ABSTRACT
Membrane-type 1 matrix metalloproteinase (MT1-MMP), which hydrolyzes type I collagen and activates MMP-2, are deeply involved in angiogenesis as well as in tumor cell invasion and metastasis. We previously screened a number of natural and synthetic compounds to obtain a specific inhibitor of MT1-MMP and observed that (-)-epigallocatechin gallate (EGCG) has a potent and distinct inhibitory activity against MT1-MMP. In the present study, we investigated the effect of EGCG on tumor angiogenesis. EGCG significantly inhibited the invasion of human umbilical vein endothelial cells (HUVECs) at the concentration of 10 microM. This effect was not due to the toxicity of EGCG since this concentration of EGCG did not affect the HUVEC growth. Furthermore, morphological change of HUVEC at this concentration of EGCG was not observed under confocal laser scanning microscopy. EGCG suppressed tube formation by HUVECs in vitro and angiogenesis in vivo by using dorsal air sac model. Finally, we observed that both colon 26 NL17 carcinoma and Meth A sarcoma growth was suppressed in these tumor-bearing mice by EGCG administration, at least partly though the inhibition of angiogenesis.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Catechin/analogs & derivatives , Catechin/therapeutic use , Colonic Neoplasms/blood supply , Metalloendopeptidases/antagonists & inhibitors , Neovascularization, Pathologic/drug therapy , Sarcoma, Experimental/blood supply , Angiogenesis Inhibitors/pharmacology , Animals , Cell Division/drug effects , Cell Movement/drug effects , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Endothelium, Vascular/ultrastructure , Humans , Matrix Metalloproteinase 14 , Matrix Metalloproteinases, Membrane-Associated , Methylcholanthrene , Mice , Neovascularization, Pathologic/prevention & control , Sarcoma, Experimental/enzymology , Sarcoma, Experimental/pathology , Tumor Cells, Cultured , Umbilical Veins , Urokinase-Type Plasminogen Activator/antagonists & inhibitorsABSTRACT
We synthesized several prodrugs of glycine and gamma-aminobutyric acid. In order to establish a screening system from the prodrugs of selective activity to MAO-A or MAO-B, we examined purification conditions such as solubilization with Triton X-100, precipitation with ammonium sulfate, gel filtration and anion exchange chromatography. MAO-B was purified from various tissues such as guinea pig brain, kidney and spleen. MAO-A from human placenta without MAO-B was unstable in above purifications and used as crude. At each purification step, we checked sensitivity of the enzyme to specific inhibitors by developing a convenient fluorescence assay, in which hydrogen peroxide produced by the enzyme was reacted with p-hydroxyphenylpropionic acid. A fluorescence microplate reader measured a fluorescence of the fluorescent product from p-hydroxyphenylpropionic acid with horseradish peroxidase. In comparison with milacemide, N,N-bis(carbamoylmethyl)-N-pentylamine was the best and exclusive substrate for MAO-B. 2-N-(phenylethylamino)-acetoamide was the good substrate for MAO-A and MAO-B same as milacemide. 4-N-(n-pentylamino)-butyric acid and 4-(N-phenylethylamino)-butyric acid were the moderate substrates for both enzymes, which should release gamma-aminobutyric acid. These drugs will be new leading compounds.
Subject(s)
Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase/metabolism , Prodrugs/pharmacology , Animals , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Female , Guinea Pigs , Humans , Mitochondria, Liver/drug effects , Mitochondria, Liver/enzymology , Monoamine Oxidase Inhibitors/analysis , Monoamine Oxidase Inhibitors/chemistry , Prodrugs/analysis , Prodrugs/chemistryABSTRACT
Matrix metalloproteinases (MMPs), especially membrane-type 1 matrix metalloproteinase (MT1-MMP), which generates an active form of MMP-2 from proMMP-2, are deeply involved in angiogenesis as well as in tumor cell migration and metastasis. To obtain a specific inhibitor for MT1-MMP, we screened a number of natural and synthetic compounds using recombinant human MMP-2, MMP-7, and soluble MT1-MMP in a fluorogenic peptide cleavage assay. (-)-Epigallocatechin 3-O-gallate (EGCG) followed by (-)-epigallocatechin 3,5-di-O-gallate and epitheaflagallin 3-O-gallate, was found to have potent and distinct inhibitory activity against MT1-MMP. Therefore, we investigated the effect of EGCG on the suppression of MMP-2 activation as determined by gelatin zymography, and observed that the active form of MMP-2 in the conditioned medium of human umbilical vein endothelial cells was decreased in the presence of EGCG. The results suggest the possibility that tea polyphenols suppress tumor growth through the suppression of angiogenesis.