6-Benzylaminopurine stimulates melanogenesis via cAMP-independent activation of protein kinase A

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. The present study was designed to determine the effects of 6-benzylaminopurine (6-BAP) on melanogenesis and elucidate the molecular events of melanogenesis induced by 6-BAP. To elucidate the pigmenting effect of 6-BAP and its mechanism, several experiments were performed in B16 melanoma cells. Melanin content, tyrosinase activity, cAMP production, and Western blots for proteins which are involved in melanogenesis were introduced in this study. Melanin content and tyrosinase activity increased in response to treatment with 6-BAP in a concentration-dependent manner. The tyrosinase, TRP-1, TRP-2 and MITF protein levels were found to increase significantly in response to 6-BAP in a time-dependent manner. In addition, Western blot analysis revealed that 6-BAP increased the phosphorylated level of CRE-binding protein. The increased melanin synthesis that was induced by treatment with 6-BAP treatment was reduced significantly in response to co-treatment with H-89 [a protein kinase A (PKA) inhibitor], whereas co-treatment with SB203580 (a p38 MAPK inhibitor) and Ro-32-0432 (a PKC inhibitor) did not attenuate the increase in melanin content levels that was induced by 6-BAP. In a cAMP production assay, 6-BAP did not increase the intracellular cAMP level. These findings suggest that 6-BAP activates PKA via a cAMP-independent pathway and subsequently stimulates melanogenesis by up-regulating MITF and tyrosinase expression.     

墨旱莲乙醇提取物动物致色素作用和对小鼠B16黑素瘤细胞黑素生成的影响

目的选择离体实验中对黑色素合成的关键酶—酪氨酸酶(tyrosinase,TYR)有激活作用的墨旱莲(旱莲草),研究其动物致色素作用和对小鼠B16黑素瘤细胞黑素生成及相关基因犤TYR、TYR相关蛋白(TRP-1/2)mRNA犦表达的影响。方法以棕色豚鼠为动物模型,用Schmorl法染色,计数含黑素的细胞;用多巴(DOPA)-氧化酶染色,计算每100个基底细胞中DOPA阳性细胞数。培养的小鼠B16黑素瘤细胞以四甲基偶氮唑盐比色(MTT)法、氢氧化钠(NaOH)法和体外氧化DOPA反应方法分别测定细胞的增殖活性、黑素生成量及TYR活性;逆转录-聚合酶链反应(RT-PCR)测定TYR及其TRP-1/2基因的表达。结果墨旱莲乙醇提取物可使豚鼠表皮基底层中含黑素颗粒细胞增多,使豚鼠表皮内DOPA阳性细胞增多(P<0.05);具有促进小鼠B16黑素瘤细胞黑素合成及TYR活性作用(P<0.05),对细胞TYR基因有上调作用,而对TRP-1/2mRNA的表达无明显作用。结论墨旱莲乙醇提取物具有促进黑素合成及上调酪氨酸酶基因表达的作用,对白癜风色素恢复有较好应用和开发的前景。     

The antimycotic agent clotrimazole inhibits melanogenesis by accelerating ERK and PI3K‐/Akt‐mediated tyrosinase degradation

Azole antimycotic agents are known to have anti‐inflammatory and anti‐cancer effects, which are mediated through their effects on the p38‐cyclooxygenase‐2 (COX‐2)‐prostaglandin E2 (PGE2) pathway, as well as anti‐oxidant effects. Furthermore, pyridinyl imidazole compounds, such as SB203580 have recently been shown to inhibit melanogenesis. Accordingly, we hypothesized that azole antifungal agents might affect skin pigmentation. We herein investigated the effect of clotrimazole, the most commonly used azole antifungal agent, on melanogenesis. Intriguingly, clotrimazole reduced the melanin content in human melanocytes and mouse melanocytes, as well as in B16F10 mouse melanoma cells. Clotrimazole reduced levels of tyrosinase protein without altering mRNA expression. Simultaneous treatment with a proteasomal inhibitor restored both the suppression of melanin synthesis, and the downregulation of tyrosinase level, by clotrimazole. Clotrimazole also induced the phosphorylation of extracellular signal‐regulated kinase (ERK) and PI3K/Akt, while each inhibitor of these two signals abolished the decrease of melanin synthesis by clotrimazole. Thus, our data suggest that clotrimazole inhibits melanin synthesis by promoting the proteasomal degradation of tyrosinase, which is mediated through activation of the ERK and Akt signaling pathways. These results may indicate a new role for clotrimazole as a molecular‐mechanism‐based, safe depigmenting agent for topical management of hyper‐pigmentary sequelae related to fungal infection, or for other skin inflammatory disorders.     

Superoxide Dismutase 1 Inhibits Alpha-Melanocyte Stimulating Hormone and Ultraviolet B-Induced Melanogenesis in Murine Skin

Background

Over the last decade, the incidence of ultraviolet B (UVB)-related skin problems has increased. Oxidative stress caused by UVB induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyperpigmentation. Therefore, increasing the antioxidant abilities of skin cells is thought to be a beneficial strategy for the development of sunscreen agents. Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that is known to exhibit antioxidant properties.

Objective

The purpose of this study was to investigate the effect of SOD1 on alpha-melanocyte stimulating hormone (α-MSH) and UVB-induced melanogenesis in B16F10 melanoma cells and HRM-2 melanin-possessing hairless mice.

Methods

The inhibitory effect of SOD1 on tyrosinase activity was evaluated in a cell-free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of SOD1 in vitro, and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of SOD1 in vivo.

Results

We found that SOD1 inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 melanoma cells. SOD1 did not inhibit tyrosinase activity under cell-free conditions. The results indicate that SOD1 may reduce pigmentation by an indirect, nonenzymatic mechanism. We also found that SOD1 decreased UVB-induced melanogenesis in HRM-2 melanin-possessing hairless mice, as visualized through hematoxylin and eosin staining and Fontana-Masson staining.

Conclusion

Our results indicate that SOD1 has an inhibitory effect on α-MSH and UVB-induced melanogenesis, indicating that SOD1 may be a promising sunscreen agent.     

Galvanic zinc–copper microparticles inhibit melanogenesis via multiple pigmentary pathways

The endogenous electrical field of human skin plays an important role in many skin functions. However, the biological effects and mechanism of action of externally applied electrical stimulation on skin remain unclear. Recent study showed that galvanic zinc–copper microparticles produce electrical stimulation and reduce inflammatory and immune responses in intact skin, suggesting the important role of electrical stimulation in non-wounded skin. The objective of this study is to investigate the biological effect of galvanic zinc–copper microparticles on skin pigmentation. Our findings showed that galvanic zinc–copper microparticles inhibited melanogenesis in a human melanoma cell line (MNT-1), human keratinocytes and melanoma cells co-cultures, and in pigmented epidermal equivalents. Treatment of galvanic zinc–copper microparticles inhibited melanogenesis by reducing the promoter transactivation of tyrosinase and tyrosinase-related protein-1 in human melanoma cells. In a co-culture Transwell system of keratinocytes and melanoma cells, galvanic zinc–copper microparticles reduced melanin production via downregulation of endothelin-1 secretion from keratinocytes and reduced tyrosinase gene expression in melanoma cells. In addition, exposure of pigmented epidermal equivalents to galvanic zinc–copper microparticles resulted in reduced melanin deposition. In conclusion, our data demonstrated for the first time that galvanic zinc–copper microparticles reduced melanogenesis in melanoma cells and melanin deposition in pigmented epidermal equivalents by affecting multiple pigmentary pathways.     

siRNA-mediated knock-down of COX-2 in melanocytes suppresses melanogenesis

Cyclooxygenase-2 (COX-2) is an enzyme induced in response to multiple mitogenic and inflammatory stimuli, including UV light. UV-induced COX-2 expression induces production of prostaglandin E2 (PGE2) in keratinocytes, which mediates inflammation and cell proliferation. Until recently, studies regarding COX-2 and PGE2 in the skin have focused on keratinocytes and skin cancer and the effect of PGs produced by keratinocytes on melanocytes. However, the effects of COX-2 itself or COX-2 inhibitors on melanogenesis are not well known. Therefore, to establish the role of COX-2 in melanogenesis, we investigated the effects of knock-down of COX-2 in melanocytes on melanin production and the expression of melanogenic molecules through silencing of COX-2 expression with COX-2 short interfering RNA (siRNA). COX-2 knock-down in melanocytes decreased the expressions of tyrosinase, TRP-1, TRP-2, gp100 and MITF and also reduced tyrosinase enzyme activity. Furthermore, COX-2 siRNA-transfected melanocytes showed markedly reduced alpha-melanocyte stimulating hormone (α-MSH)-induced melanin production. In addition, α-MSH-induced COX-2 expression in both scrambled siRNA-transfected and COX-2 siRNA-transfected melanocytes was greater than α-MSH-untreated cells. Our results suggest that COX-2 might be a candidate target for the development of anti-melanogenic agents and α-MSH-induced pigmentation could be closely associated with COX-2 expression. COX-2 inhibitors might therefore be of particular use in whitening cosmetics for hyperpigmentation disorders such as melasma, postinflammatory hyperpigmentation and solar lentigo.     

Hyperpigmentation of human skin grafted on to athymic nude mice: immunohistochemical study

Human skin grafted on to athymic nude mice (BALB/C-nu/nu) spontaneously hyperpigments. We wished to identify the morphological and molecular bases for the hyperpigmentation for this phenomenon. We present data on the relationship of healing, regeneration of melanocytes and production of some melanogenic stimuli. Biopsies were taken at preset times post-graft and studied by histological and immunohistochemical methods. DOPA-positive melanocytes first became visible 120 h post-graft and melanin deposition became visible along the basal cell layer 2 weeks post-graft and increased in quantify with time. By immunochemical stains the quantity of three melanocyte specific enzymes, i.e. tyrosinase, tyrosinase-related protein-1 (TRP-1) and DOPA-chrome tautomerase (TRP-2), was markedly enhanced 1 week after grafting and persisted until 4 weeks post-graft. α-Melanocyte-stimulating hormone and adrenocorticotrophic hormone were clearly detected in the epidermis soon after grafting. They were still strongly detected in the epidermis and in the dermis 2–4 weeks post-graft. We conclude that hyperpigmentation in the grafted skin accompanies a marked increase in the quantity of melanogenic enzymes and melanogenic peptides. The neouropeptides might be one of many factors which stimulate melanogenesis.     

Inhibitory effect of arbutin on melanogenesis--biochemical study using cultured B16 melanoma cells

Inhibitory effect of arbutin (hydroquinone-beta-D-glucopyranoside) on the melanogenesis was studied biochemically using cultured B16 melanoma cells. The maximum arbutin concentration lacking an inhibitory effect on cell growth was 5 X 10(-5) M. At this concentration, melanin content per cell was decreased significantly to about 39%, compared with that of arbutin untreated cells. Also, tyrosinase activity of arbutin treated cells was decreased significantly. When arbutin was added to B16 melanoma cell suspension, arbutin was not hydrolyzed to liberate hydroquinone. Further, tyrosinase activity in crude preparations from B16 melanoma cells was inhibited by arbutin. From these results, it is suggested that arbutin can inhibit the melanogenesis by affecting not only the synthesis but also the activity of tyrosinase rather than by killing melanocytes B16 melanoma cells. Also, it is suggested that hydroquinone is not responsible for the inhibitory effect of arbutin on the melanogenesis.