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

Impaired tumor necrosis factor receptor-1 (TNFR-1) signaling has been found in some malignant tumors with poor prognosis. However, the exact role of TNFR-1 signaling in fibrosarcoma remains unclear. Here, we explored the question by comparing the growth of TNFR-1 deficient (Tnfr1 (-)) and TNFR-1 competent (Tnfr1 (+)) fibrosarcoma FB61 cells (FB61-m and FB61-R1) in mice. TNFR-1 expression on fibrosarcoma cells delayed their growth in vivo but not in vitro. Moreover, reduced FB61-R1 tumor growth was also obtained in TNFR-1 knockout mice. The mechanism relies mainly on the TNFR-1-mediated downregulation of vascular endothelial growth factor (VEGF) production by tumor cells. Importantly, treatment of FB61-m tumors with melphalan resulted in a short delay of tumor growth, followed by a quick remission. However, when FB61-R1 tumors were treated with melphalan, tumor growth was similarly delayed at first and then completely rejected. Our results reveal evidence for TNFR-1 on tumor cells as a prerequisite in chemotherapy for fibrosarcoma, and provide novel insight into the therapeutic approach against some types of tumors using TNFR-1 angonist.
Animals ; Down-Regulation ; drug effects ; Fibrosarcoma ; drug therapy ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Melphalan ; administration & dosage ; Mice ; Molecular Targeted Therapy ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis

12E7 Antigen ; Antigens, CD ; metabolism ; Cell Adhesion Molecules ; metabolism ; Diagnosis, Differential ; Fibrosarcoma ; metabolism ; pathology ; Heart Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Humans ; Male ; Mesothelioma ; genetics ; metabolism ; pathology ; Middle Aged ; Mucin-1 ; metabolism ; Oncogene Proteins, Fusion ; metabolism ; Pericardiectomy ; Pericardium ; pathology ; Sarcoma ; metabolism ; pathology ; Sarcoma, Synovial ; genetics ; metabolism ; pathology ; surgery ; Translocation, Genetic ; Vimentin ; metabolism

This study is to investigate the tumor invasion and metastasis inhibition effects of the immunoconjugate composed of lidamycin and anti-type IV collagenase monoclonal antibody Fab' fragment. Boyden chamber assay was used to evaluate the influence of Fab'-LDM on HT-1080 cells invasion ability, gelatinase spectrum was used to measure the change of invasion factor MMP-2 and MMP-9's secretion, and RT-PCR was adopted to determine TIMP-1 mRNA expression level. The immunoconjugate inhibition of tumor in situ metastasis was also tested in nude mice. The Fab'-LDM conjugates had dose-dependent inhibition effect on HT-1080 cells' invasion. At the concentrations of 5 and 10 nmol L(-1), the Fab'-LDM inhibited the invasion by (60 +/- 12) % and (79 +/- 11) % respectively. At the concentration of 5 and 10 nmol L(-1), the Fab'-LDM inhibited the secretion of MMP-2 by (42 +/- 8) % and (54 +/- 6) % and that of MMP-9 by (57 +/- 3) % and (87 +/- 1) %, respectively. RT-PCR indicated that conjugates increased the anti-invasion factor TIMP-1 level. The in vivo experiment showed that, compared with the control group, the tumor inhibition rate in Fab', Fab'-LDM, and LDM group equaled to (30 +/- 13) %, (86 +/- 26) %, (74 +/- 22) % respectively. In conclusion, Fab'-LDM could inhibit the invasion and metastasis of tumor and it might be a new tumor biotherapy agent.
Aminoglycosides ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antibodies, Monoclonal ; immunology ; Cell Line, Tumor ; Enediynes ; pharmacology ; Fibrosarcoma ; metabolism ; pathology ; Humans ; Immunoconjugates ; pharmacology ; Immunoglobulin Fab Fragments ; pharmacology ; Matrix Metalloproteinase 2 ; immunology ; secretion ; Matrix Metalloproteinase 9 ; immunology ; secretion ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tumor Burden ; drug effects

Diagnosis, Differential ; Fetus ; Fibroma ; metabolism ; pathology ; Fibrosarcoma ; congenital ; genetics ; metabolism ; secondary ; Hemangiopericytoma ; metabolism ; pathology ; Humans ; Kidney Neoplasms ; pathology ; secondary ; Liver Neoplasms ; pathology ; secondary ; Lung Neoplasms ; pathology ; secondary ; Male ; Nerve Sheath Neoplasms ; metabolism ; pathology ; Rhabdomyosarcoma, Embryonal ; metabolism ; pathology ; Soft Tissue Neoplasms ; congenital ; genetics ; metabolism ; pathology ; Vimentin ; metabolism

OBJECTIVETo investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.

METHODSThe expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.

RESULTSIn U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.

CONCLUSIONSTAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.

Cell Line, Tumor ; Fibrosarcoma ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoprecipitation ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; genetics ; metabolism ; Interferon-alpha ; pharmacology ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Phosphorylation ; Plasmids ; STAT1 Transcription Factor ; genetics ; metabolism ; STAT2 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Transfection

OBJECTIVEThe effect of triptolide on the DNA methylation level of MMP-9 gene and the mRNA expression of tissue inhibitors of met-alloproteinases (TIMPs) were examined in human fibrosarcoma HT-1080 cells to explore the molecular mechanisms involved in the anticancer activity of triptolide.

METHODHT-1080 cells were cultured in MEM containing 10% newborn calf serum and 1% penicillin-streptomycin. Triptolide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 goL-1 and stored at -20 degrees C. Triptolide was freshly diluted with culture medium perior to use and directly added to cell cultures at the indicated concentration, and incubated for 72 hours at 37 degrees C in a humidified atmosphere with 5% CO2, with changes of reagents every 24 hours. Methylation specific PCR (MSP)was applied to assess the methylation status of MMP-9 gene promoter, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of tissue inhibitors of metalloproteinases (TIMPs) in human fibrosarcoma HT-1080 cells after 72 hours of treatment with 6 nmol x L(-1), 12 nmol x L(-1) or 18 nmol x L(-1) triptolide, respectively.

RESULTSThe methylation index of MMP-9 gene promoter was statistically elevated in HT-1080 cells after 72 hours of treatment with 18 nmol L(-1) triptolide, compared with those in controls (0.61 +/- 0.10 vs 0.39 +/- 0.10, P < 0.05), while no significant difference was noted between 6 nmol x L(-1) or 12 nmol x L(-1) triptolide treated HT-1080 cells and controls (0.40 +/- 0.15 vs 0.39 +/- 0.10, 0.46 +/- 0.20 vs 0.39 +/- 0.10, respectively, both P > 0.05). The mRNA expression of TIMP-1, -2, -3 or -4 was not significantly changed in HT-1080 cells after 72 hours of treatment with the indicated concentrations of triptolide, respectively compared with those in controls (all P > 0.05).

CONCLUSIONThe results demonstrated that triptolide upregulates the methylation level of MMP-9 gene in HT-1080 cells in vitro.

Antineoplastic Agents, Alkylating ; pharmacology ; Cell Line, Tumor ; DNA Methylation ; drug effects ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Fibrosarcoma ; drug therapy ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Phenanthrenes ; pharmacology ; Tissue Inhibitor of Metalloproteinases ; genetics ; metabolism

OBJECTIVETo investigate the genes involved in pulmonary metastasis of human fibrosarcoma HT1080 cells in nude mice.

METHODSHT1080 cells were injected into the tail vein of BALB/ C nude mice. RNA samples were extracted from pulmonary metastatic tissues and normal control lung tissues, purified using Atlas Pure Total RNA labeling System (Clonetech Laboratories). cDNA probes labeled with 32P were prepared and hybridized to a cDNA membrane constructed with spots of 1176 human cancer related genes and radioactivities on the membrane were measeured by BAS 5000. The mRNA expression of gene FN1 was determined by real time RT-PCR using TaqMan methods. Furthermore, cells with FN1 expression were localized and obtained in situ in pulmonary metastatic foci by laser captured microdissection, and the FN1 expression was quantitated by real time RT-PCR.

RESULTSOf the total 1176 genes, 27 genes (2. 3%) revealed to be apparently up-regulated and 4 genes (0. 3% ) down-regulated. Real time RT-PCR analysis verified significant up-regulation of gene FN1. Laser captured microdissection/ real time RT-PCR analysis demonstrated up-regulated gene FN1 not in stroma cells but in tumor cell nests.

CONCLUSIONGene FN1 expression in fibrosarcoma HT1080 cells may be involved in pulmonary metastasis.

Animals ; Cell Line, Tumor ; Fibronectins ; genetics ; Fibrosarcoma ; genetics ; pathology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lung ; chemistry ; Lung Neoplasms ; genetics ; secondary ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; genetics ; metabolism ; RNA, Neoplasm ; genetics ; isolation & purification ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous

OBJECTIVETo investigate the anti-tumor effect of intraportal administration of Adv-p53 in the treatment of the liver metastasis in mice.

METHODS2 x 10(5) of MCA-205 cells were injected into the mouse portal vein to establish a murine liver metastasis model. The spleen was transpositioned subcutaneously to enable the administration of Adv-p53 continually into the portal system. Different doses of Adv-p53 were injected intraportally, while HBSS and Adv-CMV were injected intraportaly in the control group. Tumors in the liver were examined on day 21 after Adv-p53 administration.

RESULTSThe liver weight in the Adv-p53 treated mice on day 0 group (1.20 +/- 0.34 g) was significantly less than that in the Adv-CMV group (2.59 +/- 0.48 g, P < 0.05). The number of metastatic nodules in the Adv-p53 treated mice on day 0 group (9.0 +/- 9.9) was significantly less than that in the Adv-CMV group (57.1 +/- 11.3, P < 0.05), indicating that intraportal administration of Adv-p53 inhibited the formation of liver metastasis. This anti-tumor effect was in a dose-dependent manner. After the liver metastasis was formed, Adv-p53 was administered intraportally. The liver weight in the Adv-p53 treated mice on day 5 group (1.22 +/- 0.09 g) was significantly less than that in the Adv-CMV group (3.98 +/- 1.01 g , P < 0.05). The number of metastatic nodules in the Adv-p53 treaed mice on day 5 group (5.5 +/- 3.5) was significantly less than that in the Adv-CMV group (113.2 +/- 5.8, P < 0.05). Repeatedly intraportal administration of Adv-p53 could enhance this anti-tumor effect.

CONCLUSIONLocal administration of Adv-p53 into the portal system would be a useful strategy for the liver metastasis treatment.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Female ; Fibrosarcoma ; pathology ; Genetic Therapy ; Liver Neoplasms, Experimental ; secondary ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Recombinant Proteins ; genetics ; therapeutic use ; Tumor Suppressor Protein p53 ; administration & dosage ; genetics ; therapeutic use

AIMTo examine the effect of inducible nitric oxide synthase (iNOS) on tumour cells chemosensitivity to mitomycin C (MMC) analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione (629) in vitro, and elucidate the possible role of iNOS in the metabolism of 629.

METHODSHuman sarcoma cells (HT1080) and its iNOS gene transfected clones (iNOS9, iNOS12) were exposed to 629 at concentrations of 1 nmol x L(-1) - 100 micromol x L(-1). 3-[4, 5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid (DNA) damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly.

RESULTS629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in G2/M phase.

CONCLUSIONiNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure P(O2), which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.

Antibiotics, Antineoplastic ; pharmacology ; Aziridines ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; DNA Damage ; Fibrosarcoma ; metabolism ; pathology ; Flow Cytometry ; Humans ; Indoles ; pharmacology ; Mitomycin ; chemistry ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Transfection