Melanogenesis In B16 Melanoma Cells Research Articles

Melanogenesis Stimulation in Murine B16 Melanoma Cells by Kava (Piper methysticum) Rhizome Extract and Kavalactones

Melanogenesis stimulation activity of aqueous ethanolic extracts obtained from several different parts of five Piper species, namely Piper longum, P. kadsura, P. methysticum, P. betle, and P. cubeba, were examined by using cultured murine B16 melanoma cells. Among them, the extract of P. methysticum rhizome (Kava) showed potent stimulatory effect on melanogenesis as well as P. nigrum leaf extract. Activity-guided fractionation of Kava extract led to the isolation of two active kavalactones, yangonin (2) and 7,8-epoxyyangonin (5), along with three inactive kavalactones, 5,6-dehydrokawain (1), (+)-kawain (3) and (+)-methysticin (4), and a glucosylsterol, daucosterin (6). 7,8-Epoxyyangonin (5) showed a significant stimulatory effect on melanogenesis in B16 melanoma cells. Yangonin (2) exhibited a weak melanogenesis stimulation activity.

Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells

Melanogenesis is a principal parameter of differentiation in melanocytes and melanoma cells. Our recent study has demonstrated that phospholipase D1 (PLD1) regulates the melanogenic signaling through modulating the expression of tyrosinase, the rate-limiting step enzyme in the melanin biosynthesis. The current study was designed to gain more insight into the involvement of PLD1 in the regulation of melanogenesis. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on melanogenesis in B16 melanoma cells. It was shown that the melanin synthesis was induced in PLD1-knockdowned cells, and also that the level of melanin synthesis was well correlated with increases in expression level of tyrosinase and its related proteins (Tyrp1 and Dct). Furthermore, the reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment malanoma cells with rapamycin, a potent inhibitor of mTOR effectively induced melanogenesis. The results obtained here provide possible evidence that PLD1 exerts a negative regulatory role in the melanogenic process through mTOR/S6K1 signaling.

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.

Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis.

Amphistin, a new melanogenesis inhibitor, produced by an actinomycete.

A new melanogenesis inhibitor, named amphistin, was isolated from the fermentation broth of an actinomycete strain KP-3052. Amphistin was purified from the culture filtrate by the combination of cation exchange, gel filtration, and aminosilyl silica gel chromatographic methods. The structure of amphistin was elucidated as gamma-(beta-histidinoalanino)homoalanine by NMR experiments including 1H-15N HMBC experiment and other spectroscopic analyses. Amphistin inhibited the melanogenesis of B16 melanoma cells at concentration of 6.8 microM.

New inhibitors of melanogenesis, OH-3984 K1 and K2. I. Taxonomy, fermentation, isolation and biological characteristics.

Melanogenesis inhibitors, OH-3984 K1 and K2 were isolated from fermentation broth of Streptomyces sp. OH-3984. OH-3984 K1 and K2 inhibited the melanogenesis of B16 melanoma cells at concentrations of 7.5 and 3.8 micrograms/ml, respectively, whereas inhibition of tyrosinase activity has not been observed. The microbial metabolites showed no antimicrobiological activities against Gram-positive and Gram-negative bacteria, fungi or yeast at a concentration of 1,000 micrograms/ml.

Quantitative cytochemical analysis by microdensitometry of spontaneous or α-MSH-stimulated melanogenesis in B16 melanoma cells cultivated in vitro

A spontaneous cyclic phenomenon characterized by successive waves of either high proliferative rate or intense melanogenesis is described in non-confluent B16 melanoma cells subcultivated during 2 months (or more). Dopaoxidase activity is quantified in individual cells after l-dopa reaction, by an original method of visible light absorption cytophotometry. A 24-hr treatment with α-MSH increases dopa-oxidase activity. This increase is also noted during the following 14 hr, in a fresh medium devoid of α-MSH, in which cell proliferation resumes after 24 hr. Phenylthiourea, cycloheximide or actinomycin D inhibit dopaoxidase activity, but also cell proliferation in α-MSH pre-stimulated cells. The effects of the two latter agents suggest that de novo synthesis of the enzyme takes place following α-MSH treatment.