Autophagy induced by resveratrol suppresses α‐MSH‐induced melanogenesis

Autophagy degrades cellular components and organelles through a cooperative process involving autophagosomes and lysosomes. Although autophagy is known to mainly regulate the turnover of cellular components, the role of autophagy in melanogenesis has not been well addressed. Here, we show that inhibition of autophagy suppresses the antimelanogenesis activity of resveratrol (RSV), a well‐known antimelanogenic agent. RSV strongly increased autophagy in melanocytes. However, the depletion of ATG5 significantly suppressed RSV‐mediated antimelanogenesis as well as RSV‐induced autophagy in melanocytes. Moreover, suppression of ATG5 retrieved the RSV‐mediated downregulation of tyrosinase and TRP1 in α‐MSH‐treated cells. Most importantly, electron microscopy analysis revealed that autophagosomes engulfed melanin or melanosomes after combined treatment of α‐MSH and RSV. Taken together, these results suggest that RSV‐mediated autophagy regulates melanogenesis.     

Modulation of Melanin Synthesis by Rengyolone Isolated from the Root of Eurya emarginata in Melan‐a Cells

In the course of screening for the melanogenesis inhibitors, rengyolone was isolated from Eurya emarginata (Thumb) Makino. Its chemical structure was determined on the basis of spectroscopic analysis including mass spectroscopy and nuclear magnetic resonance analysis. Rengyolone inhibited potent melanogenesis in melan‐a cells with an IC₅₀ value of 65 μM without cytotoxicity. Also, rengyolone showed a melanin biosynthesis inhibition zone in a culture plate of Streptomyces bikiniensis, which is commonly used as an indicator organism. Moreover, rengyolone dramatically reduced protein expression of melanogenic enzyme, tyrosinase. Furthermore, rengyolone presented inhibition on the body pigmentation in zebrafish model system and decreased melanin contents and tyrosinase activity. These results suggest that rengyolone isolated from E. emarginata may be an effective skin‐whitening agent that regulates the expression of melanogenic enzymes. Copyright © 2013 John Wiley & Sons, Ltd.     

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.     

复色1号方对小鼠B16黑素瘤细胞增殖及酪氨酸酶活性的影响

目的 探讨复色1 号方对小鼠B16 黑素瘤细胞增殖及酪氨酸酶活性的影响.方法 采用MTT 法测定不同浓度的复色1 号方对小鼠B16 黑素瘤细胞增殖的影响,多巴速率氧化法测定对酪氨酸酶活性的影响,对其结果进行统计分析.结果 复色1 号方浓度在0.001～0.01g/l,范围内对小鼠B16 黑素瘤细胞的增殖与对照组相比无统计学意义(P＞0.05),0.05～0.1g/l 浓度范围内对小鼠B16 黑素瘤细胞的增殖与对照组相比有显著意义(P＜0.05),呈现一定的抑制作用;复色1 号方在0.001～0.1g/l 浓度范围内对小鼠B16 黑素瘤细胞中酪氨酸酶活性的影响与对照组相比,各浓度含药培养基组的A 值均有显著性差异(P＜0.05),呈剂量依赖性激活作用.在0.001～ 0.01g/l 浓度范围内,复色1 号方的最佳激活浓度为0.01g/l.结论 复色1 号方没有促进小鼠B16 黑素瘤细胞增殖的作用;在一定浓度范围内(0.001～0.1g/l),对小鼠B16 黑素瘤细胞酪氨酸酶的活性呈剂量依赖型激活作用.     

Enhancement of pulmonary metastasis formation and γ-glutamyltranspeptidase activity in B16 melanoma induced by differentiation in vitro

B16 melanoma sublines (B16-F10-BL6 and B16-Fl) exhibited elevated adenosine 3,5-cyclic monophosphate (cAMP) levels when cultured in Dulbecco's modified Eagle's medium (DMEM) in comparison to cells in RPMI-1640 medium. In parallel, cells cultured in DMEM had increased tyrosinase activity, melanization and dendrite formation, all markers of melanoma differentiation. Also, the proliferative rates of both cell lines were decreased by 80–85% when cultured in DMEM relative to cells maintained in RPMI-1640 medium. In these studies, elevated levels of the melanin precursors tyrosine (Tyr) and phenylalanine (Phe) found in DMEM were shown not to be solely responsible for the phenotypic changes observed with DMEM. Both BL6 and B16-Fl cell lines formed more experimental pulmonary tumor metastasis in syngeneic C57BL/6 mice when maintained in DMEM vs RPMI-1640 medium. Analysis of metastasis formation in nude mice with normal and depleted natural killer (NK) cell activity revealed that the enhanced lung colonizing capacity of the BL6 cells maintained in DMEM was independent of the function of T-cell or NK-cell-mediated immunity. A close association between metastatic ability of tested lines and the expression of the membrane-associated enzyme -glutamyltranspeptidase (-GTPase, EC 2.3.2.2) was observed. The highly metastatic BL6 cell line had 20-fold higher levels of -GTPase activity than the weakly metastatic B16-Fl cell line. Both cell lines, when grown in DMEM, had elevated -GTPase activity that paralleled augmentation of metastatic ability. The dramatic changes in lung-colonizing capacity of the variant B16 melanoma cells maintained in DMEM in contrast to those grown in RPMI-1640 medium may serve as a useful model in understanding certain steps involved in triggering cell differentiation as well as metastasis development.     

细菌基序对黑素细胞及前列腺素E2的影响

目的：研究细菌基序（CpG-ODN）对黑素细胞形态、黑素生成及前列腺素E2（PGE2）的影响。方法：用CpG-ODN处理体外培养的B16黑素瘤细胞和melan-α黑素细胞,分别用MTT法、多巴氧化法、NaOH裂解法和ELISA法检测B16细胞活力、酪氨酸酶活性、黑素含量以及上清中PGE2的水平,另外,观察CpG-ODN干预后melan-α细胞形态的变化。结果：干预24h后,5μg/mlCpG-ODN促进B16细胞增殖,10μg/ml对细胞增殖的影响与空白对照组无差异,而15μg/mlCpG-ODN则抑制细胞增殖。48h后,不同浓度CpG-ODN组的细胞增殖率趋于稳定,与正常对照组间差异无统计学意义（P〉0.05）。10μg/mlCpG-ODN提高酪氨酸酶活性和黑素生成,作用近似于α-MSH（P〉0.05）。另外,此浓度CpG-ODN作用48h后的细胞上清液中PGE2含量也有所升高,但与对照组差异无统计学意义（P〉0.05）,推测为检测时间滞后所致。10μg/mlCpG-ODN作用于melan-α细胞24h后,细胞树突数目增加,并有二级以致三级树突出现。结论：CpG-ODN作用于鼠黑素细胞的适宜浓度为10μg/ml,此浓度对黑素细胞的作用有时间依赖性。该实验为CpG-ODN作用于正常人黑素细胞的研究奠定了基础。     

氧化苦参碱对皮肤疾病的药理作用与临床应用研究进展

氧化苦参碱能抑制疤痕成纤维细胞增殖和胶原合成,并诱导凋亡和基质金属蛋白酶表达。氧化苦参碱能抑制皮肤癌细胞增殖并诱导凋亡,也能抑制表皮角质形成细胞增殖和黑素形成。氧化苦参碱对各种免疫性和非免疫性皮肤炎症均有抗炎作用,临床上已被试用于多种皮肤病的治疗。     

中药抑制黑素生成作用的筛选研究

目的:通过研究中药对小鼠黑素细胞系Mel-Ab及melan-a黑素生成的影响,筛选出有减少黑素生成作用的中药。方法:药物处理Mel-Ab细胞后分别进行黑素含量和细胞生存率的测定。选取的中药在melan-a与鼠角质形成细胞系SP-1共培养做进一步实验。熊果苷作为阳性对照。结果:川芎、威灵仙、桂枝、麦冬、白果、蒿本、红花、白苏叶的醇提物,五倍子的水提物在细胞培养水平对黑素生成有明显抑制作用,强于相同浓度熊果苷的作用。其中五倍子、威灵仙、白果、川芎在共培养中作用效果更为显著。结论:本研究表明上述九种中药提取物对黑素生成有明显的抑制作用。