Norcantharidin: a potential antiangiogenic agent for gallbladder cancers in vitro and in vivo.
Our objective was to explore the antiangiogenic activity of norcantharidin (NCTD) as an angiogenic inhibitor for gallbladder cancers. In vitro and in vivo experiments to determine the effects of NCTD on HUVECs, chicken CAM capillaries and gallbladder cancer xenograft angiogenesis in nude mice were respectively done. The MTT method was used to assay the cytotoxicity of NCTD on HUVECs. Immunofluorescence was used to evaluate HUVEC apoptosis. The scraping line method, matrigel invasion assay and tube formation assay were used to detect the migration, invasion and tube formation of HUVECs. A digital camera was used to observe chicken CAM capillaries. Experiments with NCTD in a xenograft model were used to observe the effect of NCTD on xenograft growth and survival of mice with xenografts. CD₃₄ immunohistochemistry, flow cytometry and micro-MRA were used, respectively, to determine MVD, cell apoptosis and hemodynamic analysis of the xenografts. Immunohistochemistry and RT-PCR were used, respectively, to detect the expression of VEGF, Ang-2, TSP, TIMP-2 proteins/mRNAs of the xenografts. The xenograft MVD associated with tumor volume, the PCNA/apoptosis ratio and related-protein expression was evaluated simultaneously. We found that NCTD effectively inhibited the proliferation, migration, invasion and capillary-like tube formation of HUVECs in vitro; it reduced angiogenesis and directly destroyed the formed CAM capillaries in vivo. In the experiments in mice, NCTD not only inhibited significantly xenograft proliferation and growth, prolonged survival time of mice with xenografts, decreased the xenograft MVD and vascular perfusion, but also, similarly to ES, decreased significantly the expression of VEGF or Ang-2 protein/mRNA, increased the expression of TSP or TIMP-2 protein/mRNA. Moreover, the xenograft MVD was positively related with tumor volume, PCNA/apoptosis ratio, and VEGF or Ang-2 expression, respectively (all P<0.05), but negatively correlated with TSP or TIMP-2 expression (both P<0.05). These data showed that NCTD could serve as a potential antiangiogenic agent for gallbladder cancers.
Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, PR China.
Jing-Tao
JT
Zhang
Yue-Zu
YZ
Fan
Chun-Qiu
CQ
Chen
Zhe-Ming
ZM
Zhao
Wei
W
Sun
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D042702:Angiopoietin-2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; 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; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D005706:Gallbladder Neoplasms / Q000098:blood supply / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D018810:Magnetic Resonance Angiography; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D055420:Perfusion Imaging / Q000379:methods; D016133:Polymerase Chain Reaction; D051381:Rats; D012039:Regional Blood Flow; D019699:Thrombospondins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D047368:Tumor Burden / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D020533:Angiogenesis Inhibitors; D042702:Angiopoietin-2; D019086:Bridged Bicyclo Compounds, Heterocyclic; C560823:TIMP2 protein, human; D019699:Thrombospondins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019716:Tissue Inhibitor of Metalloproteinase-2; C069741:norcantharidin