title: Metronomic ceramide analogs inhibit angiogenesis in pancreatic cancer through up-regulation of caveolin-1 and thrombospondin-1 and down-regulation of cyclin D1.
abstract: OBJECTIVE
  To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms.
  
  
  METHODS
  Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice.
  
  
  RESULTS
  Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine.
  
  
  CONCLUSIONS
  Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.
authors.affiliation: Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy. guido.bocci@med.unipi.it
authors.forename: Guido
authors.initials: G
authors.lastname: Bocci
authors.forename: Anna
authors.initials: A
authors.lastname: Fioravanti
authors.forename: Paola
authors.initials: P
authors.lastname: Orlandi
authors.forename: Teresa
authors.initials: T
authors.lastname: Di Desidero
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Natale
authors.forename: Giovanni
authors.initials: G
authors.lastname: Fanelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Viacava
authors.forename: Antonio Giuseppe
authors.initials: AG
authors.lastname: Naccarato
authors.forename: Giulio
authors.initials: G
authors.lastname: Francia
authors.forename: Romano
authors.initials: R
authors.lastname: Danesi
mesh_terms: D059250:Administration, Metronomic; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051242:Caveolin 1 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002518:Ceramides / Q000008:administration & dosage / Q000494:pharmacology*; D019938:Cyclin D1 / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000098:blood supply* / Q000188:drug therapy / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism*; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D051242:Caveolin 1; D002518:Ceramides; D019700:Thrombospondin 1; D019938:Cyclin D1; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3