PURPOSE: We tried to select and validate the candidate gene for the prognostic marker of breast cancer by comparing the analysis of copy number variation (CNV) between normal breast tissues and breast cancer tissues by performing array comparative genomic hybridization (CGH). METHODS: Array CGH was performed with using the fresh frozen tissues of 77 breast cancer patients. We selected the clones with more than a 20% frequency of gain or loss, and the clones with gain or loss in more than 2 consecutive clones. We finally selected the clones that were statistically significant on the survival analysis. We searched for the candidate gene that belonged to the candidate clones and we selected the final candidate gene that is assumed to be most related to the carcinogenesis of breast cancer by searching for information of the individual gene. We performed RT-PCR to validate the RNA expression of the final candidate gene with using the breast tissues of another 20 breast cancer patients. RESULTS: Eleven (10 in the gain group and 1 in the loss group) clones were finally selected as candidate clones. The significant CNVs with gain were found in the regions of 1q23.1, 1q41, 1q44, 5p15.33, 8q21.3, 15q26.3, 17q12 and 21q22.3 and the significant CNV with loss was found in 14q32.33. COL18A1 (21q22.3) was selected as the final candidate gene and the RT-PCR results revealed that the expression of COL18A1 was up-regulated in the cancer tissues of 18 of the other 20 (90%) breast cancer patients. CONCLUSION: We selected COL18A1 (21q22.3) as the candidate gene for the prognostic marker of breast cancer by comparing the analysis of CNVs from the array CGH. The RNA of COL18A1 was over-expressed in breast cancer tissue, as determined by RT-PCR.
Breast ; Breast Neoplasms ; Clone Cells ; Coat Protein Complex I ; Collagen Type XVIII ; Comparative Genomic Hybridization ; DNA ; DNA Copy Number Variations ; Gene Amplification ; Humans ; Microarray Analysis ; RNA

PURPOSE: We tried to select and validate the candidate gene for the prognostic marker of breast cancer by comparing the analysis of copy number variation (CNV) between normal breast tissues and breast cancer tissues by performing array comparative genomic hybridization (CGH). METHODS: Array CGH was performed with using the fresh frozen tissues of 77 breast cancer patients. We selected the clones with more than a 20% frequency of gain or loss, and the clones with gain or loss in more than 2 consecutive clones. We finally selected the clones that were statistically significant on the survival analysis. We searched for the candidate gene that belonged to the candidate clones and we selected the final candidate gene that is assumed to be most related to the carcinogenesis of breast cancer by searching for information of the individual gene. We performed RT-PCR to validate the RNA expression of the final candidate gene with using the breast tissues of another 20 breast cancer patients. RESULTS: Eleven (10 in the gain group and 1 in the loss group) clones were finally selected as candidate clones. The significant CNVs with gain were found in the regions of 1q23.1, 1q41, 1q44, 5p15.33, 8q21.3, 15q26.3, 17q12 and 21q22.3 and the significant CNV with loss was found in 14q32.33. COL18A1 (21q22.3) was selected as the final candidate gene and the RT-PCR results revealed that the expression of COL18A1 was up-regulated in the cancer tissues of 18 of the other 20 (90%) breast cancer patients. CONCLUSION: We selected COL18A1 (21q22.3) as the candidate gene for the prognostic marker of breast cancer by comparing the analysis of CNVs from the array CGH. The RNA of COL18A1 was over-expressed in breast cancer tissue, as determined by RT-PCR.
Breast ; Breast Neoplasms ; Clone Cells ; Coat Protein Complex I ; Collagen Type XVIII ; Comparative Genomic Hybridization ; DNA ; DNA Copy Number Variations ; Gene Amplification ; Humans ; Microarray Analysis ; RNA

OBJECTIVETo investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization.

METHODSpBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Iand Sal I, by PCR reaction, by sequence, and then by alignment of PCR products with the gene Bank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO(3) and 25% KNO(3) cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs.

RESULTSpBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.

CONCLUSIONSThe plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.

Animals ; Corneal Neovascularization ; pathology ; therapy ; Endostatins ; genetics ; Genetic Therapy ; methods ; Humans ; Rats ; Rats, Wistar ; Transfection

OBJECTIVETo throw light on the superiority of the anti-angiogenesis activity of endostatin (ES) derivatives by reviewing the recent progress in the field of ES molecular structure modification.

DATA SOURCESThe data used in this article were mainly from PubMed with relevant English articles published from 1971 to May 2008. The search terms were "endostatin" and "angiothesis".

STUDY SELECTIONArticles involved in the ES molecular structure modification and the original milestone articles were selected.

RESULTSA number of ES derivatives were designed and studied to improve its clinical relevance. The modified ES with polyethylene glycol (PEG), low molecular weight heparin (LMWH) and IgG Fc domain extended the circulation half-life. Meanwhile the recombinant ESs showed more potent anti-tumor activity than native ES in mouse xenografts. Mutated ES also changed its anti-angiogenesis activity.

CONCLUSIONSThe anti-angiogenesis treatment remains a promising tumor therapeutic strategy. New ES derivatives would be a good choice to meet the future challenge on clinical application of ES.

Angiogenesis Inhibitors ; pharmacokinetics ; therapeutic use ; Animals ; Endostatins ; pharmacokinetics ; therapeutic use ; Humans ; Neovascularization, Pathologic ; drug therapy

PURPOSE: Angiogenesis is a critical determinant of tumor growth and the development of metastasis. Angiostatin and endostatin have been used in a variety of in vitro and in vivo animal models as effective inhibitors of angiogenesis. However, human angiostatin and endostatin have not been tested against an intact human tissue target in vitro to determine its ability to achieve an antiangiogenic response. We performed our study to determine if human angiostatin and endostatin would inhibit the development of an angiogenic response (initiation) and to determine the subsequent growth (angiogenic index) of human vessels in a dose-dependent manner with a human placental vein angiogenesis model (HPVAM). METHODS: We used full thickness human placental vein discs cultured in three-dimensional fibrin-thrombin clots with an overlay of liquid media. Human angiostatin and endostatin were evaluated in concentrations ranging from 10-9 M to 10-4 M. A positive control containing 20% fetal bovine serum and a negative control using heparin and hydrocortisone 21-phosphate were also tested. RESULTS: Human angiostatin did not inhibit the initiation of an angiogenic response and the subsequent development of the angiogenic response (angiogenic index) at any concentration. Human endostatin significantly inhibited the initiation rate of an angiogenic response at a concentration of 10-4 M (p<0.001) and the subsequent development of an angiogenic response (angiogenic index) from a concentrations of 10-5 M to 10-4 M (p<0.001, p<0.001, respectively). CONCLUSION: We conclude that a very high concentration of human endostatin can inhibit the angiogenic response in human vascular tissue and that human angiostatin will not inhibit angiogenesis of normal human blood vessels in vitroThese results suggest that human endostatin has a more powerful antiangiogenic effect than human angiostatin, but we need further investigations of human angiostatin against an intact human tissue target.
Angiostatins* ; Blood Vessels ; Endostatins* ; Heparin ; Humans* ; Hydrocortisone ; Models, Animal ; Neoplasm Metastasis ; Veins

OBJECTIVETo construct and express secretive endostatin eukaryotic plasmid for treatment of hepatoma.

METHODSMouse Igk signal peptide sequence was synthesized and cloned into pcDNA3.1 with endostatin gene. The supernant of BHK-21 transfected with recombinant was used to culture ECV304. The proliferation of latter was evaluated by MTT assay. H22 was inoculated intramusclely, then naked DNA of endostatin plasmid was injected into the inoculation site. Tumors were dissected and weighted after treatments. All data was analyzed by SPSS10.0.

RESULTSThe supernant of BHK-21 transfected with recombinant can inhibit the proliferation of ECV304 by 29.2%. Tumor weight lighter after injected with naked pSecES (1.34 g+/-0.96g) compared with naked pcDNA3.1 (2.70g+/-0.82g) and saline (3.73g+/-1.41g).

CONCLUSIONThe endostatin eukaryotic plasmid was constructed and it can be used for gene therapy on hepatoma.

Animals ; Endostatins ; genetics ; secretion ; Genetic Therapy ; Liver Neoplasms, Experimental ; therapy ; Male ; Mice ; Plasmids

OBJECTIVETo explore the in vitro anti-leukemia effect of endostar (recombinant human endostatin, rhES).

METHODSThe anti-leukemia effect of endostar on fresh bone marrow cell from acute leukemia patients and healthy adult was analyzed by typan-blue exclusion assay, MTT assay, transmission electron microscopy and flow cytometry with Annexin V-FITC/PI staining.

RESULTSTreatment with endostar at concentrations of 50, 100 and 200 µg/ml for 72 hours could inhibit the proliferation of HL-60 cells. The inhibition rates were 11.6%, 30.4% and 33.5%, respectively. For NB4 cells, the inhibitory rates of 100 and 200 µg/ml endostar were 12.4% and 16.4%, respectively. The inhibition of endostar on acute leukemia cells was time and dose dependent, 50 - 200 µg/ml endostar could also inhibit the growth of fresh bone marrow cell from acute leukemia patients. While 0 - 400 µg/ml endostar had no significant effect on fresh primary bone marrow cells from healthy adult. 100 µg/ml endostar could induce apoptosis of HL-60 and NB4 cells after treatment for 72 hours. The apoptotic rates of HL-60 and NB4 cells were 19.6% and 20.8%, respectively.

CONCLUSIONEndostar inhibits proliferation of leukemia cells by inducing apoptosis. It might be a potential medication for acute leukemia.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Endostatins ; HL-60 Cells ; Humans ; Leukemia

Animals ; Endostatins ; blood ; Estrogens ; therapeutic use ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Rats ; Rats, Wistar

PURPOSE: The purpose of this study was to examine whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against a human neuroblastoma cell line (designated TNB9). We employed a human neuroblastoma xenograft model, and we investigated whether continuous infusion is more effective than an intermittent administration. METHODS: In the first experiment, when the tumors on the backs of nude mice reached a weight of 90 mg, rhEndostatin was administered subcutaneously to the mice (n=5) every day for 10 consecutive days. In the second experiment, the same daily dose of rhEndostatin was administered continuously to the TNB9- bearing mice (n=6) via subcutaneous infusion pumps for 3 consecutive days with the total dose being 30% of the dose given in the first experiment. Nestin and factor VIII expression levels were assessed immunohistochemically to elucidate whether the effects of rhEndostatin was present according to the histologic evidence at day 4 in the second experiment. RESULTS: In the first experiment, the relative tumor weight in treated mice (n=5) was significantly less than that in the controls (n=12) on day 2 after treatment initiation only (P<.05). The maximum inhibition rate (MIR) of TNB9 xenograft growth by rhEndostatin was 46.4%, indicating the lack of efficacy. In the second experiment, the effects of rhEndostatin were much more marked than those noted in the first experiment, with the MIR being 60.7%. The mean relative tumor weight in the treated group (n=6) in the second experiment was significantly less than that in the controls (n=10) on days 2, 4 and 6 (P<.01), as well as on days 8 and 10 (P<.05). The nestin staining in the endothelium of the control tumors (n=2) was remarkable, whereas the nestin staining showed as a loss of fibrillar structure in the rhEndostatin-treated tumors (n=2). The number of vessels immunostained with antifactor VIII antibody was markedly reduced in the tumors (n=2) from the rhEndostatin-treated mice compared with that from the control mice (n=2). CONCLUSION: Continuous administration of rhEndostatin resulted in more significant tumor regression than an intermittent administration of the agent. This result suggests that the continuous infusion of rhEndostatin is an effective agent and administration method for treating patients with neuroblastoma in the future.
Animals ; Cell Line ; Endostatins* ; Endothelium ; Factor VIII ; Heterografts* ; Humans* ; Infusions, Subcutaneous ; Mice ; Mice, Nude ; Nestin ; Neuroblastoma* ; Tumor Burden

PURPOSE: Recently, there has been several studies to clarify the pathogenesis of Kawasaki disease (KD) and the relations of VEGF and endostatin that act on vascular endothelial cells to the coronary artery complications. In this report, we measured serum levels of VEGF and endostatin in acute and subacute phases of KD to assess the change of these levels and the relations to the development of coronary artery lesions (CAL). METHODS: Twenty six patients were diagnosed and treated for KD between January, 2001 and July, 2005 at Kangnam St. Mary's Hospital, the Catholic University of Korea. They were divided into those with and without CAL. Serum levels of VEGF and endostatin were measured during acute and subacute phases and compared to those measured in healthy and disease control groups. RESULTS: Serum levels of VEGF were increased in KD but no differences were noted in KD with and without CAL. Serum levels of endostatin were decreased in the acute phase of KD, however they were recovered in the subacute phase of KD, regardless of CAL. The VEGF/endostatin ratio was increased in KD. KD without CAL showed a relative decrease in this ratio during the subacute phase. Significant positive correlations were found between serum VEGF and WBC count, VEGF and ESR, VEGF/endostatin ratio and ESR in the acute phase of KD. CONCLUSION: Analysis of factors influencing the vascular endothelium such as VEGF and endostatin will help to clarify the etiology of KD and the pathogenesis of CAL.
Coronary Vessels* ; Endostatins* ; Endothelial Cells ; Endothelium, Vascular ; Humans ; Korea ; Mucocutaneous Lymph Node Syndrome* ; Vascular Endothelial Growth Factor A*