Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax

The pigment pattern expression resides in the chromatoblasts of the embryonic skin. The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF). We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one. Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period. Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase. The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors. The results obtained may actually reflect the resultant activity of the two factors present.     

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.     

Intrinsic Pigment-Cell Stimulating Activity in the Catfish Integument

In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus. This species has a dark dorsal surface in marked contrast to an almost white midventral surface. Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev. Biol. 129:25). A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS). When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed. The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis. Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis. A similarly conditioned medium based upon Leibovitz's L-15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens. Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores. A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results. The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument. It is suggested that the light-colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF. Thus, the expression of melanophores is inhibited while that of iridophores is enhanced. In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.     

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

Control of melanoblast differentiation in amphibia by alpha-melanocyte stimulating hormone, a serum melanization factor, and a melanization inhibiting factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide:Dev. Biol., 129:25-36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of alpha-melanocyte stimulating hormone (alpha-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the "outgrowth" and "melanization" of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW less than 30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493-1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW greater than 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system.(ABSTRACT TRUNCATED AT 400 WORDS)     

A ventrally localized inhibitor of melanization in Xenopus laevis skin

Melanophores normally differentiate in dorsal but not in ventral skin of Xenopus laevis. We have sought factors which might regulate this differentiation pattern, and we have obtained a putative melanization inhibiting factor (MIF) from ventral but not from dorsal skin. Preliminary studies reveal that MIF is destroyed by heat or trypsin treatment, indicating its protein composition, and has a molecular weight in the range of 300 kDa. The effects of MIF on the differentiation of neural crest derivatives to melanophores were examined in vitro in the presence of tyrosine and fetal calf serum (FCS). Tyrosine enhances melanophore differentiation in vitro at concentrations equivalent to those estimated in adult Xenopus blood plasma (20 microM). FCS also stimulates melanization, by way of materials other than the tyrosine contained in FCS. MIF strongly inhibits outgrowth and melanization of neural crest cells from neural tube explants. MIF also inhibits the differentiation of melanoblasts contained in cultured explants of ventral skin. Inhibition of melanization or melanophore differentiation by MIF occurs even in the presence of L-tyrosine and/or FCS. We suggest that MIF plays an important role in the establishment of dorso-ventral pigment patterns in amphibia.     

Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.     

The Amphibian Melanization Inhibiting Factor (MIF) Blocks the α-MSH Effect on Mouse Malignant Melanocytes

We have found that a melanization inhibitory factor (MIF) extracted from the ventral skin of Rana forreri has a slight inhibitory effect on the activity levels of tyrosinase and dopachrome tautomerase in B16/F10 and Cloudman S-91 murine melanoma cell lines. Furthermore, this factor appears to block the effects of α-MSH on these enzymatic activities. However, MIF treatment does not affect the melanogenic action of theophylline on the same cells, suggesting that MIF acts proximal to MSH-mediated cAMP formation, possibly by interaction with the MSH receptor. In this way, we show that this amphibian factor has biological activity on mammalian melanocytes. This suggests the existence of mammalian counterparts of amphibian MIF in the mouse integument that might regulate epidermal melanocytes. These peptides might be related to the agouti protein, as they share similar mechanisms of action. The interaction of different peptides with the MSH receptor would be a complex but general mechanism responsible for many mammalian coat color variants.     

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function

The aim of this study was to collect evidences on the role of transglutaminase (TG, E.C.2.3.2.13) in the antineoplastic properties exerted by nimesulide (NMS), a non-steroidal anti-inflammatory drug, on murine B16-F10 melanoma cells. Treatment of melanoma cells with nimesulide produces a considerable reduction of cell proliferation, paralleled by a remarkable decrease of the intracellular concentration of polyamines spermidine and spermine. NMS treatment induces cancer cell differentiation, likely through the observed enhancement of TG and tyrosinase activities and increase of melanin production, well known markers of melanocyte differentiation. The overall results highlight the possibility that nimesulide acts as antineoplastic agent likely through the induction of intracellular TG activity.     

Stimulation of Cultured Iridophores by Amphibian Ventral Conditioned Medium

That the ventral integument of adult frogs (Rana pipiens) contains factor(s) that stimulate iridophore expression (adhesion, morphologic appearance, proliferation) was demonstrated on iridophores derived from tadpoles of R. pipiens and Pachymedusa dacnicolor, and maintained in primary culture in a growth medium based upon Leibovitz's L-15. Experimental growth medium (VCM) conditioned by a one-hour exposure to pieces of ventral skin of adult R. pipiens induced iridophores to assume a broad and stellate appearance, to form confluent sheets, and to proliferate over a nine-day period. Iridophores in control medium assumed long thin profiles, detached easily, and exhibited no signs of proliferation. Unknown cells containing reflecting platelets and unusual other organelles appeared uniquely in chromatophore cultures of P. dacnicolor in VCM. The intense stimulation of iridophore expression in VCM is consistent with the known inhibitory effect of this medium on melanization and with its purported role in the determination of dorsal/ventral pigment patterns of amphibians. The results are discussed in terms of a prevailing theory about pigment cell origins and development.     

Stimulation of melanin synthesis of B16-F10 mouse melanoma cells by bufalin.

Bufalin, which is one of prominent components of Chinese toad venom, was found to decrease the rate of cell proliferation of mouse melanoma clone B16-F10 cells and a concomitant stimulation of expression of its melanotic phenotype. The effect of bufalin on melanogenesis included stimulation of tyrosinase activity and increase of cellular melanin content. These effects became apparent after 48 hr exposure to 10(-4) M bufalin and increased thereafter. Other cardiotonic steroids, such as cinobufagin and ouabain, at the concentration of 10(-4) M for 6 days, also showed the stimulatory effect on melanin synthesis of B16-F10 cells, but not digitoxigenin.     

Effects of dietary xanthophylls/astaxanthin ratios on the growth and skin pigmentation of large yellow croaker Larimichthys crocea (Richardson, 1846)

An 8‐week feeding trial was conducted to evaluate the effects of dietary xanthophylls/ astaxanthin ratio on the growth and skin color of large yellow croaker, Larimichthys crocea. Five pigment‐supplemented diets were formulated to contain 75/0, 50/25, 37.5/37.5, 25/50 and 0/75 mg kg^?1 of xanthophylls/astaxanthin. The xanthophylls contain 89.31% lutein and 6.12% zeaxanthin. A diet without pigment supplementation was used as the control. The large yellow croaker juveniles (13.80 ± 0.03 g) were randomly distributed in 18 sea cages (1.0 × 1.0 × 1.5 m) at a density of 45 fish per cage. Water temperature ranged from 21 to 31°C during the feeding trial. To obtain results, the survival rate, specific growth rate, feed conversion ratio, skin redness, skin yellowness, skin lightness, skin carotenoid content and skin melanin content were measured. The results showed that the survival rate, specific growth rate and feed conversion ratio were not significantly affected by dietary treatments (P > 0.05). The ventral skin lightness was also not affected by dietary treatments (P > 0.05); however, the dorsal skin lightness of fish fed with the control diet was significantly lower than those fed with pigment‐supplemented diets (P < 0.05). The lowest values of yellowness and carotenoid content both in the ventral skin and dorsal skin were found in the control group. Yellowness and carotenoid content increased with an increasing proportion of dietary xanthophylls in both the ventral and dorsal skin. Higher redness values were found in the compound pigment groups, either in the dorsal skin or ventral skin. Fish fed with the control diet showed a higher melanin content in the dorsal skin than those fed with pigment‐supplemented diets, although differences were not significant (P > 0.05). Lightness and yellowness were linearly related to skin carotenoid content. Meanwhile, skin yellowness and carotenoid content were linearly related to the proportion of xanthophylls in dietary pigments.     

Melanogenesis inhibitors from Rabdosia japonica

The effects of the four major ent-kaurene diterpenoids isolated from the aerial part of Rabdosia japonica (Labiatae) on murine B16-F10 melanoma cells were investigated. Among the compounds tested, oridonin and nodosin most significantly suppressed cellular melanin production when the cells were cultured with these diterpenoids. However, oridonin and nodosin exhibited cytotoxicity against the same melanoma cells with an IC(50) of 1.1μM (0.40μg/ml) and of 1.3μM (0.47μg/ml) and almost complete lethality was observed at 4.0μM and at 8.0μM, respectively, and therefore observed melanogenesis inhibition is mainly due to its melanocytotoxic effect. Morphological observation showed that oridonin or nodosin treated B16-F10 melanoma cells induced dendrite structure. Diterpenoids quickly formed adducts partly in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% of fetal bovine serum (10% FBS-DMEM) before their application to the cells. Approximately 20% of oridonin formed adducts within the first 15min. Notably, dihydronodosin exhibited inferior cytotoxicity (>85% cell viability at 100μM) but still significantly suppressed melanogenesis (>55%) when murine B16-F10 melanoma cells were cultured with this diterpenoid derivatives. Hence, dihydronodosin can be a potential melanogenesis inhibitor.     

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the <Emphasis Type="Italic">in vivo</Emphasis> metastatic potential

Summary. The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.     

Therapeutic potential of Smilax fluminensis ethanolic extract: antitumoral activity in murine melanoma cells

The aim of the study was to investigate the in vitro and in vivo antitumor activity of leaves ethanol extract from Smilax fluminensis on murine melanoma. The extract was performed by ethylic alcohol and submitted to classical chemical analysis. Cytotoxicity test were performed on neoplastic cells, where antitumor activity was expressed in GI₅₀ (concentration that inhibits 50% of cell growth) and the determination of selectivity index using a normal cell line. In addition, BALB/c mice models were used to evaluate the in vivo antitumor activity of extract in two different concentrations against B16-F10 melanoma cells. The tumor inhibition ratio was determined and the histopathological analyses of nodules and liver were compared. The chemical analysis indicated a major presence of phenolic compounds and flavonoids. Cytotoxicity test results that S. fluminensis extract was active in B16-F10 line (GI₅₀: 4.37 µg/mL), being the extract considered a promising antineoplastic agent. In the experimental model, the inhibition percentage of tumoral growth was between 78.77 and 83.49%. Histopathology analysis of nodules showed necrotic cells reduction, adipocytes presence, melanin deposition, vascularization, and inflammatory process in a concentration-dependent manner. On the liver, the animals treated with the extract on both concentrations showed normal hepatic organization, normal hepatocytes, and absence of inflammatory focus. The results indicate that S. fluminensis extract demonstrated both in vitro and in vivo antitumor activity, reducing the tumoral growth in B16-F10 and could therefore be a promising antineoplastic agent.

     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

Antioxidant and Anti-Melanogenic Effect of the Novel Synthetic Hexapeptide (SFKLRY-NH2)

The novel synthetic hexapeptide, Angio-S (SFKLRY-NH₂), induced angiogenesis in human endothelial cells and accelerated wound healing. Since the pathophysiology of a wound is similar to the skin-aging process, the antioxidant and anti-melanogenic effects of Angio-S were investigated in this study. The antioxidant effect was investigated in the dermal fibroblasts, and the skin-whitening effect was studied in melanoma B16 cells. Angio-S exhibited an antioxidant activity, which increased in a dose-dependent manner. A cell survival assay revealed that Angio-S aided dermal fibroblasts in the resistance of free radicals induced by tert-butyl hydroperoxide. In addition, activities of superoxide dismutase and glutathione peroxidase were enhanced after pre-treatment with Angio-S. Since antioxidants inhibit the chemical reactions leading to melanin formation, the anti-melanogenic effect of Angio-S was studied. Angio-S reduced the synthesis of melanin and inhibited the activity of tyrosinase in melanoma B16 cells. Although the underlying mechanism of inhibiting melanin synthesis was not fully studied, Angio-S may act as an anti-oxidant and directly inhibit tyrosinase during melanin biosynthesis. Collectively, these results indicate that Angio-S exhibits antioxidant and anti-melanogenic effects, and is a potential candidate for use as a skin rejuvenation agent considering the skin-rejuvenating effect at a relatively low concentration.     

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH₄Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state.