Effect of Adenosine on Melanogenesis in B16 Cells and Zebrafish

Background

Adenosine is a nucleoside, in which an adenine molecule is attached to a ribofuranose sugar moiety. It can be released into the microenvironment by metabolically active cells, and then fulfills a multitude of functions in regulation of cell proliferation, by activating four subtypes of G protein-coupled adenosine receptors.

Objective

In this study, we investigated the effect of adenosine on melanogenesis, using B16 melanoma cells.

Methods

The toxic effects of adenosine on B16 melanoma cells were assessed. To understand the mechanism of the effect of adenosine on melanogenesis in B16 cells, melanin content and tyrosinase activity were measured. Tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase were monitored by Western blotting. Finally, adenosine was applied to zebrafish embryos, and its in vivo effect on pigmentation investigated.

Results

At a low concentration, adenosine increased melanin content and tyrosinase activity, while a high dose of adenosine resulted in inhibition of tyrosinase activity. Western blotting showed that adenosine increased tyrosinase protein levels slightly, while high-dose adenosine decreased the expression of tyrosinase. In zebrafish tests, adenosine slightly inhibited body pigmentation.

Conclusion

In this study, we investigated the effect of adenosine on melanogenesis, using the well-established B16 melanoma cell and zebrafish models. The results suggest that adenosine may inhibit pigmentation, through negative regulation of tyrosinase.     

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.     

The Inhibitory Effect of Phytoclear-EL1 on Melanogenesis

Background

Phytoclear-EL1, an extract from Euphorbia lathyris seeds, has a whitening effect due to inhibition of tyrosinase activity.

Objective

The purpose of this study was to investigate the inhibitory effect of phytoclear-EL1 on melanogenesis.

Methods

Cultured B-16 melanoma cells and 30 human volunteers were used for in vitro and in vivo studies, respectively. Phytoclear-EL1 was added to the cultured B-16 melanoma cells, and applied to UVB-induced hyperpigmented lesions of human volunteers twice daily for 7 weeks. Changes in the number of B-16 melanoma cells, as well as changes in morphology, melanin content, and tyrosinase activity, were measured and then compared with the normal control and the 10^-3M arbutin groups. Also, the effect of phytoclear-EL1 on UVB-induced hyperpigmented lesions was examined through subjective and objective measurements.

Results

In the in vitro study (p<0.05), the number, melanin content, and tyrosinase activity of cultured B-16 melanoma cells were decreased in the 5µg/ml phytoclear-EL1 group compared to the control group. On objective assessment with a chromameter, the 0.2% phytoclear-EL1 group had a larger difference in the mean L values before and 7 weeks after applying phytoclear-EL1 as compared to the other groups. On subjective assessment by both the researchers and subjects 7 weeks after applying experimental materials, the 0.2% phytoclear-EL1 group and positive control (3% arbutin) had higher scores than the placebo groups. These results demonstrated that phytoclear-EL1 in vivo and in vitro had an inhibitory effect on melanogenesis.

Conclusion

Phytoclear-EL1 may be a candidate extract in the control of hyperpigmentary disorders.     

威灵仙等4种中药抑制黑素生成作用的机制研究

目的：研究威灵仙等4种中药对melan-a小鼠黑素细胞酪氨酸酶（Tyr）活性、Tyr及酪氨酸酶相关蛋白（TRP）基因转录与蛋白表达的影响,对4种中药抑制黑素生成的作用机制进行初步探讨.方法：用中药提取物处理melan-a小鼠黑素细胞后进行黑素含量测定,左旋多巴染色、实时荧光定量反转录（RT）-PCR和Western blot分别测Tyr及TRP-1、TRP-2的mRNA和蛋白表达水平.以熊果苷作为阳性对照.结果：4种中药在20 μg/mL浓度时具有明确的抑制黑素生成的作用,并且都不同程度地抑制Tyr的氧化活性.威灵仙、五倍子可以同时降低Tyr及TRP-1、TRP-2的基因转录和蛋白表达量.而麦冬、白苏叶可以降低Tyr及TRP-1、TRP-2的合成,但是对基因表达无影响.结论：威灵仙及五倍子提取物可以通过抑制Tyr、TRP-1、TRP-2的基因表达及蛋白合成和Tyr的多巴氧化活性这三方面实现其抑制黑素产生的作用;麦冬、白苏叶在基因转录后的酶蛋白水平发挥抑制黑素生成的作用.     

女贞子对培养的黑素细胞酪氨酸酶活性和黑素合成的影响

目的 研究女贞子单体酪醇和齐墩果酸对人黑素细胞的增殖、酪氨酸酶活性及黑素合成的影响。方法 采用MTT法测定细胞增殖情况,多巴氧化法测定酪氨酸酶活性,氢氧化钠裂解法测定黑素含量,半定量RT-PCR检测药物对黑素细胞酪氨酸酶和酪氨酸酶相关蛋白-1 mRNA表达的影响。结果 加入酪醇72h后,各组黑素细胞的酪氨酸酶活性和合成黑素能力明显增强,且呈浓度依赖性;酪氨酸酶和酪氨酸酶相关蛋白-1 mRNA表达量分别比空白对照增加39.10%和99.26%;齐墩果酸也能明显激活酪氨酸酶(P<0.01),促进黑素合成(P<0.05),并使黑素细胞酪氨酸酶和酪氨酸酶相关蛋白-1mRNA表达量分别比加入无水乙醇的对照增加31.88%和17.73%。结论 酪醇和齐墩果酸可能通过上调酪氨酸酶和酪氨酸酶相关蛋白-1 mRNA的表达而增强正常人黑素细胞的黑素合成和酪氨酸酶活性:两者可能是女贞子中影响黑素细胞生物学活性的主要成分。     

Possible Existence of Melanocytes or Melanoblasts in Human Sebaceous Glands

Background

Melanocytes are present in both basal epidermis and hair follicles. Melanocyte stem cells have been found in hair follicle bulge. During embryogenesis, the outer cells of the bulge differentiate into the sebaceous gland (SG) and proliferate.

Objective

To identify and determine the distribution and morphological characteristics of melanocytes in human SGs.

Methods

A total of 171 biopsy specimens of face and scalp were studied. Of these specimens, 103 samples contained SGs. We conducted a retrospective review of slides stained with H&E, F-M, anti-S100, anti-c-kit, anti-HMB-45, anti-CD1a, anti-MITF, and anti-tyrosinase. The presence and distribution of melanocytes in human SGs was also evaluated by electron microscopy. In addition, melanocytes were isolated from SGs for primary culture.

Results

S-100-positive cells were observed mainly at the periphery of SGs in 34 of 54 specimens. We did not find F-M-positive and HMB-45-positive cells in SGs. CD1a-positve cells were identified in two specimens. We also found c-kit-, MITF-, and tyrosinase-positive cells in SGs. Electron micrograph showed the presence of melanocytes in the suprabasal portion of SGs. These melanocytes showed fewer melanin-containing granules than the melanocytes of basal epidermis. However, the individually distributed melanosomes in suprabasal melanocytes were larger than those in epidermal melanocytes. Primary culture of melanocytes derived from SGs showed morphologically homogeneous, slender cell bodies with few dendrites.

Conclusion

Our study confirms the presence of non-melanogenic melanocytes and Langerhans cells in human SGs. In addition, the characteristics of the melanocytes in SGs were found to be different from those of the epidermal melanocytes.     

全反式维甲酸和阿达帕林对中波紫外线诱导的melan-a黑素细胞黑素合成的影响

目的 探讨外用维甲酸脱色素作用的机制.方法 利用体外培养的melan-a黑素细胞,观察全反式维甲酸和阿达帕林对中波紫外线(UVB)诱导的melan-a黑素细胞黑素合成的影响.结果 二者可剂量依赖性抑制UVB诱导的细胞酪氨酸酶活性和黑素合成,使其回复到基础水平,但对基础黑素合成无明显影响,10-5M的全反式维甲酸可抑制黑素细胞增殖,阿达帕林对细胞增殖无明显影响.全反式维甲酸和阿达帕林对细胞黑素合成的影响无明显差异.结论提示维甲酸可直接作用于黑素细胞抑制其黑素合成,这可能是维甲酸脱色素作用的机制之一.     

Comparison of Gene Expression Profiles between Keratinocytes,Melanocytes and Fibroblasts

Background

The skin has many important functions such as protection, preservation, temperature regulation, and vitamin D synthesis. It is composed of a variety of cell types including keratinocytes, melanocytes and fibroblasts.

Objective

We attempted to compare the gene expression profiles between keratinocytes, melanocytes and fibroblast, using cDNA microarray.

Methods

Keratinocytes, melanocytes and fibroblasts were primary cultured from five foreskin specimens. Total RNAs were extracted and pooled to reduce the individual variations, and then used for cDNA microarray.

Results

Total 12,028 genes were selected as the reliable genes whose expression was detected in at least one of the three cell types. By comparing the relative expression levels with cutoff limitation as a fourfold change, we obtained 126 fibroblast-specific, 179 keratinocyte-specific and 173 melanocyte-specific genes, many of which are known to be characteristically expressed in each cell type. In addition, we identified many genes whose skin-specific functions have not yet been determined.

Conclusion

Our data provide important information on which to base further investigation into the specification of skin cell types.     

Expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant

Background

Cytosolic NADP⁺-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione.

Objective

To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant.

Methods

The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked.

Results

The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H₂O₂ compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H₂O₂.

Conclusions

This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.     

Melanoma inhibitory activity in Brazilian patients with cutaneous melanoma

BACKGROUND:

Melanoma inhibitory activity is a protein secreted by melanoma cells and has been used as a tumor marker. Increased Melanoma inhibitory activity serum levels are related to metastatic disease or tumor recurrence. Currently there are no studies on Melanoma inhibitory activity and cutaneous melanoma involving Brazilian patients.

OBJECTIVE:

To evaluate the performance and feasibility of measuring Melanoma inhibitory activity levels in Brazilian patients with cutaneous melanoma.

METHODS:

Blood was obtained from ten patients with proved metastatic cutaneous melanoma (Group 1), 15 patients resected for cutaneous melanoma without metastasis (Group 2) and 5 healthy donors (Group 3). Melanoma inhibitory activity was measured using a commercially available ELISA kit.

RESULTS:

There was a statistically significant difference of Melanoma inhibitory activity levels between patients with and without metastasis (p=0.002), and between patients with metastasis and healthy donors (p=0.002). There was no difference between patients without metastasis and healthy donors (p=0.443).

CONCLUSION:

Melanoma inhibitory activity is a tumor marker for cutaneous melanoma and the Melanoma inhibitory activity-ELISA test can be easily performed. Patients with metastasis have increased Melanoma inhibitory activity serum levels when compared to patients without metastasis and healthy donors.     

苦参碱及肉桂酸对体外培养小鼠黑素细胞黑素生成的影响

目的:观察苦参碱和肉桂酸对melan-a小鼠黑素细胞黑素生成的影响。方法:用药物干预melan-a小鼠,观察黑素细胞的形态并测定各组黑素量及酪氨酸酶的活性。结果:肉桂酸干预melan-a小鼠黑素细胞2d后就表现出明显的促进树突生成的作用。肉桂酸和苦参碱在高、中、低浓度(1.00、0.10、0.01mmol/L)有抑制melan-a小鼠黑素细胞酪氨酸酶活性的作用(除外0.01mmol/L苦参碱),其抑制作用均弱于对照药氢醌(P<0.01)。苦参碱(0.001～1.000mmol/L)和肉桂酸(0.001～1.000mmol/L)均能使melan-a小鼠黑素细胞黑素量减少,但其抑制黑素合成的作用明显低于同浓度氢醌。Fontana-Masson染色表明,经苦参碱干预后的melan-a小鼠黑素细胞内黑素颗粒少于空白对照组。结论:苦参碱和肉桂酸均有抑制melan-a小鼠黑素细胞酪氨酸酶活性的作用及使黑素合成量减少的作用,且苦参碱作用强于肉桂酸,而肉桂酸有促进黑素细胞树突形成的作用。     

淫羊藿苷抑制正常黑素细胞黑素合成的研究

目的:观察中药单体淫羊藿苷对体外培养的正常黑素细胞黑素合成和酪氨酸酶活性的影响。方法:选择高、中、低3个浓度的中药单体作用于体外培养的黑素细胞,测定细胞酪氨酸酶活性、黑素含量和细胞增殖率。采用间接免疫荧光染色法对药物作用的黑素细胞和对照的酪氨酸酶(tyrosinase熏TYR)、酪氨酸酶相关蛋白-1(tyrosinaserelatedprotein-1熏TRP-1)、TRP-2进行标记,然后采用激光共聚焦显微镜半定量分析淫羊藿苷作用后细胞内3种成分表达量的变化。结果:淫羊藿苷明显抑制酪氨酸酶活性和黑素的生成(P<0.01),且呈浓度依赖性,但淫羊藿苷抑制黑素生成的作用较氢醌弱(P<0.01);淫羊藿苷以浓度依赖的方式促进细胞的增殖,氢醌则抑制了细胞的增殖;淫羊藿苷对黑素细胞内TYR和TRP-2的表达没有明显影响,但是对TRP-1的表达却明显增加了。结论:淫羊藿苷能够抑制黑素合成和酪氨酸酶活性,这种抑制可能主要通过抑制酪氨酸酶活性起作用。     

The Efficacy and Safety of 4-n-butylresorcinol 0.1% Cream for the Treatment of Melasma: A Randomized Controlled Split-face Trial

Background

Melasma is a common acquired symmetrical hypermelanosis that occurs on sun-exposed areas, and it is frequently observed among women. Various treatment modalities have been tried, but none are completely satisfactory. 4-n-butylresorcinol, which is a resorcinol derivative that has an inhibitory effect on both tyrosinase and tyrosinase-related protein-1, was introduced in 1995 and it has received increasing attention as a new hypopigmenting agent. However, the hypopigmenting effect of 4-n-butylresorcinol in human subjects has only been shown in a few studies.

Objective

The aim of this study was to investigate the hypopigmenting efficacy and safety of 4-n-butylresorcinol 0.1% cream for the treatment of melasma.

Methods

Twenty patients with melasma were enrolled to this randomized, double-blind, vehicle-controlled, split-face comparative study. The patients were instructed to apply 4-n-butylresorcinol 0.1% cream or vehicle to each side of the face twice daily for 8 weeks. Mexameter measurements were performed along with photography at baseline, 4 weeks and 8 weeks. Adverse events were observed and recorded throughout the study.

Results

All the patients completed the study. Mexameter measurements demonstrated that the melanin index of the treated side showed a significant decrease when compared with that of the vehicle-treated side after 4 weeks (p=0.006) and after 8 weeks (p<0.0005). All the adverse reactions were mild and transient.

Conclusion

4-n-butylresorcinol 0.1% cream showed rapid efficacy and it was well tolerated when used for the treatment of melasma.     

1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)propane inhibits melanin synthesis by dual mechanisms

Background

1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)propane (DP) was reported as a novel tyrosinase inhibitor by Nesterov et al. In previous study, we showed that DP is an antioxidant and accelerates the fading of UVB-induced tan in human skin but details of inhibiting mechanism of DP in melanogenesis remain incomplete.

Objective

To clarify additional mechanisms of DP inhibition of melanogenesis, we studied the effect of DP on tyrosinase processing and degradation.

Methods

Tyrosinase inhibition was assessed using mushroom and human tyrosinase. The effect of DP on mRNA and protein levels as well as glycosylation and degradation of tyrosinase was examined using normal human epidermal melanocytes (NHEM).

Results

DP was 200 times more potent than that of kojic acid in inhibiting mushroom tyrosinase activity. In contrast, DP (IC₅₀ = 200 μM) was significantly less effective at inhibiting tyrosinase from NHEM. DP decreased melanin content in cultured NHEM after 7th day (IC₅₀ = 10 μM). The IC₅₀ for DP against human tyrosinase activity was found to be at least 20 times higher than that of melanin synthesis. At a non-cytotoxic concentration DP did not decrease tyrosinase mRNA however protein level decreased by 46% after 48 h treatment. DP did not alter the ratio of mature and immature tyrosinase assayed by endo H cleavage. Tyrosinase degradation assays revealed that DP accelerated tyrosinase degradation in NHEM.

Conclusions

We found that DP acts through dual mechanisms to reduce melanin synthesis; by inhibition of tyrosinase activity via an anti-oxidant effect, and, more importantly, by the acceleration of tyrosinase degradation.     

In Vitro Antimicrobial Activities of Fusidic Acid and Retapamulin against Mupirocin- and Methicillin-Resistant Staphylococcus aureus

Background

The in vitro activities of retapamulin and fusidic acid against clinical isolates of mupirocin-resistant and methicillin-resistant Staphylococcus aureus (MRSA) from Korea are not well understood.

Objective

This study aimed to determine the activities of retapamulin and fusidic acid against clinical isolates of mupirocin-resistant MRSA.

Methods

Clinical isolates of mupirocin-resistant MRSA were collected from two tertiary hospitals. The minimal inhibitory concentrations of mupirocin, fusidic acid, and retapamulin were determined using agar dilution method. Polymerase chain reaction was used to confirm the identity of the species and the presence of resistance genes. Pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNA were used to determine the genetic similarity of high-level mupirocin-resistant isolates.

Results

Of the 497 MRSA isolates tested, 22 (4.4%) were mupirocin-resistant. Of these, 9 (1.8%) and 13 (2.6%) had high-level and low-level mupirocin resistance, respectively. Analysis of the PFGE patterns of the high-level mupirocin-resistant MRSA isolates identified five clusters. All 13 of the low-level mupirocin-resistant isolates were resistant to fusidic acid but susceptible to retapamulin. However, among the 9 high-level mupirocin-resistant isolates, 56% were resistant to fusidic acid, and all were susceptible to retapamulin.

Conclusion

Retapamulin is highly active in vitro against Korean clinical isolates of high-level mupirocinand methicillin-resistant Staphylococcus aureus with different genetic backgrounds. Fusidic acid is more active against high-level mupirocin-resistant MRSA than low-level mupirocin-resistant MRSA.     

Recipient site preparation for epidermal graft in stable vitiligo by a special fraise

BACKGROUND

The only approach used in the refractory lesions of stable vitiligo is the surgical supply of melanocytes. Suction Blistering Epidermal Graft is one of the most common and effective techniques. There are multiple modalities, including the motor-driven diamond fraise, for the preparation of recipient sites in suction blistering epidermal graft with different repigmentation rates and complications.

OBJECTIVES

To evaluate preparation of recipient site by a motor-driven dental lab finishing carbide bur.

METHODS

Sixty-one stable, depigmented lesions were selected in 14 patients (9 women and 5men), aged 16-29 years, of which 9, 3 and 2 had localized, generalized and segmental vitiligo, respectively. Recipient site was prepared by a motor-driven dental lab finishing carbide bur.

RESULT

Excellent repigmentation at the recipient site was observed in 53 out of 61 (86.9%) grafted lesions. Postinflammatory hyperpigmentation and perigraft halo were seen in 11 (18%) and 17 (27.9%) patients at the recipient site, respectively.

CONCLUSION

Using a motor-driven dental lab finishing carbide bur to prepare the recipient site of suction blistering epidermal graft technique is reliable and effective, removing only the depigmented epidermis in a simple and safe manner, even on complex-shaped lesions and scar-prone sites.