| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 97 === Vascular endothelial growth factor-C (VEGF-C) which induces lymphangiogenesis through VEGF receptor 3 (VEGFR3) can be produced from tumor cells. In addition, VEGFR3 is also found in various tumor cells (such as A549 cells). Up to date, it is known that activation of VEGFR3 by VEGF-C induced cell migration of A549 through a p38/MAPK pathway. However, it is not clear whether VEGF-C induced tumor cell proliferation. Since our preliminary data showed the increase of cell counts by VEGFC at 150 ng/ml, the purpose of this study was to explore the action mechanism of VEGFC on cell proliferation by combination of biochemical, functional and quantitative proteomic assays. To exclude the possibility that the increase of cell counts by VEGF-C was due to the increase of cell survival, the effect of VEGF-C on Akt phosphorylation was further examined. FAScan analysis, immunofluorescent analysis, and western blotting assay showed VEGF-C-induced increases in cell population at G1/S phase, BrdU-positive cells and Rb phosporylation. The data confirmed the induction of cell cycle shift from G0 to G1 phase by VEGF-C. Finally, subcellular proteomic analysis coupled with quantitative technology revealed about 42 nuclear proteins which might be involved in VEGF-C-induced shift in cell cycle. For example, cell cycle and apoptosis regulatory protein 1 (CARP-1), histone H2A and H2B were decreased by VEGF-C; histone H1, catenin delta-1, serine/ threonine-protein kinase 4, and ADP-ribosylation factor-like protein 2 were increased by VEGF-C. VEGFR3 antagonists (Cpd. 1250 and VEGFR3/Fc) reversed VEGF-C-induced increases in CARP-1 expression, BrdU-positive cell, cell population in apoptotic phase. Cpd. 1250 inhibited cell proliferation and also increased pAkt and Ebp-1 to maintain cell survival. Both proteomic and biochemical results suggest that high concentrations of VEGF-C accelerated the progression of cell cycle from G0 to G1/S through a CARP-1/Rb-mediated pathways, histone-mediated chromatin remodeling. Unlike VEGFR3/Fc, Cpd. 1250 was a multitarget antagonist that inhibited cell proliferation through a VEGFR3-dependnet pathway and increased phosphorylation of Akt and expression of Ebp-1 to maintain cell survival through a VEGFR3-independent pathway in A549 cells. In addition, VEGFR3 antagonists (Cpd. 1250 and VEGFR3/Fc) may be a good current therapy to inhibit malignant cancer proliferation and metastasis at local sites with no cytotoxicity.
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