BR55 is an ultrasound contrast agent targeting vascular endothelial growth factor receptor 2,which can be used to detect tumor neovascularization and improve the diagnostic accuracy.Overseas researchers have used BR55 for human ultrasound molecular imaging,which showed good safety and tolerance.We reviewed the research progress on BR55 applied in the evaluation of tumor neovascularization from the composition,characteristics,animal experiments,and clinical studies of BR55.
Animals ; Contrast Media ; Humans ; Microbubbles ; Molecular Imaging/methods* ; Neovascularization, Pathologic/diagnostic imaging* ; Ultrasonography/methods* ; Vascular Endothelial Growth Factor Receptor-2/analysis*

OBJECTIVETo study the molecular risk factors of lymph node metastasis in stage T1 and T2 colorectal cancers by tissue microarray and immunohistochemistry techniques.

METHODSTwo hundred and three patients with stage T1 and T2 colorectal carcinoma who underwent radical surgery from 1999 to 2010 in our department were included in this study. Their clinicopathological data were retrospectively analyzed. Expression of the following 14 molecular markers were selected and assayed by tissue microarray and immunohistochemistry: VEGFR-3, HER2, CD44v6, CXCR4, TIMP-1, EGFR, IGF-1R, IGF-2, IGFBP-1, ECAD, MMP-9, RKIP, CD133, MSI. Chi-squared test and logistic regression were used to evaluate the variables as potential risk factors for lymph node metastasis.

RESULTSThe positive expression rates of biomarkers were as following: VEGFR-3 (44.3%), EGFR (30.5%), HER-2 (28.1%), IGF-1R (63.5%), IGF-2 (44.8%), IGFBP-1 (70.9%), ECAD (45.8%), CD44v6 (51.2%), MMP-9 (44.3%), TIMP-1 (41.4%), RKIP (45.3%), CXCR4 (40.9%), and CD133 (49.8%). The positive rate of MSI expression was 22.2%. Both univariate and multivariate analyses showed that VEGFR-3, HER-2, and TIMP-1 were significant predictors of lymph node metastasis. Univariate analysis showed that CD44v6 and CXCR4 were significant significant predictors of lymph node metastasis.

CONCLUSIONSVEGFR-3, HER2 and TIMP-1 are independent factors for lymph node metastasis in stage T1 and T2 colorectal cancers.

Aged ; Biomarkers, Tumor ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Female ; Humans ; Hyaluronan Receptors ; metabolism ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Microsatellite Instability ; Middle Aged ; Neoplasm Staging ; Oligonucleotide Array Sequence Analysis ; Receptor, ErbB-2 ; metabolism ; Receptors, CXCR4 ; metabolism ; Rectal Neoplasms ; metabolism ; pathology ; Retrospective Studies ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; metabolism

Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody (AK404R-Fc), this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody (Mab-04). Firstly, the light chain (L-chain) and heavy chain (H-chain) were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids (pcDNA3.1-L-chain and pcDNA3.1-H-chain) were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody (1 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x L(-1)). These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.
Animals ; Antibodies, Monoclonal, Humanized ; analysis ; CHO Cells ; Cricetulus ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; immunology ; Plasmids ; Recombinant Fusion Proteins ; analysis ; genetics ; Single-Chain Antibodies ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology

OBJECTIVE: Supplementation with vitamin E is able to protect bone against free radical-induced elevation of bone-resorbing cytokines. We examined gene expression by microarray analysis during the differentiation of human mesenchymal stem cells treated with vitamin E into osteoblasts in vitro. METHODS: Human bone marrow stem cells were cultured in osteogenic differentiation medium and vitamin E was added. A colorimetric immunoassay for the quantification of cell proliferation was used to measure osteoblast differentiation. Gene expression was analyzed using a microarray technique. We also used a real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It was found that vitamin E enhanced cell proliferation when compared to cells cultured in media without vitamin E. We focused on 68 genes which are related to osteogenesis and osteoclastogenesis. Alkaline phosphatase, transforming growth factor-beta 1, fibroblast growth factor receptor 1, matrix metalloproteinase 2, muscle segment homeobox 2, bone morphogenetic protein 1, biglycan, vascular endothelial growth factor B, dentin sialophosphoprotein, cartilage oligomeric matrix protein, runt-related transcription factor 2, fibroblast growth factor receptor 3, and SMAD2 were upregulated > 2-fold compared to the control. Conversely, osteopetrosis-associated transmembrane protein 1, microphthalmia-associated transcription factor, and epidermal growth factor receptor were downregulated > 2-fold compared to the control. Vitamin E produced a 1.5-fold increase in the expression of runt-related transcription factor 2 and transforming growth factor-beta 1 as determined by real time RT-PCR. CONCLUSION: Vitamin E had a positive effect on the gene expressions regarding osteogenic differentiation of mesenchymal stem cells.
Alkaline Phosphatase ; Biglycan ; Bone Marrow ; Bone Morphogenetic Protein 1 ; Cartilage ; Cell Proliferation ; Cytokines ; Dentin ; Durapatite ; Extracellular Matrix Proteins ; Gene Expression ; Genes, Homeobox ; Glycoproteins ; Humans ; Immunoassay ; Matrix Metalloproteinase 2 ; Mesenchymal Stromal Cells ; Microarray Analysis ; Microphthalmia-Associated Transcription Factor ; Muscles ; Osteoblasts ; Osteogenesis ; Phosphoproteins ; Receptor, Epidermal Growth Factor ; Receptor, Fibroblast Growth Factor, Type 1 ; Receptor, Fibroblast Growth Factor, Type 3 ; Sialoglycoproteins ; Stem Cells ; Transcription Factors ; Vascular Endothelial Growth Factor B ; Vitamin E ; Vitamins

We investigated the patterns of pretreatment expression of the epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) by immunohistochemical staining and determined their correlation with treatment response and survival in 44 patients with esophageal squamous cell carcinoma (ESCC) treated with definitive concurrent chemoradiotherapy (CCRT). The definitive CCRT consisted of a median dose of 54 Gy (range: 40.0-68.4 Gy) and two cycles of concurrent administration of mostly 5-fluorouracil + cisplatinum. High expression of EGFR, VEGF, and COX-2 was found in 79.5%, 31.8%, and 38.6%, respectively. The Cox regression analysis for overall survival (OS) showed that both the treatment response and COX-2 expression were significant. The 3-yr OS rates of patients that achieved a complete response and those that did not were 46.7% and 5.3%, respectively (P = 0.006). The logistic regression analysis for treatment response with various parameters showed that only a high expression of VEGF was significantly associated with a complete response. Unlike other well-known studies, higher expression of VEGF was significantly correlated with a complete response to CCRT in this study. However, higher expression of COX-2 was significantly associated with shorter survival. These results suggest that VEGF might be a predictive factor for treatment response and COX-2 a prognostic factor for OS in patients with ESCC after definitive CCRT.
Aged ; Antineoplastic Agents/therapeutic use ; Carcinoma, Squamous Cell/drug therapy/*mortality/radiotherapy/*therapy ; Cisplatin/therapeutic use ; Combined Modality Therapy ; Cyclooxygenase 2/*metabolism ; Drug Therapy, Combination ; Esophageal Neoplasms/drug therapy/*mortality/radiotherapy/*therapy ; Female ; Fluorouracil/therapeutic use ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Neoplasm Staging ; Predictive Value of Tests ; Radiation Dosage ; Receptor, Epidermal Growth Factor/metabolism ; Regression Analysis ; Survival Rate ; Vascular Endothelial Growth Factor A/*metabolism

In carcinoma ex pleomorphic adenoma (CXPA), pleomorphic adenoma (PA) and diverse carcinoma components showing luminal (ductal) or non-luminal (myoepithelial) differentiation coexist. To elucidate the clinicopathological implications of cellular differentiation in CXPA and the potential role of p53, vascular endothelial growth factor (VEGF), c-erbB-2, c-kit, and glucose transporter 1 (Glut-1) in carcinogenesis, we analyzed 11 CXPAs with luminal differentiation (CXPAs-LD) and 6 CXPAs with non-luminal differentiation (CXPAs-NLD) and compared protein expressions in residual PAs and carcinomas by immunohistochemistry. Among the CXPAs-LD, 5 were invasive and 8 were histologically high-grade tumors. The 5-year survival rate was 72.7%. P53, c-erbB-2, VEGF, and Glut-1 were more immunoreactive in carcinoma components than in PAs (P = 0.008, 0.004, 0.002, and 0.024, respectively); c-erbB-2 overexpression was associated with high histological grade (P = 0.024). Carcinoma components frequently lacked c-kit expression (P = 0.009). CXPAs-NLD were all low-grade and invasive with a larger mean tumor size (5.2 cm) than CXPAs-LD (3.3 cm) (P = 0.040). The patients remained disease-free without significant immunohistochemical expression. The immunoprofiles and clinical course of CXPA differed according to cellular differentiation. Therefore, it is important to report the histological subtype and to assess potential biomarkers in diagnostic and therapeutic trials.
Adenoma, Pleomorphic/*immunology/metabolism/*pathology ; Adult ; Aged ; Carcinoma/*immunology/metabolism/*pathology ; Cell Differentiation ; Female ; Glucose Transport Proteins, Facilitative/metabolism ; Humans ; Male ; Middle Aged ; Proto-Oncogene Proteins c-kit/metabolism ; Receptor, erbB-2/metabolism ; Salivary Gland Neoplasms/*immunology/metabolism/*pathology ; Tumor Markers, Biological/*analysis ; Tumor Suppressor Protein p53/metabolism ; Vascular Endothelial Growth Factor A/metabolism

This study is to investigate the binding capability of lidamycin apoprotein (LDP), an enediyne-associated apoprotein of the chromoprotein antitumor antibiotic family, to human breast cancer and normal tissues, the correlation of LDP binding capability to human breast cancer tissues and the expression of tumor therapeutic targets such as VEGF and HER2. In this study, the binding capability of LDP to human breast cancer tissues was detected with tissue microarray. The correlation study of LDP binding capability to human breast tumor tissues and relevant therapeutic targets was performed on breast cancer tissue microarrays. Immunocytochemical examination was used to detect the binding capability of LDP to human breast carcinoma MCF-7 cells. As a result, tissue microarray showed that LDP staining of 73.2% (30/41) of breast cancer tissues was positive, whereas that of 48.3% (15/31) of the adjacent normal breast specimens was positive. The difference between the tumor and normal samples was significant (Chi2 = 4.63, P < 0.05). LDP immunoreactivity in breast cancer correlated significantly with the overexpression of VEGF and HER2 (P < 0.001 and < 0.01, r = 0.389 and 0.287, respectively). Determined with confocal immunofluorescent analysis, LDP showed the binding capability to mammary carcinoma MCF-7 cells. It is demonstrated that LDP can bind to human breast cancer tissues and there is significant difference between the breast cancer tissues and the corresponding normal tissues. Notably, the binding reactivity shows positive correlation with the expression of VEGF and HER2 in breast carcinoma tissues. The results imply that LDP may have a potential use as targeting drug carrier in the research and development of new anticancer therapeutics. This study may provide reference for drug combination of LDM and other therapeutic agents.
Aminoglycosides ; metabolism ; Antibiotics, Antineoplastic ; metabolism ; Apoproteins ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Enediynes ; metabolism ; Female ; Humans ; Protein Binding ; Receptor, ErbB-2 ; metabolism ; Tissue Array Analysis ; methods ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo construct, express and identify the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma encoding bifunctional protein sflk1-IFN-gamma (soluble fetal liver kinase 1 and interferon-gamma).

METHODSsflk1 and IFN-gamma gene fragments were cloned by RT-PCR, and then inserted into pcDNA3.1(+) plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma. The recombinant sflk1-IFN-gamma transiently expressed in COS-7 cells was detected by ELISA and Western blotting. Bioactivities of sflk1-IFN-gamma fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay.

RESULTSpcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in COS-7 cells. Concentrations of sflk1 and IFN-gamma in culture supernatants of pcDNA3.1(+)/sflk1-IFN-gamma transfected COS-7 cells were (20.85+/-2.48) ng/ml and (1.08+/-0.09) ng/ml, respectively. Western blotting showed that the molecular weight of sflk1-IFN-gamma fusion protein was about 130 kDa, while that of sflk1 was 115 kDa. The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes, demonstrating that sflk1-IFN-gamma fusion protein possessed the bioactivities of both sflk1 and IFN-gamma.

CONCLUSIONThe constructed plasmid pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in eukaryotes. The expressed sflk1-IFN-gamma fusion protein has the biological activities of both sflk1 and IFN-gamma.

Animals ; COS Cells ; Cercopithecus aethiops ; Female ; Interferon-gamma ; biosynthesis ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; Recombinant Proteins ; analysis ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

OBJECTIVETo investigate the roles of human fetal liver stromal cells (hFLSCs) and human fetal bone marrow stromal cells (hFBMSCs) in the hematopoietic differentiation of human embryonic stem cells and analyze their gene expression profile changes.

METHODSThe embryonic bodies on day 4 were cocultured with hFLSCs or hFBMSC in the presence of cytokines. Flow cytometry was performed after 8 days of induction to detect the expressions of the hemangioblast markers KDR and CD34, and the differential gene expression profiles between hFBMSC and hFLSCs were examined by cDNA microarray analysis.

RESULTSEight days after the induction, (1.06-/+0.20)% of the hFLSCs and (8.8-/+1.49)% of the hFBMSCs were positive for KDR, with the positivity rates for CD34 of (1.25-/+0.16)% and (9.17-/+2.10)%, respectively. In hFLSCs and hFBMSCs cultures, 0.9-/+0.36 and 10.6-/+0.63 hemagioblast-like cell colonies were found, respectively. cDNA microarray analysis showed that 240 genes were highly expressed in hFBMSCs, and 21 genes related to secreted cytokines, cell adhesion molecules and extracellular matrix proteins were highly expressed.

CONCLUSIONThe microenvironment including the cell matrix protein and cytokines secreted by the hFBMSCs might play an important role in hemangioblastic differentiation of human bone marrow stromal cells in vitro.

Antigens, CD34 ; genetics ; metabolism ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Coculture Techniques ; Embryonic Stem Cells ; cytology ; Fetus ; Gene Expression Profiling ; Hematopoietic Stem Cells ; cytology ; Humans ; Liver ; cytology ; Oligonucleotide Array Sequence Analysis ; Stromal Cells ; physiology ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo study the effects of prolonged 75% oxygen exposure on the expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2) in the neonatal rat lungs and to elucidate the effects of prolonged exposure of high concentration of oxygen on lung vascular development and its relationship with bronchopulmonary dysplasia (BPD).

METHODSForty eight Sprague-Dawley rat pups were randomly exposed to air (control group) and 75% oxygen (experimental group) 12 hrs after birth. The rats were sacrificed 7, 14 and 21 days after exposure and their lungs were sampled. The lung sections were stained with hematoxylin and eosin for histological evaluation. Expression of VEGF, VEGFR1 and VEGFR2 protein and mRNA was detected by immunohistochemistry and RT-PCR.

RESULTSAfter being exposed to 75% oxygen for 21 days, lung tissues had pathological changes as 'new' BPD. Expressions of VEGF protein (10.9 + or - 2.7 vs 30.8 + or - 6.4), VEGFR1 protein (5.4 + or - 1.4 vs 15.6 + or - 3.4) and VEGFR2 protein (11.3 + or - 2.6 vs 21.7 + or - 4.5) on day 21 in the experimental group decreased significantly as compared with the control group (p<0.05). The expression of VEGF mRNA (1.6 vs 3.3), VEGFR1 mRNA (0.4 vs 6.6) and VEGFR2 mRNA (0.5 vs 4.9) on day 21 in the experimental group also decreased significantly as compared with the control group (p<0.05).

CONCLUSIONSProlonged exposure of high concentration of oxygen may cause BPD possibly by inhibiting lung vascular development in neonatal rats.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; etiology ; Female ; Humans ; Infant, Newborn ; Lung ; blood supply ; Male ; Oxygen ; toxicity ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; Vascular Endothelial Growth Factor Receptor-1 ; analysis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; analysis ; genetics