No abstract available.
Animals ; Capsaicin* ; Colon* ; Cytokines* ; Lung* ; Melanoma, Experimental* ; Mice* ; RNA, Messenger*

BACKGOUND: Imiquimod enhances both acquired and innate immune responses by inducing the synthesis of IFN-alpha and other cytokines (TNF-alpha, IL-1, 6, 8, 12, GM-CSF). Imiguimod was investigated in relation to melanoma, a highly resistant cancer in its immunogenic nature, but also an attractive target for immunotherapy. OBJECTIVE: The purpose of this study was to investigate the anti-tumor effect of 5% imiquimod cream in vivo. METHOD: C57BL/6 mice were inoculated intradermally into the left flank with 2X103 murine B16 melanoma cells. Five groups (including vehicle only, imiquimod cream, imiquimod cream with dimethyl sulfoxide (DMSO), vehicle with ultrasound, and imiquimod cream with ultrasound) were treated once daily at the inoculation site of the melanoma cells. The tumor growth was evaluated daily by using a caliper. RESULTS: The difference in tumor growth was seen around day 12 of treatment, and only the group of 5% imiquimod cream with ultrasound showed a decrease in tumor growth when compared to the control group. CONCLUSION: These results indicate that topical application of 5% imiquimod cream on mouse skin melanomas induces inhibition of tumor growth, and imiquimod cream could be used for the combination treatment of malignant melanoma.
Animals ; Cytokines ; Dimethyl Sulfoxide ; Immunity, Innate ; Immunotherapy ; Interleukin-1 ; Melanoma* ; Melanoma, Experimental ; Mice* ; Skin ; Ultrasonography

BACKGROUND: Numerous reports suggest the role of oxygen in melanogenesis. However, little has been reported on the effect of a hypoxic environment on cellular melanogenesis. OBJECTIVE: The effect of low oxygen tension on cellular melanogenesis was investigated in B16 murine melanoma cells. METHODS: Using cells cultured under an ambient (21% O2) or hypoxic (5% O2) condition, melanin content and tyrosinase activity were measured spectrophotometrically. The expression of tyrosinase, tyrosinase-related protein (TRP)- 1, and TRP-2 were analyzed by RT-PCR and Western blot. RESULTS: Culture of cells under hypoxic conditions caused significant inhibition of isobutylmethylxanthine (IBMX)- induced increase of melanin content. No cytotoxicity was observed during the hypoxic culture periods. Decreased melanin content occurred through the decrease of tyrosinase protein and activity (p<0.01). The mRNA levels of tyrosinase and TRP-2 were also decreased by hypoxia, while that of TRP-1 was unchanged. Similar inhibitions of melanin content and tyrosinase activity were observed in the cells stimulated with dibutyryl-cAMP. CONCLUSION: IBMX-induced melanogenesis in B16 cells was significantly inhibited under hypoxic culture conditions, suggesting the important role of oxygen tension in cellular melanogenesis.
Anoxia* ; Blotting, Western ; Melanins ; Melanoma ; Melanoma, Experimental* ; Monophenol Monooxygenase ; Oxygen ; RNA, Messenger

BACKGROUND: The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), was reported to induce apoptosis of some cancer cells and neurons, although it is generally known to exert mitogenic and antiapoptotic effects. Recently, it was described that S1P induced time- and dose-dependent apoptosis in B16 melanoma cells, that was not associated with cell membrane receptors for S1P but ERK and caspase-3 activation. OBJECTIVE: In this study, we aimed to investigate the exact mechanism of apoptosis by S1P using cultured B16 melanoma cells with caspase activity assay and Western blot assays for p-ERK, Fas, Bcl-2 family and cytochrome C proteins. METHODS: We cultured B16 melanoma cells and treated S1P with various concentrations and time. Caspases (3, 8, and 9) activity assay and Western blot assays for phosphor-ERK, Fas, Bcl-2 family and cytochrome C proteins were performed. RESULTS: We observed S1P induced caspase-3, -8, and -9 activations in our results. S1P also induced prolonged activation of ERK in 72 hours. S1P concomitantly increased Bcl-2 protein expression in the early 12 hours of the treatment. Neither fas nor cytochrome C were affected by S1P. CONCLUSION: In conclusion, we propose that S1P may induce apoptotic signal on B16 melanoma cells by prolonged ERK activation and caspase-8, -9, -3 activations. S1P also appears to exert antiapoptotic signal by increasing Bcl-2 protein.
Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 8 ; Caspases ; Cell Membrane ; Cytochromes c ; Humans ; Lysophospholipids ; Melanoma ; Melanoma, Experimental ; Neurons ; Proteins ; Sphingosine

The half-dried leaves of Stewartia. pseudocamellia were extracted with hot water (SPE) and partitioned with n-hexane (SPEH), dichloromethane (SPED), and ethyl acetate (SPEE) successively. SPE and SPEE showed significant inhibitory effects against melanogenesis and tyrosinase activities. By bioassay-guided isolation, ten phenolic compounds were isolated by column chromatography from SPEE. The whitening effect of the isolated compounds from SPEE were tested for the inhibitory activities against melanogenesis using B16 melanoma cells, in vitro inhibition of tyrosinase, and L-3,4-dihydorxy-indole-2-carboxylic acid (L-DOPA) auto-oxidation assay. A cytotoxic activity assay was done to examine the cellular toxicity in Raw 264.7 macrophage cells. Of the compounds isolated, gallic acid and quercetin revealed significant inhibitory activities against melanogenesis compared to arbutin. In particular, quercetin exhibited similar inhibitory activities against tyrosinase and L-DOPA oxidation without cytotoxicity. These results suggested that SPE could be used as a potential source of natural skin-whitening material in cosmetics as well as in food products.
Arbutin ; Chromatography ; Gallic Acid ; Levodopa ; Macrophages ; Melanoma, Experimental ; Methylene Chloride ; Monophenol Monooxygenase ; Phenol* ; Quercetin ; Water

OBJECTIVETo study melanoma cell fusion and find a highly efficient fusion method for tumor cells.

METHODSMelanoma cells were labeled with green fluorescent protein and red fluorescent protein, respectively, and fused by a modified phytohaemagglutinin (PHA)-ECM830 fusion method. Melanoma fusion cells were selected by the fluorescence activated cell sorting. DNA content was determined by propidium iodide staining.

RESULTSMelanoma cells were labeled with green fluorescent protein and red fluorescent protein markers and successfully fused through PHA-ECM830 fusion method. The fusion efficiency (7.18%) was much higher compared with ECM830 electricfusion method (0.50%). Melanoma fusion cells were successfully obtained by the fluorescence activated cell sorting.DNA content was doubled in melanoma fusion cells compared to B16-F10 melanoma cells. The proliferation rate of melanoma fusion cells was significantly decreased in vitro and in vivo.

CONCLUSIONSWe successfully obtained the melanoma fusion cells by the improved PHA-ECM830 fusion method. The proliferation rate of melanoma fusion cells dramatically decreases.

Animals ; Cell Fusion ; methods ; Cell Line, Tumor ; Cell Proliferation ; Melanoma, Experimental ; pathology ; Mice ; Phytohemagglutinins ; pharmacology

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.
Adenine ; Adenosine* ; Blotting, Western ; Cell Proliferation ; Embryonic Structures ; Melanins ; Melanocytes ; Melanoma, Experimental ; Monophenol Monooxygenase ; Pigmentation ; Receptors, Purinergic P1 ; Zebrafish*

BACKGROUND: Plant extracts have been widely used as important therapeutic drugs for many centuries all over the world. There have been many reports that natural products have various kinds of biological activities such as anti-allergy, anti-inflammatory and antimicrobial activities. Recently, the screening for the efficacy and safety of natural products has been extensively performed. OBJECTIVE: This study was carried out to find a beneficial plant extract possessing excellent antioxidative and anti-melanogenic activities. We have found that the leaf of Robinia pseudo-acacia L. has active substances which are involved in those activities. METHODS: To confirm the antioxidative activity of the extract obtained from the leaves of Robinia pseudo-acacia L., scavenging ability of the extract on DPPH free radicals and its inhibitory effects on lipid autoxidation and peroxidation were investigated. In addition, inhibitory effects of the extract on mushroom tyrosinase as well as melanin biosynthesis in cultured B16 melanoma cells were evaluated. RESULTS: The acacia extract showed not only powerful antioxidative activity but also antimelanogenic acitivity as strong as that of arbutin which is a well known inhibitor of melanogenesis. CONCLUSION: These resulis suggest that the extract from the leaves of Robinia pseudo-acacia L. could be used as a 4ghtening and antioxidative agent for the skin.
Acacia ; Agaricales ; Arbutin ; Biological Products ; Free Radicals ; Mass Screening ; Melanins ; Melanoma, Experimental ; Monophenol Monooxygenase ; Plant Extracts ; Plants ; Robinia* ; Skin

The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics ; Animal ; Cell Differentiation ; Cyclic AMP/*metabolism ; Forskolin/*pharmacology ; Isoenzymes/genetics ; Melanoma, Experimental/*enzymology/*pathology ; Mice ; Signal Transduction

Animals ; Apoptosis ; Cell Line, Tumor ; Down-Regulation ; Melanoma, Experimental ; pathology ; virology ; Mice ; Receptor, IGF Type 1 ; physiology ; Sendai virus ; pathogenicity