title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.
abstract: Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.
authors.affiliation: Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome 00158, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Gabellini
authors.forename: Teresa
authors.initials: T
authors.lastname: De Luca
authors.forename: Daniela
authors.initials: D
authors.lastname: Trisciuoglio
authors.forename: Marianna
authors.initials: M
authors.lastname: Desideri
authors.forename: Marta
authors.initials: M
authors.lastname: Di Martile
authors.forename: Daniela
authors.initials: D
authors.lastname: Passeri
authors.forename: Antonio
authors.initials: A
authors.lastname: Candiloro
authors.forename: Mauro
authors.initials: M
authors.lastname: Biffoni
authors.forename: Maria Giulia
authors.initials: MG
authors.lastname: Rizzo
authors.forename: Augusto
authors.initials: A
authors.lastname: Orlandi
authors.forename: Donatella
authors.initials: D
authors.lastname: Del Bufalo
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D064593:Heterografts; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008175:Lung Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000556:secondary*; D008545:Melanoma / Q000098:blood supply / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology*; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D014945:Wound Healing
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A