Human Melanoma Cell Lines Show Little Relationship Between Expression of Pigmentation Genes and Pigmentary Behaviour In Vitro

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with α-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by α-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; α-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or α-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinaserelated genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.     

Synthesis and bioactivity of novel isoxazole chalcone derivatives on tyrosinase and melanin synthesis in murine B16 cells for the treatment of vitiligo

A new series of chalcone derivatives 1–18, bearing isoxazole moieties were designed and synthesized, and biologically evaluated for their activity on mushroom tyrosinase and melanin synthesis in murine B16 cells. The result indicated that most of prepared compounds 1–18 showed potent activating effect on tyrosinase, especially for 1–2, 4, 6–7, 9 and 15. Among them, compounds 2, 4 and 9 demonstrated the best activity with EC₅₀ = 1.3, 2.5 and 3.0 μmol·L⁻¹ respectively, much better than the positive control 8-methoxypsoralan (8-MOP, EC₅₀ = 14.8 μmol·L⁻¹); In B16 cells, all the tested compounds exhibited a stronger activity on melanogenesis than 8-MOP (with the value of 115%). It was interesting that derivatives substituted with halogen (1, 2, 4, 5, 7, 9) were generally more potent. Compounds 2 (463%) and 18 (438%) with 3 and 4-fold potency compared with 8-MOP respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.     

The Identification and Partial Cloning by PCR of the Gene for Tyrosinase-Related Protein-1 in the Mexican Axolotl

The tyrosinase gene family is currently composed of three members, tyrosinase and two tyrosinase-related proteins, TRP-1 and TRP-2. These three gene products have all been found to act in the synthesis of melanin pigments with the enzyme tyrosinase catalyzing the initial rate-limiting steps. Thus far these genes have primarily been analyzed in higher vertebrates. We have used degenerate PCR primers to isolate a large fragment of an axolotl tyrosinase-related protein. Sequence analysis of the entire 1,057-bp fragment isolated indicates a high degree of similarity to the mouse TRP-1, the product of the brown locus. Phylogenetic analysis supports the conclusion that the fragment isolated corresponds to the axolotl TRP-1 homolog. This is the first TRP-1 gene to be identified in an amphibian species.     

Effect of Penicillin-Streptomycin and Other Antibiotics on Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

Penicillin and streptomycin, the most widely used antibiotics in mammalian cell cultures, caused a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity of B16/F10 melanoma cells at the routine concentration (100 units/ml penicillin and 100 μg/ml streptomycin) used for preventing bacterial growth in cultured animal cells. At these concentrations, tyrosinase activities and melanin content augmented with time during the first 24–48 hr. The opposite effect acted on cell viability. After withdrawal of the antibiotics from the culture medium, the recovery of melanogenic parameters to normal values was fully reached after few hours (around 10), and it was already noticeable as soon as 4 hr after removal. Other antibiotics used in cell culture, like kanamycin, gentamicin, and the antimicotic nystatin, exerted similar low effects at the recommended concentrations, always lower than two-fold and thus lower than those reported for amphotericin B. Taking into account these relatively low effects, and the high risk of contamination of mammalian cells culture without antibiotics, penicillin and streptomycin may still be routinely used in experiments leading to explore the melanogenic activity of malignant melanocytes in culture, unless very precise studies and strict conditions were needed.     

The Presence of Tyrosinase and Related Proteins in Human Epidermis and Their Relationship to Melanin Type

The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP-1 and TRP-2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP-1 and TRP-2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase-positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.     

Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic,In Vitro,In Vivo,and In Silico Studies

Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti‐melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC₅₀ 7.895 ± 0.24 μm ) against tyrosinase as compared to the standard drug kojic acid (IC₅₀ 16.84 ± 0.64 μm ) and kinetic analyses showed that ACZ is a non‐competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 μm of ACZ with or without 50 μm of l ‐DOPA. Western blot results showed that ACZ significantly (P < 0.05) decreased the expression level of tyrosinase at 40 μm . Zebrafish embryos were treated with 10, 20 or 40 μm of ACZ and of positive control kojic acid. At 72 h of treatment with ACZ and kojic acid, ACZ significantly (P < 0.001) decreased the embryos pigmentation to 40.8% of untreated embryos at the dose of 40 μm of ACZ while kojic acid decreased only 25.0% of pigmentation at the same dose of kojic acid. In silico docking were performed against tyrosinase using PyRx tool. Docking studies suggested that His244, Asn260 and His85 are the major interacting residues in the binding site of the protein. In conclusion, our results suggest that ACZ is a good candidate for the inhibition of melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening.     

Synthesis and biological activity of hydroxy substituted phenyl-benzo[d]thiazole analogues for antityrosinase activity in B16 cells

In this study, we synthesized hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives using substituted benzaldehydes and 2-aminothiophenol in MeOH. The structures of these compounds were established by ¹H and ¹³C NMR and mass spectral analyzes. All synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity. Out the 12 generated compounds, 2a and 2d exhibited much higher tyrosinase inhibition activity (45.36-73.07% and 49.94-94.17% at 0.01-20 μM, respectively) than kojic acid (9.29-50.80% at 1.25-20 μM), a positive control.The cytotoxicity of 2a and 2d was evaluated using B16 cells and the compounds were found to be nontoxic. Compounds 2a and 2d were also demonstrated to be potent mushroom tyrosinase inhibitors, displaying IC₅₀ values of 1.14 ± 0.48 and 0.01 ± 0.0002 μM, respectively, compared with kojic acid, which has an IC₅₀ value of 18.45 ± 0.17 μM. We also predicted the tertiary structure of tyrosinase, simulated the docking with compounds 2a and 2d and confirmed that the compounds strongly interact with mushroom tyrosinase residues. Kinetic plots showed that 2a and 2d are competitive tyrosinase inhibitors. Substitutions with a hydroxy group at R³ or both R³ and R¹ of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase. We further found that compounds 2a and 2d inhibit melanin production and tyrosinase activity in B16 cells. These results may assist in the development of new potent tyrosinase inhibitors against hyperpigmentation.     

Stereological Studies of the Effects of α-MSH and cAMP on Melanosomes in Melanoma Cells

The effects of α-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing α-MSH or cAMP harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V_Vpm,cell’V_pm) and mature melanosomes (V_Vmm,cell’V_mm) and the number of mature melanosomes (N_mm). Both stimulators also caused declines in the volume of individual mature melanosomes (_Vimm) the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAME The effect cAMP on some parameters occurred 24 hours prior to α-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enchanced melanogenesis by increasing the V_Vpm,cell’V_Vmm,cell’V_pm, V_mm and N_mm. The melanogenic level did not depend on the _Vimm and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAME It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to α-MSH.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.     

Brief history of the Society for Melanoma Research: A community of clinicians,researchers, and friends

To commemorate the 20th Anniversary of the Society of Melanoma Research and the first International Melanoma Research Congress held in June of 2003, we have described in brief, how the Society for Melanoma Research (SMR) began, the purpose, goals, and governance of the SMR, and how the society has evolved to support new melanoma researchers. In celebration of the immense progress in treating melanoma patients over the last 20 years and the impact of the SMR on these advances, we have highlighted memories and insight from early SMR members and founders.     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.     

Design,Synthesis, Molecular Docking,and Biological Evaluation of New Emodin Anthraquinone Derivatives as Potential Antitumor Substances

The emodin anthraquinone derivatives are generally used in traditional Chinese medicine due to their various pharmacological activities. In the present study, a series of emodin anthraquinone derivatives have been designed and synthesized, among which 1,3‐dihydroxy‐6,8‐dimethoxyanthracene‐9,10‐dione is a natural compound that has been synthesized for the very first time, and 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione is a compound that has never been reported earlier. Interestingly, while total seven of these compounds showed neuraminidase inhibitory activity in influenza virus with inhibition rate more than 50 %, specific four compounds exhibited significant inhibition of tumor cell proliferation. The further results demonstrate that 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione showed the best anticancer activity among all the synthesized compounds by inducing highest apoptosis rate to HCT116 cancer cells and arresting their G0/G1 cell cycle phase, through elevation of intracellular level of reactive oxygen species (ROS). Moreover, the binding of 1,3‐dimethoxy‐5,8‐dimethylanthracene‐9,10‐dione with BSA protein has thoroughly been investigated. Altogether, this study suggests the neuraminidase inhibitory activity and antitumor potential of the new emodin anthraquinone derivatives.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.