Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Specific protein production during melanogenesis in B16/C3 melanoma cells

The mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone-supplemented serum-free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, 5-bromodeoxyuridine, and acidic pH inhibit this process. Using two-dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of tyrosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B16/C3 melanoma cells.     

Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.     

Inhibitory effects of whisky congeners on melanogenesis in mouse B16 melanoma cells

We examined the effect of whisky congeners, substances other than ethanol in whisky, on melanogenesis in mouse B16 melanoma cells. Treatment with whisky congeners significantly blocked melanogenesis. Our results indicate that the inhibitory effects of whisky congeners on melanogenesis is due to direct inhibition of tyrosinase activity and to suppression of tyrosinase protein levels.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells

A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in anin vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 μg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 μg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C₂ ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.     

Effects of salicylic acid on mushroom tyrosinase and B16 melanoma cells

Salicylic acid slightly inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase noncompetitively without being oxidized. In contrast, 4-hydroxybenzoic acid did not inhibit this enzymatic oxidation if a longer reaction time was observed, although it suppressed the initial rate of the oxidation to a certain extent. Neither acid showed noticeable effects on cultured murine B16-F10 melanoma cells except weak cytotoxicity.     

Inhibitory effects of whisky polyphenols on melanogenesis in mouse B16 melanoma cells

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.     

Synthesis of quercetin glycosides and their melanogenesis stimulatory activity in B16 melanoma cells

4′-O-β-d-Glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyra-noside (3) was isolated from Helminthostachys zeylanica root extract as a melanogenesis acceleration compound and was synthesized using rutin as the starting material. Related compounds were also synthesized to understand the structure–activity relationships in melanin biosynthesis.Melanogenesis activities of the glycosides were determined by measuring intracellular melanin content in B16 melanoma cells. Among the synthesized quercetin glycosides, quercetin-3-O-β-d-glucopyranoside (1), quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (2), and 3 showed more potent intracellular melanogenesis acceleration activities than theophyline used as positive control in a dose-dependent manner with no cytotoxic effect.     

Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

MSH stimulates adenylate cyclase and tyrosinase in cultivated melanoma cells in the presence of cytochalasin B

MSH and cholera toxin increase intracellular levels of cyclic AMP (cAMP) and tyrosinase activity in cultivated mouse melanoma cells in the presence of cytochalasin B (CB) at a concentration sufficient to prevent pinocytosis and cytokinesis. The data suggest that MSH and cholera toxin exert their effects without entering the cell.     

Stimulation of melanogenesis by the citrus flavonoid naringenin in mouse B16 melanoma cells

Naringenin is a naturally occurring citrus flavanone. In this study, we examined the effect of naringenin on melanogenesis in mouse B16 melanoma cells. Melanin contents and tyrosinase activities were strongly increased by naringenin. Naringenin was found to cause marked increases in the expression levels of melanogenic enzymes.     

Induction of B16 melanoma melanogenesis by a serum-free synthetic medium.

Cultured murine B16 melanoma cells normally grow as spindle-shaped cells firmly attached to tissue culture flasks. Pellets obtained from harvested B16 melanoma cells are white to grey in color. When the same cells were grown in synthetic, serum-free AIM V medium, cellular morphology and pigmentation were radically altered. Within 3 days of subculture in AIM V, cells rounded up and grew in clusters in suspension. Melanin content increased to greater than 30 times and tyrosinase activity was found to be 10-50 times higher in cells grown in AIM V medium compared to those cultured in normal medium. A concomitant increase in the level of immunoreactive tyrosinase was also induced. The individual growth factors and hormones present in AIM V medium were examined to determine which component(s) stimulates melanogenesis. Only those cells grown in the presence of 2.5% human albumin were stimulated to synthesize melanin. These findings suggest that albumin, or a component associated with albumin, has a major effect upon the regulation of melanogenesis in these cells.