title: Suppression of lymphangiogenesis in human lymphatic endothelial cells             by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 with norcantharidin.
abstract: Lymph node metastasis of tumors is a crucial early step in the metastatic             process. Tumor lymphangiogenesis plays an important role in promoting tumor metastasis             to regional lymph nodes. Norcantharidin (NCTD) has been reported to possess potent             anti-angiogenesis and antitumor properties in several cell lines and xenograft             tumor models. However, its role in tumor-associated lymphangiogenesis and lymphatic             metastasis remains unclear. Here, we investigated the effect of NCTD on proliferation,             apoptosis, migration, invasion and the lymphatic tube formation, lymphangiogenesis,             of human lymphatic endothelial cells (HLECs) in vitro by MTT, proliferation assay,             Hoechst staining and flow cytometry, scraping line method, Matrigel invasion assay,             inverted or fluorescence microscope and transmission electron microscope. Moreover,             the underlying mechanisms, such as VEGF-C, VEGF-D, VEGFR-3 at protein and mRNA             levels in lymphangiogenesis using 3-dimensional (3-D) culture of HLECs were measured             by immunohistochemistry, western blotting and real-time polymerase chain reaction             (RT-PCR). It was shown that NCTD inhibited proliferation, migration, invasion             and lymphatic tube formation (forming-lymphatic and/or formed-lymphatic) of HLECs,             induced HLEC apoptosis (all P<0.01) significantly, in a dose- and time-dependent             manner (IC50 6.8 µg/ml); and downregulated the expression of VEGF-C, VEGF-D and             VEGFR-3 at protein or/and mRNA levels (P<0.01) in HLEC lymphatic tube formation.             Thus, we identified for the first time that NCTD inhibited HLEC lymphangiogenesis             by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 in vitro. The present findings             may be of importance to explore the therapeutical target or strategy of NCTD for             tumor lymphangiogenesis and lymphatic metastasis.
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, PR China.
authors.forename: Zhong-Yan
authors.initials: ZY
authors.lastname: Liu
authors.forename: Hai-Ou
authors.initials: HO
authors.lastname: Qiu
authors.forename: Xiao-Jian
authors.initials: XJ
authors.lastname: Yuan
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Ni
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Sun
authors.forename: Wei
authors.initials: W
authors.lastname: Jing
authors.forename: Yue-Zu
authors.initials: YZ
authors.lastname: Fan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004729:Endothelium, Lymphatic / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D042583:Lymphangiogenesis / Q000187:drug effects*; D042582:Vascular Endothelial Growth Factor C / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D042643:Vascular Endothelial Growth Factor D / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D042582:Vascular Endothelial Growth Factor C; D042643:Vascular Endothelial Growth Factor D; C069741:norcantharidin; D040321:Vascular Endothelial Growth Factor Receptor-3