OBJECTIVE: To explore the inhibitory effects of Nardostachys Jatamansi DC. volatile oil (NJVO) on psychological factors substance P (SP)/cortisol (CORT)-induced hyperpigmentation. METHODS: The model of psychologically-induced hyperpigmentation of B16F10 cells was created using SP (10 nmol/L) + CORT (10 µmol/L) for 72 h. The levels of melanin content, tyrosinase (TYR) activity using NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation methods were assessed, respectively. The effect of NJVO on SP/CORT-induced normal human skin tissue pigmentation was detected by Masson staining. Protein expressions of tyrosinase-related protein 1 (TRP-1), tyrosinase-relative protein 2 (DCT), and microphthalmia-associated transcription factor were determined using Western blot. The melanosome number, maturation, and melanosomal structure changes were detected through transmission electron microscopy and immunofluorescence experiments. In vivo, zebrafish pigment content was evaluated in SP/CORT-induced zebrafish hyperpigmentation model. RESULTS: NJVO significantly reduced the melanin content (P<0.01) and inhibited tyrosinase activity (P<0.01), the pigmentation of the normal skin tissue in the NJVO group was significantly lower than that in the SP/CORT group (P<0.05). And NJVO considerably downregulated expressions of melanogenesis-related proteins (TYR, TRP-1, DCT) in cells (P<0.01). In addition, the number of melanosomes was decreased and the dentrites formation of B16F10 cells was inhibited after NJVO treatment (P<0.01). In vivo, NJVO significantly reduced the pigment content in the zebrafish body (P<0.01). CONCLUSION NJVO effectively reversed SP/CORT-induced hyperpigmentation by suppressing the activity and expression of TYR and TRPs and inhibiting melanosome maturation in mouse B16F10 melanoma cells.
Animals ; Hyperpigmentation/psychology* ; Zebrafish ; Oils, Volatile/therapeutic use* ; Melanins/metabolism* ; Humans ; Monophenol Monooxygenase/metabolism* ; Mice ; Nardostachys/chemistry* ; Substance P ; Hydrocortisone ; Skin Pigmentation/drug effects* ; Cell Line, Tumor ; Melanosomes/ultrastructure* ; Microphthalmia-Associated Transcription Factor/metabolism* ; Melanoma, Experimental ; Oxidoreductases/metabolism* ; Intramolecular Oxidoreductases/metabolism*

Ferritin is considered as an ideal delivery system due to its precise targeting, reversible self-assembly, high biocompatibility, and easy modification. this study aims to express, purify, and identify three fusion ferritin proteins, and explore their tumor targeting. Three fusion ferritin genes were synthesized and cloned into prokaryotic expression vectors, and the recombinant proteins were purified by affinity chromatography with nickel columns. The fusion ferritin proteins were identified by native polyacrylamide gel electrophoresis (native-PAGE), Western blotting, and circular dichroism. Fluorescein 5-isothiocyanate (FITC) was used to react with fusion ferritin, and confocal laser scanning microscopy was employed to evaluate the tumor targeting of fusion ferritin. The reaction system of sulfo-cyanine7 (Cy7-SE) with fusion ferritin was injected into the tail vein of melanoma mice for in vivo tumor imaging to explore the tumor targeting of fusion ferritin. The results showed that soluble fusion ferritin proteins of about 21 kDa were expressed under the induction by isopropylthio-β-d-galactoside (IPTG), and the recombinant proteins with high purity were obtained. Western blotting showed that the recombinant proteins could be recognized by the corresponding antibodies. The target proteins were identified as multimers with α helixes by native-PAGE and circular dichroism. In vitro and in vivo tumor uptake experiments demonstrated that fusion ferritin was taken up by tumor cells and tumor tissue. This study successfully expressed, purified, and identified fusion ferritin, and verified its tumor uptake in vitro and in vivo, which laid a foundation for the application of ferritin in biomedicine.
Animals ; Mice ; Recombinant Fusion Proteins/isolation & purification* ; Ferritins/metabolism* ; Escherichia coli/metabolism* ; Melanoma, Experimental/metabolism* ; Humans

OBJECTIVES: Malignant melanoma is highly aggressive, prone to early metastasis, and associated with extremely poor prognosis, posing a serious threat to human health. Identifying molecular mechanisms that inhibit metastasis is of great significance for improving treatment and prognosis. Interleukin-37 (IL-37), an anti-inflammatory cytokine, has not only been linked to various inflammatory diseases but also exhibits anti-tumor properties. This study aims to explore the effect of IL-37 on melanoma metastasis in vivo by establishing a murine model of pulmonary metastasis. METHODS: Mouse melanoma B16F1 cells were transfected with either IL-37 overexpression plasmid (IL-37 oe) or empty vector. Three groups were set: An IL-37 oe group (transfection reagent+IL-37 oe plasmid), a Vector group (transfection reagent+vector plasmid), and a Blank group (transfection reagent only). C57 mice were randomly divided into 3 groups (n=3 per group) and injected intravenously with logarithmic-phase B16F1 cells under sterile conditions. Mice were weighed every 3 days. After 1 month, mice were euthanized by cervical dislocation, and organs including lungs, heart, liver, spleen, and kidneys were harvested. Lung metastases were photographed and counted. Organs were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). RESULTS: Western blotting confirmed successful plasmid transfection. There were no significant differences in body weight among the 3 groups over the 28-day period (P>0.05). Lung tumors were observed upon dissection, indicating successful metastasis modeling. HE staining showed no morphological differences in the heart, liver, spleen, and kidneys between groups. The numbers of lung metastases in the Blank, Vector, and IL-37 oe groups were (24.00±2.08), (24.67±0.88), and (5.33±1.45), respectively. The IL-37 oe group had significantly fewer lung metastases than the other 2 groups (P<0.05), while no difference was observed between the Blank and Vector groups. CONCLUSIONS IL-37 significantly inhibits lung metastasis of malignant melanoma cells in mice without affecting body weight or major organs. It may serve as a potential molecular target for gene therapy or immunotherapy of malignant melanoma.
Animals ; Mice ; Interleukin-1/genetics* ; Lung Neoplasms/secondary* ; Melanoma, Experimental/pathology* ; Mice, Inbred C57BL ; Cell Line, Tumor ; Transfection ; Melanoma/pathology* ; Female ; Male

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

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

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

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

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

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