synthesis of melanosomes and melanin. L-tyrosine and L-dopa appeared to elicit these responses through separate but overlapping regulatory pathways. Here we show an additional effect of L-tyrosine: stimulation of MSH binding capacity. Cells cultured for 24–48 hours in the presence of 200 M L-tyrosine display a 3–4 fold increase in their ability to bind¹²⁵l--MSH. L-dopa did not stimulate MSH binding under the same conditions. In control experiments neither L-tyrosine nor L-dopa had any effect on insulin binding. The amelanotic cells respond to MSH with increased dendrite formation, increased tyrosinase activity without melanin production, and decreased growth rate.     

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.     

Biochemical characterization of three hamster melanoma variants--II. Glycolysis and oxygen consumption

Lactate production and oxygen consumption were studied in single cell suspension prepared from solid tumours of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) melanomas of hamster. Aerobic lactase production was about 5 times higher in the fast growing Ab melanoma than in the slow growing Ma and MI melanomas. Aerobic lactate production in both melanotic hamster melanomas was stimulated by 3-(3,4-dihydroxyphenyl)-L-alanine. This compound was without effect on the cells isolated from amelanotic hamster melanoma. L-Phenylalanine, a known competitive tyrosinase inhibitor reduced the stimulatory effect of L-DOPA on the the lactate production. Oxygen consumption was similar in all three melanomas. The oxygen consumption was inhibited completely by 1 mM potassium cyanide in the Ab melanoma but only in about 1/3 in the Ma and MI melanomas. The Pasteur effect was higher in relative terms and lower in absolute terms in the melanotic melanomas than in the Ab melanoma only . The Crabtree effect was present in the Ab melanoma only. Thus glycolysis measured by aerobic and anaerobic lactic acid formation, and cell respiration, measured by oxygen consumption sensitive to KCN, were both higher in the more malignant, less differentiated Ab melanoma than in the Ma and MI melanomas. The suggestion is presented that the process of melanogenesis influences both aerobic glycolysis and the KCN insensitive consumption in the melanotic hamster melanomas.     

Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis

The composition of the gangliosides of hamster melanoma cells is closely related to their cellular growth and degree of differentiation, with slow-growing, highly differentiated melanotic melanoma MI cells expressing GM3 and fast-growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O-acetyl-GD3. To study the putative function of O-acetyl-GD3, we established stably transfected AbC-1 amelanotic hamster melanoma cells with O-acetylesterase gene from influenza C virus to hydrolyze the O-acetyl group from O-acetyl-GD3. The content of O-acetyl-GD3 in the transfected cells expressing O-acetylesterase gene was reduced by >90%. These O-acetyl-GD3-depleted cells differed from the parental ones in their cellular morphology, growth behavior, and melanogenesis activity. The absence of O-acetyl-GD3 in the transfected cells was accompanied by increased thick dendrite formation with an enlarged cell body, which is in striking contrast to the control cells, which were rounded and flattened, with few processes. Their growth was significantly slower than that of the control cells. They also demonstrated significantly lower tyrosinase activity and melanogenic potential. We suggest that the enhanced expression of melanoma-associated O-acetyl-GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation but not melanogenesis.     

The effect of intrastriatal injection of liposome-entrapped tyrosinase on the dopamine levels in the rat brain.

Parkinson's disease is a neurodegenerative disorder which is mainly characterized by degeneration of the dopaminergic cells in the nigro-striatal system. Due to a lowered L-tyrosine 3-monooxygenase activity, L-tyrosine is not sufficiently transformed to L-DOPA. To date the most common therapy is the administration of the dopamine precursor L-DOPA, with severe collateral effects. Therefore, the substitution of the lacking tyrosine hydroxylase with tyrosinase might be a novel therapeutical approach that would generate specifically L-DOPA from L-tyrosine. We present here evidence that stereotaxic injection of liposome-entrapped tyrosinase is able to significatively increase the levels of dopamine in the rat brain. The catecholamines L-DOPA, dopamine, L-epinephrine, L-norepinephrine were extracted by acid treatment from the brains and detected by HPLC.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.     

Molecular mechanism of tyrosinase regulation by L-dopa in hamster melanoma cells

Exposure of hamster amelanotic melanoma cells to L-dihydroxyphenylalanine (L-DOPA) resulted in a time dependent increase of cell pigmentation, tyrosinase concentration and activity with peak after 24 hours. Northern blot analysis showed a small but reproducible increase of tyrosinase mRNA after 3 hours and a decrease below the control level after 9 hours. After 24 and 48 hours tyrosinase mRNA was undetectable. It is suggested that L-DOPA or its oxidation products can stimulate intracellular tyrosinase concentration and regulate tyrosinase mRNA level both in positive and negative fashion.     

Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells

The biosynthesis of dopamine (DA) in catecholaminergic neurons is regulated by tyrosine hydroxylase, which converts tyrosine into 3, 4-dihydroxyphenylalanine (L-DOPA). In melanocytes, tyrosinase catalyzes both the hydroxylation of tyrosine and the consequent oxidation of L-DOPA to form melanin. Although it has been demonstrated that tyrosinase is also expressed in the brain, the physiological role of tyrosinase in the brain is still obscure. In this study, to investigate the role of tyrosinase in catecholaminergic neuronal cells, we examined the effects of tyrosinase inhibition on the viability of CATH.a and SH-SY5Y cells using tyrosinase inhibitors-specifically, phenylthiourea (PTU) and 5-hydroxyindole (5-HI)-and the transfection of antisense tyrosinase cDNA. Both inhibitors significantly reduced the cell viability of CATH.a cells in a dose-dependent manner. PTU also specifically enhanced DA-induced cell death, but 5-HI did not. This discrepancy in cell death is probably due to the inhibitors' different mechanism of action: 5-HI inhibits the hydroxylation of tyrosine as a competitor for the substrate to induce cell death that may be due to depletion of DA, whereas PTU mainly inhibits the enzymatic oxidation of L-DOPA and DA rather than tyrosine hydroxylation to increase consequently autooxidation of DA. Indeed, the intracellular DA content in CATH.a cells was enhanced by PTU exposure. In contrast, PTU showed no enhancing effects on DA-induced cell death of SH-SY5Y cells, which express little tyrosinase. Furthermore, transfection with antisense tyrosinase cDNA into CATH.a cells dramatically reduced cell viability and significantly enhanced DA-induced cell death. These results suggest that tyrosinase controls the intracellular DA content by biosynthesis or enzymatic oxidation of DA, and the dysfunction of this activity induces cell death by elevation of intracellular DA level and consequent gradual autooxidation of DA to generate reactive oxygen species.     

Tyrosinase activity in primary cell culture of amelanotic melanoma cells

After transfer of the Ab amelanotic melanoma cells from in vivo to in vitro growth conditions tyrosinase activity in their soluble fraction rapidly increased. This increase lasted to the middle of the logarithmic phase of growth and was followed by a decrease of tyrosinase activity, which was accompanied by accumulation of melanin in the cells. Calf serum stimulated simultaneously tyrosinase activity, melanin synthesis, and proliferation of the melanoma cells. Acrylamide-gel electrophoresis patterns of soluble tyrosinase from the Ab melanoma cells cultured in vitro consisted of two bands, similarly as soluble tyrosinase from the Ma melanotic melanoma cells freshly isolated from solid tumors.     

Biochemical characterization of three hamster melanoma variants--I. Tyrosinase activity and melanin content

Tyrosinase activity in the soluble fraction of the cells and melanin content in the whole cells of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) hamster melanomas were studied. The activity of the soluble tyrosinase was highest in MI lower in Ma, and very low in Ab melanoma. Melanin content was greatest in the Ma, lower in MI, and none in Ab melanoma. Acrylamide gel electrophoretic pattern of the soluble tyrosinase consisted of 2 bands in Ma and MI melanomas, and of 1 band in Ab melanoma.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.     

The in vitro effect of new muramyl peptide derivatives on cytotoxic activity of NK (Natural Killer) cells from hamsters bearing AB Bomirski Melanoma

The modulation of NK activity by muramyl dipeptides derivatives against Ab (amelanotic) Bomirski melanoma and human erythroleukemia K562 cells was studied in vitro. The stimulatory effect was observed for 3 of 7 muramyl dipeptides: MDP(L-Ala)C921, MDPC857 and L18-MDP(Ala) in relation to cytotoxic activity of NK cells obtained from peripheral blood and spleen of healthy and Ab Bomirski melanoma bearing hamsters. An increased of cytotoxic activity NK cells isolated from animals before and during the transplantable phase of the tumor against K562 was found. A similar stimulation was received for NK cells obtained from animals against their own melanoma cells. The most significant influence of examined MDP derivatives on the cytotoxic activity of NK cells were obtained from animals between 10 to 12 days of tumor growth. The extent of the modulation of cytotoxic activity of NK cells was dependent on its initial value both in healthy control and Ab Bomirski melanoma bearing hamsters. If natural cytotoxic activity was high the stimulatory effect of the examined MDP derivatives was only slightly expressed.     

Inhibition of -tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells

It was previously found that -tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM -tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. -Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of -tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high -tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of -tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM -tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from -tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of -tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.     

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 μM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 μM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.     

D‐tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity

Although L‐tyrosine is well known for its melanogenic effect, the contribution of D‐tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L‐tyrosine, D‐tyrosine dose‐dependently reduced the melanin contents of human MNT‐1 melanoma cells and primary human melanocytes. In addition, 500 μM of D‐tyrosine completely inhibited 10 μM L‐tyrosine‐induced melanogenesis, and both in vitro assays and L‐DOPA staining MNT‐1 cells showed that tyrosinase activity is reduced by D‐tyrosine treatment. Thus, D‐tyrosine appears to inhibit L‐tyrosine‐mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D‐tyrosine inhibited melanogenesis induced by α‐MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D‐tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D‐tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte‐derived cells.