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 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

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

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

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

Though the malignancy of a tumor is generally postulated to be affected by the degree of differentiation of tumor cells, the relationship between differentiation and malignancy of tumors has not been clearly elucidated. Using in vitro established mouse(B16) and human(IGR3) melanoma cell lines, we performed various in vitro and in vivo experiments to clarify the relationship between melanogenicity and malignancy. High and low melanin-producing clones were selected from both cell lines by the limiting dilution technique and their melanogenicities were confirmed by the determination of melanin quantity and tyrosinase activity along with electron microscopic examination. Selected clones from both cell lines revealed that low melanin-producing clones showed a slightly broader chromosomal distribution, a shorter doubling time with a higher DNA synthesis and a greater colony forming capacity in semi-solid agar medium than those of high melanin-producing clones. The low melanin-producing clone derived from B16 also had a lower tumor-take dose and a more rapid tumor-growth rate than the high melanin-producing counterpart following transplantation into syngeneic mice. These results support the concept that the melanogenicity and other biological characteristics associated with malignancy are inversely related in malignant melanoma cells.
Animal ; Cell Line ; Human ; Melanins/*metabolism ; Melanoma/*pathology ; Melanoma, Experimental/*pathology ; Mice ; Mice, Inbred Strains ; Microscopy, Electron ; Neoplasm Transplantation ; Skin Neoplasms/*pathology ; Tumor Cells, Cultured/*pathology ; Tumor Stem Cell Assay

OBJECTIVETo provide ideal animal models for exploring pathogenesis and experimental therapy of primary malignant melanoma of the small intestine.

METHODSThe histologically intact primary and liver metastatic fragments derived from surgical specimens of one patient with metastatic malignant melanoma of the small intestine were orthotopically implanted in the small intestinal mucous layer of nude mice. The take rate, invasion and liver metastasis were observed. Morphology (light microscopy, electron microscopy), immunophenotype analysis, flow cytometry and karyotype analysis were applied for the original human tumors and the transplanted tumors.

RESULTSThe primary and liver metastatic fragments of malignant melanoma of the small intestine were successfully implanted in nude mice. After continuous passages in nude mice,an orthotopic model of human primary malignant melanoma of the small intestine(from the primary focus)in nude mice (termed HSIM-0501) and a liver metastatic model of human primary malignant melanoma of the small intestine (from the liver metastatic focus) in nude mice (termed HSIM-0502) were established. Histological examination of transplanted tumors revealed high-grade melanoma. S-100 protein and HMB45 were positive. Massive melanin granules and melanin complex were seen in cytoplasm of tumor cells.Chromosomal modal number was between 55 and 59. DNA index (DI) was 1.49-1.61, representing heteroploid. HSIM-0501 and HSIM-0502 were maintained for 25 and 27 passages in nude mice respectively. Three hundred and seventeen nude mice were used for transplantation. Both the take rate after transplantation and resuscitation rate of liquid nitrogen cryopreservation were 100%. HSIM-0501 exhibited 46.2% liver metastasis and 36.7% lymph node metastases. In HSIM-0502, both liver and lymph node metastases were 100%.The transplanted tumors autonomically and invasively grew in the small intestines of nude mice and hematogenous metastasis, lymph node metastasis and celiac planting metastasis occurred.

CONCLUSIONTwo nude mice liver metastatic models of human primary malignant melanoma of the small intestine are successfully established, which provide ideal animal models for the research of pathogenesis,metastasis biology and anti-metastatic experimental therapy of primary malignant melanoma of the small intestine.

Animals ; Female ; Humans ; Intestinal Neoplasms ; pathology ; Intestine, Small ; Liver Neoplasms, Experimental ; secondary ; Male ; Melanoma ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Metastasis ; Neoplasm Transplantation

OBJECTIVETo investigate the effect of antisense VEGF165 infection on the growth of A375 cells in nude mice.

METHODSA375 cells were injected s.c into the axilla of the nude mouse. After the tumor formed, we cut it into 16 pieces equally, then transplanted into another 15 nude mice. There were three groups: Group PBS, Group Ad-GFP, and Group Ad-aVEGF. Four weeks after interfere, the mice were sacrificed and their tumors were excised for naked eye and histological observation. The VEGF expression was checked with ISH and immunohistochemistry staining. The micro-vessel density (MVD) in tumor mass was counted by VIII factor immunohistochemistry staining.

RESULTSThe visible and palpable nodules had developed at all the injected sites. Tumor growth speed was more slowly in Group Ad-aVEGF than that in other groups. GFP gene could express effectively in tumor mass. Ad-aVEGF infection could suppress the growth of tumors, and there were no obvious side effects. Ad-aVEGF resulted more tissue necrosis, but it had no obvious effect on cell apoptosis. VEGF expression was inhibited significantly in Group Ad-aVEGF, and MVD was decreased accordingly.

CONCLUSIONSAd-aVEGF interfere may be a new method against human malignant melanoma, whose main mechanism is to induce ischemia, but not apoptosis.

Adenoviridae ; Animals ; Genetic Therapy ; Genetic Vectors ; Humans ; Male ; Melanoma ; genetics ; pathology ; Melanoma, Experimental ; genetics ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; genetics ; Skin Neoplasms ; genetics ; Vascular Endothelial Growth Factor A ; genetics

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

Six hundred and eight fungi strains were isolated from seventy-eight samples of mangrove plants and soil that collected from Qinglan harbor. Cyctotoxic activity was detected by observing the growth inhibition or killing of the tumor cells under microscope. The result showed that 81 strains (about 13.32% of the total strains isolated) displayed cytotoxic activity against B16 tumor cell. The most fungi strains were isolated from mangrove plant Sonneratia alba, and most of cytotoxic active fungi strains were isolated from mangrove plant Heritiera littoralis.
Animals ; China ; Cytotoxicity Tests, Immunologic ; Fungi ; isolation & purification ; physiology ; Melanoma, Experimental ; pathology ; Mice ; Plant Roots ; microbiology ; Rhizophoraceae ; microbiology ; Soil Microbiology ; Tumor Cells, Cultured