title: A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 "multi-points priming" mechanisms in vitro and in vivo.
abstract: BACKGROUND
  Tumor lymphangiogenesis plays an important role in promoting growth and metastasis of tumors, but no antilymphangiogenic agent is used clinically. Based on the effect of norcantharidin (NCTD) on lymphangiogenesis of human lymphatic endothelial cells (LECs), we firstly investigated the antilymphangiogenic activity of NCTD as a tumor lymphangiogenic inhibitor for human colonic adenocarcinomas (HCACs).
  
  
  METHODS
  In vivo and in vitro experiments to determine the effects of NCTD on tumor growth and lymphangiogenesis of the in-situ colonic xenografts in nude mice, and lymphatic tube formation of the three-dimensional (3-D) of the co-culture system of HCAC HT-29 cells and LECs were done. Proliferation, apoptosis, migration, invasion, Ki-67, Bcl-2 and cell cycle of LECs and the co-culture system in vitro were respectively determined. Streparidin-peroxidase staining, SABC, western blotting and RT-PCR were respectively used to examine the expression of LYVE-1, D2-40, CK20 (including their LMVD), and VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in vitro and in vivo.
  
  
  RESULTS
  NCTD inhibited tumor growth and lymphangiogenesis of the in-situ colonic xenografts in vivo, and these observations were confirmed by facts that lymphatic tube formation, proliferation, apoptosis, migration, invasion, S-phase cell cycle, and Ki-67 and Bcl-2 expression in vitro, and LYVE-1, D2-40, CK20 expression and their LMVD in vitro and in vivo were inhibited and affected. Furthermore, the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 at protein/mRNA levels in the process of lymphatic tube formation in vitro and tumor lymphangiogenesis in vivo was downregulated; NCTD in combination with mF4-31C1 or Sorafenib enhanced these effects.
  
  
  CONCLUSIONS
  NCTD inhibits tumor growth and lymphangiogenesis of HCACs through "multi-points priming" mechanisms i.e. affecting related malignant phenotypes, inhibiting Ki-67 and Bcl-2 expression, inducing S-phase cell cycle arrest, and directly or indirectly downregulating VEGF-A,-C,-D/VEGFR-2,-3 signaling pathways. The present finding strongly suggests that NCTD could serve as a potential antilymphangiogenic agent for tumor lymphangiogenesis and is of importance to explore NCTD is used for antitumor metastatic comprehensive therapy for HCACs.
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. northernguy@163.com.
authors.forename: Xin-Ping
authors.initials: XP
authors.lastname: Li
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. surgeon-jing@hotmail.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Jing
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. 13916653283@163.com.
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Sun
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. liuzyno1@163.com.
authors.forename: Zhong-Yan
authors.initials: ZY
authors.lastname: Liu
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. drzhangjingtao826@aliyun.com.
authors.forename: Jing-Tao
authors.initials: JT
authors.lastname: Zhang
authors.affiliation: Department of Surgery, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, 200072, People's Republic of China. tjshdoc_sw@aliyun.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Surgery, Dahui Hospital, Postgraduate on-the-job in 2011 Grade, Tongji University School of Medicine, Shanghai, 200237, People's Republic of China. zhuzhululu@yeah.net.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Tongji University, Shanghai, 200065, People's Republic of China. fanyuezu@hotmail.com.
authors.forename: Yue-Zu
authors.initials: YZ
authors.lastname: Fan
mesh_terms: D020533:Angiogenesis Inhibitors / Q000008:administration & dosage* / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D042783:Endothelial Cells / Q000166:cytology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019073:HT29 Cells; D006801:Humans; D066298:In Vitro Techniques; D042583:Lymphangiogenesis / Q000187:drug effects*; D008207:Lymphatic Metastasis / Q000517:prevention & control*; D051379:Mice; D008819:Mice, Nude; D040262:Receptors, Vascular Endothelial Growth Factor / Q000037:antagonists & inhibitors; D042442:Vascular Endothelial Growth Factors / Q000037:antagonists & inhibitors; D023041:Xenograft Model Antitumor Assays
chemical_list: D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D042442:Vascular Endothelial Growth Factors; C069741:norcantharidin; D040262:Receptors, Vascular Endothelial Growth Factor