Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1β (IL-1β) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.
Animal Fur ; Animals ; Color ; Female ; Melanins ; Melanocytes ; enzymology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Monophenol Monooxygenase ; metabolism ; Pigmentation ; Skin ; physiopathology ; Stress, Physiological

OBJECTIVE: To investigate whether interleukin-12 (IL-12) over-expression in malignant melanoma B16 cells affects the expression level of programmed death-1 (PD-1) on T cells in mice during immune microenvironment reconstruction. METHODS: B16 cells were transfected with an IL-12 expression lentiviral vector, and IL-12 over-expression in the cells was verified qPCR and ELISA. Plate cloning assay was used to compare the cell proliferation activity between B16 cells and B16/IL-12 cells. The expression of IL-12 protein in B16/IL-12 cells-derived tumor tissue were detected by ELISA. C57BL/6 mice were inoculated with B16 cells or B16/IL-12 cells, and 14 days later the proportion of T cells with high expression of PD-1 in the tumor-draining lymph nodes was detected by flow cytometry. Mouse models of immune reconstitution established by 650 cGy X-ray radiation were inoculated with B16 (B16+RT group) or B16/IL-12 (B16/IL-12+RT group) cells, with the mice without X-ray radiation prior to B16 cell inoculation as controls. Tumor growth in the mice was recorded at different time points, and on day 14, flow cytometry was performed to detect the proportion of T cells with high PD-1 expression in the tumor-draining lymph nodes and in the tumor tissue. RESULTS: B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12 ( < 0.001) without obvious changes in cell viability (>0.05). B16/IL-12 cells expressed higher levels of IL-12 than B16 cells ( < 0.01). In the tumor-bearing mouse models, the proportion of CD4 PD-1 T cells was significantly lower in B16/IL-12 group than in B16 group ( < 0.01). In the mice with X-ray radiation-induced immune reconstitution, PD-1 expressions on CD4 T cells ( < 0.05) and CD8+ T cells ( < 0.01) were significantly higher in B16+ RT group than in the control mice and in B16/IL-12+RT group ( < 0.01 or 0.001); the tumors grew more slowly in B16/IL-12+RT group than in B16 + RT group ( < 0.001). CONCLUSIONS During immune microenvironment reconstruction, overexpression IL-12 in the tumor microenvironment can reduce the percentage of PD-1 T cells and suppress the growth of malignant melanoma in mice.
Animals ; CD8-Positive T-Lymphocytes ; Cell Line, Tumor ; Immune Reconstitution ; Interleukin-12 ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Tumor Microenvironment

BACKGROUND: Many researchers have sought to identify safe, natural herbal extracts that exert an anti-melanogenesis effect. Cinnamomi cortex has been widely used as a herbal medicine in Asia and Europe. OBJECTIVE: To confirm the inhibitory effects of Cinnamomi cortex extract against melanogenesis and inflammation and to elucidate the underlying mechanism of these actions. METHODS: Effects of Cinnamomi cortex extract on melanin synthesis and tyrosinase activity in B16F10 melanoma cells were evaluated using an ELISA reader. Tyrosinase and MITF protein expression was determined using western blotting. Nitric oxide production in RAW 264.7 cells was measured using Griess reaction. PGE₂ was assayed with an ELISA kit. RESULTS: Cinnamomi cortex extracts inhibited melanin synthesis, tyrosinase activity, and MITF and tyrosinase expression through regulation of the ERK and CREB genes in α-MSH-induced B16 melanoma cells. In addition, Cinnamomi cortex extracts inhibited the expression of NO, PGE₂, and pro-inflammatory cytokines in lipopolysaccharide-induced RAW 264.7 cells. CONCLUSION: We suggest that Cinnamomi cortex may be a potentially useful agent for treating inflammatory skin diseases such as hyperpigmentation based on its inhibitory effects against melanin synthesis and inflammation response in vitro.
Anti-Inflammatory Agents ; Asia ; Blotting, Western ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Europe ; Herbal Medicine ; Hyperpigmentation ; In Vitro Techniques ; Inflammation ; Melanins ; Melanoma ; Melanoma, Experimental ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Nitric Oxide ; RAW 264.7 Cells ; Skin Diseases

To study the effects of the anthocyanin single component cyanidin-3-O-glucoside (Cy-3-glu) on the proliferation and migration of mouse melanoma cells and to elucidate the underlying mechanisms, B16-F10 cells were treated with different concentrations of Cy-3-glu. Cell viability was analyzed by a CCK-8 method. Cell migration was determined by the callus scratching technique. Cell cycle was measured by the flow cytometry. The expression levels of genes involved in cell cycle regulation were detected by real-time PCR. Protein expression levels of p-AKT, E-cadherin, N-cadherin and vimentin were analyzed by Western blot. The growth and migration of B16-F10 cells in C57BL/6J mice were monitored by the cryogenically cooled IVIS-imaging system. The results showed that Cy-3-glu significantly inhibited the growth (P < 0.001) and migration (P < 0.01) of B16-F10 cells, and arrested the cell cycle in the S phase. After Cy-3-glu treatment, the expression levels of p-AKT (P < 0.05), N-cadherin and vimentin (P < 0.001) were decreased significantly, and the expression level of E-cadherin was dramatically increased (P < 0.05). The size and weight of tumors and tumor metastasis in mice fed with a diet containing Cy-3-glu were significantly reduced (P < 0.05). In conclusion, Cy-3-glu inhibits proliferation and migration of B16-F10 cells by inhibiting the PI3K/AKT signaling pathway, cell adhesion and migration signals.
Animals ; Anthocyanins ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glucosides ; pharmacology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; metabolism

OBJECTIVETo detect the expression of protein 4.1 family members in mouse melanoma cell lines and evaluate their effect on cell proliferation.

METHODSPCR and Western blot were used to detected to the expression of protein 4.1 family members (4.1R, 4.1B, 4.1G, and 4.1N) at the mRNA and protein levels in B16 and B16-F10 cell lines. The expression plasmid vector pEGFP-N1-EPB41L3 carrying 4.1B gene sequence amplified from genomic RNA of mouse embryo fibroblasts was constructed and transiently transfected into mouse melanoma cells. The change in cell proliferation was assessed using MTT assay.

RESULTSThe mRNA and protein expressions of all the protein 4.1 family members, with the exception of 4.1B, were detected in both B16 and B16-F10 cells. Transfection of cells with the eukaryotic expression vector pEGFP-N1-EPB41L3 markedly inhibited cell proliferation as compared with the non-transfected cells.

CONCLUSIONThe eukaryotic expression vector carrying EPB41L3 sequence is capable of inhibiting the proliferation of mouse melanoma B16 and B16-F10 cells.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; Neuropeptides ; metabolism ; Plasmids ; Transfection

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 investigate the effect of high-intensity focused ultrasound (HIFU) on tumor metastasis in mouse model bearing melanoma xenograft.

METHODSMice bearing murine melanoma B16-F10 cell xenograft were randomized for sham-HIFU or HIFU exposure when the tumors grew to a maximum diameter of 7-10 mm, and the tumor size was measured every 3 days. The cumulative survival rate of the mice and tumor metastasis rate were calculated, and the circulating melanoma cells were detected using qRT-PCR. At 14 days after HIFU treatment, B16-F10 cells were retransplanted via the tail vein and the pulmonary metastatic nodules were counted.

RESULTSThe median survival time of the mice was 19.00 days (95% CI 17.14-20.86 days) in the sham group and 26.00 days (95%CI 24.76-27.25 days) in HIFU group. The cumulative survival rate in the HIFU group was significantly higher than that in sham-HIFU group (P<0.01), and the tumor size was significantly smaller in HIFU group at 20, 23, and 26 days after HIFU treatment (P<0.05). Compared with the sham-HIFU group, HIFU group had significantly lower levels of MAGE-A3, MART1 and PAX3 at 7 days after HIFU (P<0.05) with still lower MAGE-A3 level at 14 days (P<0.05). HIFU group showed a significantly smaller number of pulmonary metastatic nodules following tumor cell retransplantation than in sham-HIFU group (P<0.01) with a metastasis inhibition rate of 42.4%.

CONCLUSIONHIFU treatment can inhibit tumor metastasis in melanoma-bearing mice possibly by reducing tumor cell detachment from the primary tumor site and suppressing colonization of the circulating melanoma cells.

Animals ; High-Intensity Focused Ultrasound Ablation ; Melanoma, Experimental ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; prevention & control ; Survival Rate

OBJECTIVETo test the effect of bifunctional molecule IL2-GMCSF in promoting the activation of dendritic cells (DCs) cultured in tumor conditioned medium.

METHODSWe prepared a tumor conditioned medium using mouse melanoma cell line B16F10 supplemented with IL2-GMCSF, GM-CSF, IL-2, or the combination of the latter two. After culturing mouse DC cell line DC2.4 in the conditioned medium for 24 h, the DCs were examined for phagocytosis, proliferation, maturation phenotype, cytokine secretion, and signal pathway activation.

RESULTSDC2.4 cells displayed characteristics of immature DCs. After cell culture in the conditioned medium, the cells showed enhanced phagocytosis but significantly suppressed cell proliferation activity. Culture in the conditioned medium also promoted DC cell maturation and secretion of macrophage-derived chemokine (MDC), but inhibited IL-12 secretion. Supplementation of the conditioned medium with IL2-GMCSF promoted phagocytosis, proliferation, maturation, and cytokine (including both IL-12 and MDC) secretion of DC2.4 cells. Compared with GM-CSF, IL2-GMCSF induced a higher level of NF-κB signal pathway activation but suppressed STAT3 activation.

CONCLUSIONCompared with GM-CSF, IL2-GMCSF can better promote DC activation in the context of tumor-induced immune suppression, and thus shows potentials in anti-tumor therapy.

Animals ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Chemokine CCL22 ; metabolism ; Culture Media, Conditioned ; chemistry ; Dendritic Cells ; cytology ; drug effects ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Immune Tolerance ; Interleukin-12 ; metabolism ; Interleukin-2 ; pharmacology ; Melanoma, Experimental ; pathology ; Mice ; NF-kappa B ; metabolism ; Phagocytosis ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

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