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

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

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: 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

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

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

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*

BACKGROUNDAdoptive cell transfer (ACT) immunotherapy has been used clinically for years to treat malignancies. Improving the killing efficiency of effector cells, such as tumor-specific cytotoxic T lymphocytes (CTLs), is an important component for enhancing the clinical response of cancer immunotherapy. Hence, we explored a novel method for preparing cancer-specific CTLs using naive T lymphocytes.

METHODSC57BL/6 mice bearing B16 melanoma tumors were pretreated with cyclophosphamide (CTX) by peritoneal injection. The immunosuppressive influence of CTX on tumor regression and the tumor microenvironment was assessed. Naive T cells and T cell pools were isolated via negative selection using immunomagnetic beads. The proliferative potential and cytokine production of different T cell subpopulations were evaluated in vitro. Tumor-specific CTLs derived from naive T cells (naive CD4+ T cells: naive CD8+ T cells = 2:1) and pooled T cells were generated in vitro, respectively. B16 melanoma-bearing C57BL/6 mice were pretreated with CTX, followed by ACT immunotherapy using dendritic cell-induced CTLs. The homing abilities of the effector cells and interleukin-2 (IL-2), interferon-γ, granzyme B, and perforin mRNA levels in tumor tissues were evaluated, and the change in tumor volume was measured.

RESULTSMice receiving CTX peritoneal pretreatment injections did not display tumor regression compared with control mice. However, a significant downregulation of splenic Tregs and tumor growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) serum levels was observed (P < 0.05). Naive T cells showed a stronger proliferative capacity and elevated cytokine production than did pooled T cells (P < 0.05). In addition, effector cells generated from naive T cells displayed more potent antitumor activity in vivo than those derived from pooled T cells (P < 0.05).

CONCLUSIONEffector cells derived from the naive T cells possess a stronger proliferative potential, homing capacity, and enhanced cytokine production, which leads to a superior antitumor response.

Animals ; Cell Line, Tumor ; Cells, Cultured ; Female ; Flow Cytometry ; Immunotherapy, Adoptive ; methods ; Melanoma, Experimental ; therapy ; Mice, Inbred C57BL

OBJECTIVETo study the inhibitory effect of arsenic trioxide (As2O3) on the pulmonary metastasis of melanoma B16 cells.

METHODSMice melanoma cells B16 were injected into the vein of the eye socket of C57BL/6J mice. The lung tissue weight and the B16 melanoma lung metastasis nodules were examined after intraperitoneal injection of As2O3. The microvessel density in the pulmonary metastatic tumor nodules was observed using HE staining and immunohistochemistry analysis for VIII-R Ag. The cell adhesion rate was detected using CellTiter 96 Aqueous One reagent.

RESULTSAs2O3 could significantly inhibit the pulmonary metastasis of B16 melanoma. The lung weight, the pulmonary metastasis nodules, and microvessels per visual field of the experimental group and the control group were 0.139+/-0.013 g and 0.353+/-0.070 g, 20.42+/-1.78 and 61.42+/-3.09, 3.25+/-0.75 and 7.50+/-1.45, respectively (all P<0.01). As2O3 showed significant effect on the cell adhesion rate, showing statistical difference between the two groups (P<0.01).

CONCLUSIONSAs2O3 had significant antitumor metastasis effect. It might be correlated with inhibiting angiogenesis and enhancing the cell adhesion.

Animals ; Arsenicals ; pharmacology ; Cell Line, Tumor ; Lung Neoplasms ; secondary ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oxides ; pharmacology

OBJECTIVETo investigate the effect of mouse melanoma cell line B16F10-derived conditioned medium on the apoptosis of B16F10 cells.

METHODSB16F10 cells were cultured in high-glucose DMEM in the presence of 10% fetal bovine serum, and upon cell confluence, the growth medium was replaced with serum-free high-glucose DMEM. After 8 h, the medium was collected and infiltrated to serve as the conditioned medium. B16F10 cells cultured in normal growth medium or the conditioned medium were exposed to 10 mmol/L sodium L-ascorbate, and the cell apoptosis was analyzed. The ingredients in the conditioned medium with relative molecular mass less or more than 5 000 were extracted to assess their effect on sodium L-ascorbate-induced cell apoptosis.

RESULTSThe conditioned medium for B16F10 cells significantly inhibited cell apoptosis induced by sodium L-ascorbate, and the effective ingredients in the medium showed a relative molecular mass below 5,000.

CONCLUSIONMouse melanoma cell line B16F10-derived conditioned medium can suppress sodium L-ascorbate- induced apoptosis of B16F10 cells, and the ingredients with relative molecular mass less than 5 000 are responsible for this effect.

Animals ; Apoptosis ; drug effects ; Ascorbic Acid ; antagonists & inhibitors ; Cell Line, Tumor ; Culture Media, Conditioned ; pharmacology ; Melanoma, Experimental ; pathology ; Mice