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Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

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World Journal of Gastroenterology 2005年第40期11卷 6385-6388页

作者： Li-Bo YU Xin-Shu Dong Wen-Zhou sun Dong-Lu Zhao Yue Yang Department of GeneralSurgery the Affiliated Tumor Hospital of Harbin MedicalUniversity Harbin 150040 Heilongiiang Province China Cancer Research Institute of HarbinMedical University Harbin 150040 Heilongjiang Province China

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

关键词： Colorectal Neoplasms Neoplasm Invasiveness Cell Line, Tumor Cell Movement Dose-Response Relationship, Drug Female gelatinase a Humans Male NG-Nitroarginine Methyl Ester Nitric Oxide Nitric Oxide Synthase inhibitors Tissue Inhibitor of Metalloproteinase-2

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Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

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World Journal of Gastroenterology 2001年第4期7卷 532-536页

作者： Li Hou Ying Li Yong-Hua Jia Bo Wang Yi Xin Mao-Ying Ling Shen Lü Department of Pathology,Dalian Medical University,Dalian 116027,Liaoning Province,ChinaDepartment of Biochemistry,Dalian Medical University,Dalian 116027,Liaoning Province,China Department of Pathology Dalian Medical University Dalian 116027 China. houli72@***

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P<0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P <0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression

关键词： Animals DNA Fragmentation Flow Cytometry gelatinase a gelatinase B Liver Neoplasms, Experimental Lymph Nodes Lymphatic Metastasis Macrophages Membrane Glycoproteins Mice Mice, Inbred Strains Research Support, Non-U.S. Gov't Tumor Cells, Cultured

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Electroporative interleukin-10 gene transfer ameliorates carbon tetrachlo-ride-induced murine liver fibrosis by MMP and TIMP modulation

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Acta Pharmacologica Sinica 2006年第4期27卷 469-476页

作者： Tsung-hsing LEE Allan M CONCEJERO Departments of Anesthesiology Departments of Surgery Kaohsiung Chang Gung Memorial HospitalTaipeiChina

Aim:Liver fibrosis represents a process of healing and scarring in response tochronic liver *** therapies for liver fibrosis are ***-10（IL-10）is a cytokine that downregulates pro-inflarnmatory responses and has amodulatory effect on hepatic *** aim of this study was to investi-gate whether electroporative IL-10 gene therapy has an hepatic fibrolytic effect ***:Hepatic fibrosis was induced by administering carbon tetrachlo-ride（CCl4）for 10 weeks in *** human IL-10 expression plasmid was deliv-ered via electroporation after hepatic fibrosis was ***,reverse transcription polymerase chain reaction（RT-PCR）,immunoblotting,andgelatin zymography were used to investigate the possible mechanisms of actionof ***:Human IL-10 gene therapy reversed CCl4-induced liver fibrosisin ***-PCR revealed that IL-10 gene therapy attenuated liver TGF-β1,col-lagen α1,fibronectin,and cell adhesion molecule mRNA *** transfer,both the activation of(x-smooth muscle actin and cyclooxygenase-2were significantly ***,IL-10 significantly inhibited matrixmetalloproteinase-2（MMP-2）and tissue inhibitors of matrix metalloproteinase（TIMP）activation after ***:We demonstrated that IL-10gene therapy attenuated CCl4-induced liver fibrosis in ***-10 preventedupregulated fibrogenic and pro-inflammatory gene *** collagenolyticeffect may be attributed to MMP and TIMP ***-10 gene therapy maybe an effective therapeutic modality against liver fibrosis with potential clinicaluse.

关键词： interleukin-10 gene therapy liver cirrhosis gelatinase a tissue inhibitor of metalloproteinases cyclooxygenase 2

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Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

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World Journal of Gastroenterology 2001年第3期7卷 335-339页

作者： Qing Wang~1 Zhi Ying Lin~2 Xiao Li Feng~3 ~1Department of Microbiology,Medical Center of Fudan University.the former Shanghai Medical University,Shanghai 200032,China ~2Liver Cancer Institute,Zhongshan Hospital,Shanghai 200032,China ~3Shanghai Institute of Biochemistry,Academy Sinica,Shanghai 200031,ChinaQing Wang earned master degree from Shanghai Medical University in 1996,now a senior lecturer of microbiology,specialized in the role of oncogcncs on tumor metastasis,having 8 papers published. Department of Microbiology Medical Center of Fudan University Shanghai 200032 China. zmswq@***

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

关键词： Carcinoma, Hepatocellular Cell Adhesion Cell Movement gelatinase a gelatinase B Gene Expression Regulation, Neoplastic Genes, ras Humans In Vitro Liver Neoplasms Phenotype Predictive Value of Tests Receptor, Epidermal Growth Factor Transfection

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Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

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World Journal of Gastroenterology 2001年第5期7卷 647-651页

作者： Ping-Sheng Chen~(1,2) Wei-Rong Zhai~1 Xiao-Mei Zhou~3 Jin-Sheng Zhang~1 Yue-E Zhang~1 Yu-Qin Ling~1 Ying-Hong Gu~1 1 Department of Pathology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China2 Ping-Sheng Chen now works in the Department of Pathology,School of Basic Medical Sciences the (former Nanjing Railway Medical College),Southeast University,Nanjing 210009,China3 Institute of Cancer Research,Shanghai 200032,China Department of Pathology School of Basic Medical Sciences Fudan University 138 Yixueyuan Road Shanghai 200032 China.

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P<0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P < 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P < 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P < 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P < 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

关键词： Animals Cell Division Cell Hypoxia Cells, Cultured gelatinase a Gene Expression Regulation, Enzymologic Hepatocytes Hyperoxia Metalloendopeptidases RNA, Messenger Rats Rats, Sprague-Dawley Research Support, Non-U.S. Gov't Tissue Inhibitor of Metalloproteinase-2

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Prognostic value of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in bladder carcinoma

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Chinese Medical Journal 2002年第5期115卷 743-745,155页

作者：许克新侯树坤杜志军北京大学人民医院泌尿外科北京100044

OBJECTIVES: To detect the level of matrix metalloproteinase-2 (MMP-2) mRNA and the tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in bladder transitional cell carcinoma (BTCC), and to estimate the prognosis for bladder tumor based on the quality and quantity of MMP-2 and TIMP-2 mRNA. METHODS: Thirty-five samples of human BTCC and 15 normal fresh bladder tissues were studied by RT-PCR analysis followed by computer-assisted image analysis. RESULTS: The level of the MMP-2 mRNA in BTCC was significantly increased compared with that in normal bladder epithelium. The positive rates of MMP-2 and TIMP-2 mRNA were 71.4% and 65.7% in BTCC, and 66.7% and 60.0% in the normal bladder wall. The expression intensity of the MMP-2 mRNA by image analysis tended to increase with tumor grading and staging, which showed statistical significance. Similarly, the MMP-2 to TIMP-2 ratio also showed statistically significant difference between normal bladder tissue and bladder carcinoma (P

关键词： Adult Aged Aged, 80 and over Bladder Bladder Neoplasms Female gelatinase a Gene Expression Regulation, Neoplastic Humans Male Middle Aged Neoplasm Staging Predictive Value of Tests Prognosis RNA, Messenger Research Support, Non-U.S. Gov't Tissue Inhibitor of Metalloproteinase-2

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