title: Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.
abstract: Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.
authors.affiliation: Centre Pluridisciplinaire d'Oncologie, University of Lausanne Medical School, CH-1011 Lausanne, Switzerland.
authors.forename: Manuela
authors.initials: M
authors.lastname: Bezzi
authors.forename: Meriem
authors.initials: M
authors.lastname: Hasmim
authors.forename: Grégory
authors.initials: G
authors.lastname: Bieler
authors.forename: Olivier
authors.initials: O
authors.lastname: Dormond
authors.forename: Curzio
authors.initials: C
authors.lastname: Rüegg
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004002:Clodronic Acid / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D004164:Diphosphonates / Q000494:pharmacology*; D018274:Electroporation; D042783:Endothelial Cells / Q000187:drug effects* / Q000473:pathology*; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D039081:Integrin alpha5beta1 / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D000077211:Zoledronic Acid
chemical_list: D020533:Angiogenesis Inhibitors; D017304:Annexin A5; D004396:Coloring Agents; D004164:Diphosphonates; D007093:Imidazoles; D039081:Integrin alpha5beta1; D039302:Integrin alphaVbeta3; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; D004002:Clodronic Acid; D000077211:Zoledronic Acid; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt