Investigation of the Different Effects and Mechanisms of Pyrrolidine Dithiocarbamate on Matrix Metalloproteinases Activation between ECV304 and HUVEC Cells

• Main Authors: Chiao-Ling Chu, 朱喬伶
• Other Authors: George Hsiao
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/38675865106216329846

碩士 === 臺北醫學大學 === 藥理學研究所 === 92 === Matrix metalloproteinases (MMPs) can catalyze and degrade extracellular matrix (ECM), including ground substances and connecting fibers, which have their function to maintain tissue structure. Thus, MMPs play several important roles in tissue remodeling, repairing and destroys. The levels and activities of MMPs are strictly regulated and controlled in various ways. Many evidences indicate that human endothelial cells, cancer cells, monocyte and macrophages synthesize and secrete several MMPs which participate in the degradation of ECM components in rheumatoid arthritis tissues or atherosclerosis or during cancer growth and metastasis. In general, inflammatory cytokines and several growth factors can stimulate MMPs gene expression and biosynthesis. In our study, we used two endothelial cells to investigate the mechanisms of MMPs production from stimulated endothelial cells which inflammatory mediators affect and look for the difference of characters and drug responses between the endothelial cell and endothelial cell line. However, according to previous experiments, we found Pyrrolidine dithiocarbamate (PDTC), regard as an antioxidant、a metal chelator and NF-kB inhibitor so far. Thus, we used spontaneously transformed human umbilical vein endothelial cell line (ECV304) and primary human umbilical vein endothelial cell (HUVEC) as our experimental cells and then treated tumor necrosis factor- (TNF-) and phorbol-12-myristate- 13-acetate (PMA) (10 ng/ml) as stimulators. By using gelatin zymography analysis, we found TNF- and PMA (10 ng/ml) both significantly stimulate the expression of MMP-9 on ECV304 cells and PDTC could significantly enhance TNF--induced or PMA-induced MMP-9 activities especially at 20M. By western bolt and reverse transcription-polymerase chain reaction (RT-PCR) method, we also found PDTC increased MMP-9 protein and mRNA on TNF--induced ECV304. This indicated that PDTC have effect on the protein expression of MMP-9 and deeper influence on the level of MMP-9 transcription. Further more, we investigated the mechanism of action of PDTC in various signaling pathways. According to results, we presume the mechanism that PDTC enhance the expression of MMP-9 on TNF-a-induced ECV304 through the JNK or PI-3 kinase signal pathways. On the other hand, we found PMA induce active form expression of MMP-2 on HUVEC by zymography analysis. Then, the expression of MMP-2 would be inhibited by PDTC on a concentration manner. By western blot method, we also found PDTC decrease MMP-2 protein on PMA-induced HUVEC. The mechanism that PDTC inhibit the expression of MMP-2 on PMA-induced HUVEC might through the protein kinase C (PKC) signal pathway . According to our results, ECV304 and HUVEC cells have somehow difference in the expression of MMPs resulted from responding to stimulators or drugs by different mechanisms. The different characters between ECV304 and HUVEC would be a resource of research the future.