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.     

Inhibitory Effects of Whisky Polyphenols on Melanogenesis in Mouse B16 Melanoma Cells

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.     

Development of Cutaneous Amelanotic Melanoma in the Absence of a Functional Tyrosinase

Lack of characteristic pigmentation and a wide range of clinical presentations account for the diagnostic challenge associated with amelanotic malignant melanoma. Experimental studies of this important human cancer have been hampered by the lack of an appropriate animal model. We previously described a transgenic mouse line (TG‐3) that spontaneously develops pigmented cutaneous melanoma. F1 crosses were generated with TG‐3 and several albino strains, and backcrosses were then made with the albinos. In the present report, we describe the restricted development and characterization of cutaneous amelanotic melanoma in these albino transgenic backcrosses. The incidence and behavior of melanoma in these mice were monitored. A high incidence (80–100%) of spontaneous amelanotic melanoma was observed in albino transgenic mice derived from backcrosses with A, AKR, FVB, and SJL strains. The lowest incidence (30%) was obtained in BALB/c‐derived crosses. No tumors were observed in non‐transgenic mice. Immunohistochemical and western blot analyses using antibodies against three melanocyte‐specific markers of the tyrosinase family of proteins confirmed that the tumors were composed of amelanotic melanocytes. Furthermore, the presence of numerous premelanosomes observed by electron microscopy further supported the melanocytic origin of these tumors. Previous in vitro studies on human melanoma have suggested that cutaneous amelanotic melanoma was evolving from pre‐existing pigmented cutaneous melanoma. However, our results indicate that it can occur directly, as evidenced by the appearance of cutaneous amelanotic melanoma in the tyrosinase‐deficient albino mice. These mice represent a potentially valuable model for studying the mechanistic, diagnostic, and therapeutic features of this highly malignant neoplasm.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 μg/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173–175) for tyrosine hydroxylase.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.     

Anti-oxidant adaptation in the AML cells supersensitive to hydrogen peroxide

The purpose of this study was to investigate the adaptive mechanisms of hydrogen peroxide-supersensitive AML cells against the reactive oxygen species (ROS). Their scavenging capacity against ROS was determined using a fluorometric probe in the doxorubicin-resistant AML-2/DX100 cell characterized by the down-regulation of catalase. AML-2/DX100 cells had more scavenging capacity against endogenous pro-oxidants than did the parental cells AML-2/WT, suggesting that an anti-oxidant adaptation against ROS occurred. cDNA microarrays for 8000 human genes revealed that among 21 anti-oxidant genes, each four gene was up- and down-regulated more than 1.5-fold in AML-2/DX100 compared with AML-2/WT. The mRNA expression of glutathione S-transferase Pi, peroxiredoxin 2, thioredoxin 2, and glutaredoxin was elevated whereas that of peroxiredoxin 3, metallothionein-1F, superoxide dismutase 2, and thioredoxin reductase 1 was depressed. The result indicates that the down-regulation of certain anti-oxidant mechanisms can be compensated for by the up- and down-regulation of the other anti-oxidant mechanisms.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Natural variation of carotenoids in the eggs and gonads of the echinoid genus, Strongylocentrotus: implications for their role in ultraviolet radiation photoprotection

We examined variability in carotenoid concentration in the gonads and eggs of four sea urchin species (Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, Strongylocentrotus pallidus and Strongylocentrotus droebachiensis) to explore the possible role of carotenes as photoprotectants. Carotene concentrations were measured in gonads and gametes of each species, while in eggs the ultraviolet radiation (UV-R) sensitivity and self-shading capacity by carotenes were calculated. Mean concentrations of carotenes in gonads ranged from 0.13±0.017 mg g⁻¹ dw (S. purpuratus), 0.14±0.019 mg g⁻¹ dw (S. franciscanus), 0.29±0.079 mg g⁻¹ dw (S. pallidus) to 0.36±0.06 mg g⁻¹ dw (S. droebachiensis). In eggs, concentrations ranged from 0.026±0.003 to 0.09±0.034 mg g⁻¹ dw. UV-R sensitivity in eggs was quantified by measuring UV-R induced first-cleavage delay. Intra-specifically, cleavage delay varied significantly between individuals, and could be correlated with carotene concentration. Interspecific differences in cleavage delay and carotene concentrations were not correlated. Using the observed concentration of β, β-echinenone (which makes up between 82.4% and 94.9% of the total carotene concentration in the eggs) and a molar extinction coefficient of ε=13.7×10³ mol⁻¹ cm⁻¹ at 334 nm, we calculated self-shading efficiency in the eggs. Self-shading capacity (J₃₃₄) indicated that the eggs could only screen from 4.6% (J₃₃₄=0.046) down to 1.5% (J₃₃₄=0.015) of UV-R at 334 nm. While not sunscreens, we suggest that carotenes can photoprotective in echinoid eggs, probably by mitigating the effects of reactive oxygen species.     

十种苯丙氨酸衍生物对小鼠黑色素瘤细胞系B16的作用研究

该文研究了十种苯丙氨酸（Phe）的对位衍生物Fp、Clp、Bp、Ip、Ap、Np、Sp、Mp、Pp、Cp对小鼠黑色素瘤细胞系B16的细胞毒性、诱导细胞凋亡作用和抑制细胞成集落作用。结果表明,它们的细胞毒性大小依次为Pp、Fp、Mp、Ip、Sp、Ap、Bp。其中Mp和Ip的毒性相近,Sp和Ap的毒性相近。它们诱导细胞凋亡的作用强弱依次为Fp、Mp、Ip。其中Mp和Ip的作用相近。它们抑制细胞成集落的作用大小依次为Pp、Fp、Ip、Mp、Sp、Bp、Np、Ap。其中Pp、Fp、Ip、Mp的作用相近,Sp、Bp、Np的作用相近。初步的细胞毒理分析表明,Fp、Mp、Ip能够诱导B16细胞凋亡和抑制B16细胞形成集落。     

Inhibitory Effects of Bakuchiol,Bavachin, and Isobavachalcone Isolated from Piper longum on Melanin Production in B16 Mouse Melanoma Cells

An EtOH extract of fruits of Piper longum was found to exhibit a potent inhibitory effect against α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 mouse melanoma cells. Bioassay-directed fractionation led to the isolation of prenylated phenolic compounds bakuchiol, bavachin, and isobavachalcone. These compounds and the crude extract of the fruits of P. longum may have suppressive effects against pigmentation by melanin in the skin.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.