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

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

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.
Animals ; Body Weight ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; Interleukin-15 ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Neoplasm Transplantation ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

Tumors are believed to consist of a heterogeneous population of tumor cells originating from rare cancer stem cells (CSCs). However, emerging evidence suggests that tumor may also originate from non-CSCs. To support this viewpoint, we are here to present definitive evidence indicating that the number of tumorigenic tumor cells is greater than that of CSCs in tumor, and tumor can also derive from non-CSCs. To achieve this, an idealized mathematical model was employed in the present study and theoretical calculation revealed that non-CSCs could initiate the occurrence of tumor if their proliferation potential was adequate. Further, experimental studies demonstrated that 17.7%, 38.6% and 5.2% of tumor cells in murine B16 solid melanoma, H22 hepatoma and Lewis lung carcinoma, respectively, were potentially tumorigenic. Thus, based on the aforementioned findings, we propose that the scarce CSCs, if exist, are not the sole source of a tumor.
Algorithms ; Animals ; Carcinogenesis ; pathology ; Carcinoma, Lewis Lung ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Liver Neoplasms, Experimental ; pathology ; Melanoma, Experimental ; pathology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Biological ; Neoplasms, Experimental ; pathology ; Neoplastic Stem Cells ; pathology ; Time Factors ; Tumor Stem Cell Assay ; methods

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146(EC-KO)) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146(EC-KO) mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells (ECs) of CD146(EC-KO) mice. Mechanistic studies further confirmed that VEGF-induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/NF-κB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146(EC-KO) mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.
Animals ; CD146 Antigen ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Female ; Fibrosarcoma ; metabolism ; pathology ; Male ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-kappa B ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Retinal Vein ; growth & development ; pathology ; Signal Transduction ; drug effects ; Transplantation, Homologous ; Vascular Endothelial Growth Factor A ; pharmacology ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo evaluate the therapeutic effect of MHSP65-TCL on melanoma and its effect on the activity of the immunocytes.

METHODSMHSP65-TCL was prepared by mixing MHSP65 with TCL derived from B16 melanoma cell lysate by repeated freezing and thawing. The MHSP65-TCL vaccine was administered in mice bearing B16 melanoma, and the changes in melanoma growth was observed. To investigate the influence of TCL in MHSP65-TCL on the activity of the immunocytes, we co-cultured TCL and mouse spleen cells in vitro, and analyzed CD69 expression on the cells, cell apoptosis, and levels of IL-10 and IFN-γ in the cell culture supernatant.

RESULTSThe MHSP65-TCL vaccine showed an anti-melanoma effect in the tumor-bearing mice. In the in vitro experiment, TCL in MHSP65-TCL strongly stimulated the activation of mouse spleen cells while causing apoptosis in some of the immunocytes and promoting cellular IL-10 secretion, but not IFN-γ.

CONCLUSIONSMHSP65-TCL derived from B16 melanoma cells has an anti-melanoma effect mediated by the activation of immunocytes. TCL in MHSP65-TCL also has immunosuppressive effect on immunocytes possibly due to the presence of suppressive components in TCL, and identifying and eliminating these components may potentially improve the anti-tumor actovoty of MSHP65-TCL vaccine.

Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Apoptosis ; Bacterial Proteins ; administration & dosage ; immunology ; Cancer Vaccines ; Cell Extracts ; administration & dosage ; immunology ; Cell Line, Tumor ; Chaperonin 60 ; administration & dosage ; immunology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Lectins, C-Type ; metabolism ; Melanoma, Experimental ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Spleen ; cytology ; immunology ; metabolism ; Tumor Burden ; immunology

iRGD-modified sterically stabilized liposomes loaded doxorubicin (iRGD-SSL-DOX) were prepared and their cellular toxicity and anti-tumor efficacy were evaluated, comparing to doxorubixin loaded sterically stabilized liposomes (SSL-DOX) and RGD modified doxorubixin loaded sterically stabilized liposomes (RGD-SSL-DOX). The iRGD peptide, with both tumor targeting and cell penetrating functions, was conjugated to DSPE-PEG-NHS and DSPE-PEG-iRGD was obtained. DSPE-PEG-RGD was gained in the same way. iRGD-SSL-DOX, RGD-SSL-DOX and SSL-DOX were prepared by ammonium sulfate gradient method. The size and zeta potential of the liposomes were characterized by dynamic laser light scattering. The cellular toxicity study was done on B16 melanoma cell line and the anti-tumor efficacy study was carried on B16 cell line bearing C57BL/6 mice. The results showed that the particle sizes of liposomes were all around 90-100 nm. DOX entrapment efficiency was above 95%. The formulations were with good preparation reproducibility. iRGD-SSL-DOX showed no significant difference in B16 cellular toxicity with SSL-DOX and RGD-SSL-DOX, but the anti-tumor efficacy on B16 melanoma bearing C57BL/6 mice was significantly better than that of SSL-DOX, similar as that of RGD-SSL-DOX. Therefore, iRGD modified liposomes loaded DOX would be a promising drug delivery system for tumor therapy.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Doxorubicin ; administration & dosage ; pharmacology ; Drug Carriers ; Drug Delivery Systems ; Liposomes ; Male ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Molecular Weight ; Neoplasm Transplantation ; Oligopeptides ; chemistry ; pharmacology ; Particle Size ; Phosphatidylethanolamines ; chemistry ; Polyethylene Glycols ; chemistry ; Tumor Burden ; drug effects

The anti-melanogenesis effect of glyceollins was examined by melanin synthesis, tyrosinase activity assay in zebrafish embryos and in B16F10 melanoma cells. When developing zebrafish embryos were treated with glyceollins, pigmentation of the embryos, melanin synthesis and tyrosinase activity were all decreased compared with control zebrafish embryos. In situ expression of a pigment cell-specific gene, Sox10, was dramatically decreased by glyceollin treatment in the neural tubes of the trunk region of the embryos. Stem cell factor (SCF)/c-kit signaling pathways as well as expression of microphthalmia-associated transcription factor (MITF) were determined by western blot analysis. Glyceollins inhibited melanin synthesis, as well as the expression and activity of tyrosinase induced by SCF, in a dose-dependent manner in B16F10 melanoma cells. Pretreatment of B16F10 cells with glyceollins dose-dependently inhibited SCF-induced c-kit and Akt phosphorylation. Glyceollins significantly impaired the expression and activity of MITF. An additional inhibitory function of glyceollins was to effectively downregulate intracellular cyclic AMP levels stimulated by SCF in B16F10 cells. Glyceollins have a depigmentation/whitening activity in vitro and in vivo, and that this effect may be due to the inhibition of SCF-induced c-kit and tyrosinase activity through the blockade of downstream signaling pathway.
Animals ; Embryo, Nonmammalian/drug effects ; Melanins/*biosynthesis ; Melanoma, Experimental/metabolism/pathology ; Mice ; Monophenol Monooxygenase/metabolism ; Phosphorylation/drug effects ; Pigmentation/drug effects ; Proto-Oncogene Proteins c-kit/*metabolism ; Pterocarpans/chemistry/*pharmacology ; SOXE Transcription Factors/metabolism ; Sesquiterpenes/chemistry/*pharmacology ; Signal Transduction/*drug effects ; Soybeans/*chemistry ; Stem Cell Factor/*pharmacology ; Zebrafish/embryology/metabolism

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

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