Zoledronate Sensitizes Endothelial Cells to Tumor Necrosis Factor-induced Programmed Cell Death
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m308114200
ISSN1083-351X
AutoresManuela Bezzi, Meriem Hasmim, Grégory Bieler, Olivier Dormond, Curzio Rüegg,
Tópico(s)Bone Metabolism and Diseases
ResumoBisphosphonates 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 αVβ3, but not α5β1, 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) γ, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNγ. Phosphorylation of c-Src and activation of NF-κB 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. 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 αVβ3, but not α5β1, 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) γ, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNγ. Phosphorylation of c-Src and activation of NF-κB 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. Bisphosphonates are pyrophosphate analogues in which the oxygen is replaced by a carbon atom with various side chains (1Fleisch H. Breast Cancer Res. 2002; 4: 30-34Crossref PubMed Scopus (362) Google Scholar). They efficiently accumulate in bone due to their high affinity for calcium. Bisphosphonates are potent inhibitors of bone resorption used for the treatment of diseases characterized by excessive bone loss, such as Paget's disease, osteoporosis, and osteolytic tumor bone metastases (2Rogers M.J. Watts D.J. Russell R.G. Cancer. 1997; 80: 1652-1660Crossref PubMed Google Scholar, 3Brown D.L. Robbins R. J. Clin. Pharmacol. 1999; 39: 651-660Crossref PubMed Scopus (45) Google Scholar, 4Green J.R. Curr. Opin. Oncol. 2002; 14: 609-615Crossref PubMed Scopus (44) Google Scholar). Bisphosphonates inhibit osteoclast activity at multiple levels; they prevent differentiation of macrophages into osteoclasts, block the activity of mature osteoclasts, and induce osteoclast apoptosis (5Rogers M.J. Gordon S. Benford H.L. Coxon F.P. Luckman S.P. Monkkonen J. Frith J.C. Cancer. 2000; 88: 2961-2978Crossref PubMed Google Scholar, 6Rodan G.A. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 375-388Crossref PubMed Scopus (244) Google Scholar). The exact molecular mechanisms of action of bisphosphonates are only partially understood and appear to differ among different families of bisphosphonates (6Rodan G.A. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 375-388Crossref PubMed Scopus (244) Google Scholar, 7Fleisch H. Endocr. Rev. 1998; 19: 80-100Crossref PubMed Scopus (0) Google Scholar). Non-nitrogen containing bisphosphonates (e.g. clodronate and etidronate) can be incorporated into non-hydrolyzable ATP analogs, which accumulate intracellularly and thereby suppress ATP-dependent enzymes (8Rogers M.J. Brown R.J. Hodkin V. Blackburn G.M. Russell R.G. Watts D.J. Biochem. Biophys. Res. Commun. 1996; 224: 863-869Crossref PubMed Scopus (114) Google Scholar). Nitrogen-containing bisphosphonates (e.g. zoledronate and pamidronate) inhibit critical enzymes of the mevalonate pathway, in particular farnesyl diphosphate synthase, required for the synthesis of farnesyl diphosphate and geranylgeranyl diphosphate, and thereby suppress prenylation of small GTPases essential for many cellular functions (9Bergstrom J.D. Bostedor R.G. Masarachia P.J. Reszka A.A. Rodan G. Arch. Biochem. Biophys. 2000; 373: 231-241Crossref PubMed Scopus (373) Google Scholar, 10Dunford J.E. Thompson K. Coxon F.P. Luckman S.P. Hahn F.M. Poulter C.D. Ebetino F.H. Rogers M.J. J. Pharmacol. Exp. Ther. 2001; 296: 235-242PubMed Google Scholar, 11Luckman S.P. Hughes D.E. Coxon F.P. Graham R. Russell G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 581-589Crossref PubMed Scopus (1077) Google Scholar, 12van Beek E. Pieterman E. Cohen L. Lowik C. Papapoulos S. Biochem. Biophys. Res. Commun. 1999; 255: 491-494Crossref PubMed Scopus (193) Google Scholar). Clinical and experimental evidence indicates that bisphosphonates suppress progression of bone metastases, and recent observations suggest that this effect may be independent of the inhibition of bone resorption (13Neville-Webbe H.L. Holen I. Coleman R.E. Cancer Treat. Rev. 2002; 28: 305-319Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Green J.R. Cancer. 2003; 97: 840-847Crossref PubMed Scopus (286) Google Scholar). Tumor progression and metastasis formation are critically dependent on tumor angiogenesis (15Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7531) Google Scholar). Anti-angiogenic treatments suppress tumor progression in animal models, and many anti-angiogenic substances are currently being tested in clinical trials for their therapeutic efficacy against human cancer (16Carmeliet P. Nat. Med. 2003; 9: 653-660Crossref PubMed Scopus (3505) Google Scholar). Recent evidence indicates that zoledronate possesses anti-angiogenic activities. Zoledronate was shown to inhibit serum-, fibroblast growth factor-2-, or vascular endothelial growth factor-stimulated proliferation and to modulate adhesion and migration of cultured endothelial cells (17Wood J. Bonjean K. Ruetz S. Bellahcene A. Devy L. Foidart J.M. Castronovo V. Green J.R. J. Pharmacol. Exp. Ther. 2002; 302: 1055-1061Crossref PubMed Scopus (722) Google Scholar). These effects were paralleled by a reduced vascular sprouting in the aortic ring and chicken chorioallantoic membrane (CAM) angiogenesis assays and by the suppression of fibroblast growth factor-2-induced angiogenesis in mice (17Wood J. Bonjean K. Ruetz S. Bellahcene A. Devy L. Foidart J.M. Castronovo V. Green J.R. J. Pharmacol. Exp. Ther. 2002; 302: 1055-1061Crossref PubMed Scopus (722) Google Scholar). Furthermore, zoledronate suppressed testosterone-stimulated vascular re-growth in the ventral prostate of castrated rats (18Fournier P. Boissier S. Filleur S. Guglielmi J. Cabon F. Colombel M. Clezardin P. Cancer Res. 2002; 62: 6538-6544PubMed Google Scholar). Osteoclast-mediated bone resorption and angiogenesis are critically dependent on integrin-mediated cell adhesion and signaling (19Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3104) Google Scholar, 20Varner J.A. Cheresh D.A. Curr. Opin. Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (468) Google Scholar, 21Rüegg C. Mariotti A. Cell. Mol. Life Sci. 2003; 60: 1135-1157Crossref PubMed Scopus (161) Google Scholar). Integrins are αβ heterodimeric cell surface complexes acting as the main receptors for extracellular matrix proteins with bi-directional signaling activity (21Rüegg C. Mariotti A. Cell. Mol. Life Sci. 2003; 60: 1135-1157Crossref PubMed Scopus (161) Google Scholar, 22Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). Ligand binding function is tightly regulated by cell signaling events, and in turn, ligated integrins activate multiple signaling pathways essential for cell migration, proliferation, and survival. Integrin αVβ3 is highly expressed in osteoclasts and is strongly up-regulated in angiogenic endothelial cells (23Teitelbaum S.L. J. Bone Miner. Metab. 2000; 18: 344-349Crossref PubMed Scopus (100) Google Scholar, 24Eliceiri B.P. Cheresh D.A. Cancer J. 2000; 6: 245-249PubMed Google Scholar). Pharmacological antagonists of αVβ3 efficiently inhibit bone resorption (25Hartman G.D. Duggan M.E. Expert Opin. Investig. Drugs. 2000; 9: 1281-1291Crossref PubMed Scopus (66) Google Scholar) and angiogenesis, including tumor angiogenesis, in many different animal models (21Rüegg C. Mariotti A. Cell. Mol. Life Sci. 2003; 60: 1135-1157Crossref PubMed Scopus (161) Google Scholar, 24Eliceiri B.P. Cheresh D.A. Cancer J. 2000; 6: 245-249PubMed Google Scholar). Recent results demonstrate, however, that developmental and tumor angiogenesis can proceed in the absence of αVβ3 integrin, thus demonstrating that additional integrins contribute to angiogenesis (26Reynolds L.E. Wyder L. Lively J.C. Taverna D. Robinson S.D. Huang X. Sheppard D. Hynes R.O. Hodivala-Dilke K.M. Nat. Med. 2002; 8: 27-34Crossref PubMed Scopus (556) Google Scholar, 27Hynes R.O. Hodivala-Dilke K.M. Thromb. Haemostasis. 1999; 82: 481-485Crossref PubMed Scopus (33) Google Scholar). We raised the hypothesis that bisphosphonates may exert their anti-angiogenic effects, at least in part, by interfering with vascular integrin function or signaling. To test this hypothesis, we exposed cultured human umbilical vein endothelial cells (HUVEC) 1The abbreviations used are: HUVEC, human umbilical vein endothelial cell(s); TNF, tumor necrosis factor; IFN, interferon; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; LIBS, ligand-induced binding site; PKB, protein kinase B; PARP, poly(ADP-ribose) polymerase; FAK, focal adhesion kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Z-, benzyloxycarbonyl; PCD, programmed cell death. to zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen containing bisphosphonate, and tested the effects on integrin-dependent cell adhesion and migration and on cell survival. Reagents and Antibodies—Bovine gelatin, human plasma fibronectin, and vitronectin, leupeptin, wortmannin, propidium iodide, sodium orthovanadate FITC-phalloidin, Hoechst 33258, and crystal violet were purchased from Sigma. Human recombinant tumor necrosis factor (TNF) (5 × 107 units/mg) and interferon (IFN)-γ (3 × 107 units/mg) were gifts of Dr. G. Adolf (Boehringer Ingelheim, Vienna, Austria). Zoledronate and clodronate were obtained from Novartis AG (Basel, Switzerland). ZVAD was purchased from Apotech (Epalinges, Switzerland). mAbs Lia1/2 (anti-β1 subunit), SAM-1 (anti-α5 subunit), and HUTS-21 (anti-β1 ligand-induced binding site (LIBS)) were from Beckman Coulter; mAb LM609 (anti-αVβ3 integrin) was from Chemicon (Temecula, CA); mAb LIBS-1 (anti-β3 LIBS) was kindly provided by Dr. M. Ginsberg, The Scripps Research Institute (La Jolla, CA); mAb 11C81 (anti-ICAM-1) was from R&D Systems Europe Ltd (Abington, UK). Anti-phospho-I-κB, anti-I-κB, anti-phospho-protein kinase B (PKB)/Akt (Ser-473), anti-PKB/Akt, and anti-PARP antibodies were purchased from Cell Signaling (Beverly, MA). Anti-phospho-focal adhesion kinase (FAK) (Tyr-397), -FAK, -phospho-c-Src (Tyr-418), and -c-Src antibodies were from BIOSOURCE International (Camarillo, CA). The anti-FAK (C20) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-paxillin (clone 349) and anti-caspase-3 (clone 19) mAbs were purchased from Transduction Laboratories (Basel, Switzerland). The FITC-conjugated goat anti-mouse Ig was purchased from Dako (Zug, Switzerland). The Cy3-conjugated goat anti-mouse antiserum was from Caltag (South San Francisco, CA), and the goat-anti-rat-HRP conjugated antiserum was from BIOSOURCE International. The plasmids mpPKB and wtPKB were kindly provided by Dr. B. Hemmings (FMI, Basel, Switzerland), whereas the AdΔNI-kB and AdLacZ vectors were from Dr. Ch. Esslinger (Ludwig Institute for Cancer Research, Epalinges, Switzerland). Cell Culture, Electroporation, and Adenoviral Infection—HUVEC were prepared and cultured as previously described (28Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (272) Google Scholar). For electroporation, sub-confluent HUVEC were collected and incubated on ice 5 min with 20 μg of the specific plasmid DNA in serum-free M199 medium and electroporated with a Gene Pulser (Bio-Rad). HUVEC were resuspended in complete medium and cultured for 36 h before use in the experiments. Electroporation efficiency (between 70 and 80%) was routinely assessed by FACS® analysis of enhanced green fluorescent protein expression from a co-transfected pEGFP-C1 plasmid (Clontech, La Jolla, CA). HUVEC infected with AdΔNI-kB or AdLacZ (multiplicity of infection 100) were used 24 h post-infection. Cell Adhesion Assays—Maxisorp II Nunc enzyme-linked immunosorbent assay plates (Roskilde, Denmark) were coated with 10 μg/ml fibronectin, vitronectin, or gelatin overnight at 4 °C and blocked with bovine serum albumin for 2 h at room temperature. Assays were done as previously described (28Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (272) Google Scholar). Briefly, untreated cells or cells treated for 24 h with clodronate, zoledronate, or EDTA (100 μm each) were collected and seeded in serum-free M199 medium at a concentration of 104 cells/well. After 1 h at 37 °C wells were gently washed with phosphate-buffered saline, and attached cells were fixed in 4% paraformaldehyde (Fluka Chemie, Buchs, Switzerland) and stained with 0.5% crystal violet. Absorbance of each well was read at 620 nm in a plate reader (Packard Spectra Count, Meriden, CT). Results are expressed as mean value of triplicate determinations ± S.D. Migration Assay—Confluent HUVEC monolayers were cultured in complete medium alone or in the presence of clodronate, zoledronate, or EDTA (100 μm). After 16 h, wells were washed with serum-free medium, and one wound per well was applied with a plastic tip. Cells were further cultured in serum free human endothelial basal growth medium (Invitrogen) in the absence or presence of clodronate, zoledronate, or EDTA (100 μm). Ten hours later, cultures were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Migration was assessed by taking pictures of three different regions of each wound and counting the number of cells migrated inside of the wound area. Results are expressed as the mean of the number of migrated cells/1000 μm2 wound ± S.D. Cell Viability Assays—HUVEC (104 cells/well) were plated on fibronectin-coated (10 μg/ml) Maxisorp II Nunc enzyme-linked immunosorbent assay plates in complete medium alone or in the presence of zoledronate, clodronate, or EDTA at the indicated concentrations. Eight hours later TNF (200 ng/ml or as otherwise indicated) or medium alone was added to the wells. Sixteen hours later viability was determined by MTT assay (29Yilmaz A. Bieler G. Spertini O. Lejeune F.J. Ruegg C. Int. J. Cancer. 1998; 77: 592-599Crossref PubMed Scopus (36) Google Scholar) or by staining of adherent cells with crystal violet. Stained cells were lysed in Me2SO (for MTT reading) or in 0.1 m sodium citrate, 50% ethanol (for crystal violet reading). Absorbance of each well was measured at 620 nm (MTT) or 570 nm (crystal violet) in a plate reader (Packard Spectra Count). Results are expressed as mean value of triplicate determinations ± S.D. Immunofluorescence—HUVEC were cultured in complete medium or in medium supplemented with clodronate, zoledronate, or EDTA (100 μm each) for 24 h and fixed in 4% parafomaldehyde for 10 min at room temperature. After permeabilization with 0.1% Triton X-100 (Sigma) cells were blocked in 1% bovine serum albumin, washed, and incubated for 1 h with an anti-paxillin mAb (5 μg/ml). After washing, cells were incubated with a Cy3-conjugated goat anti-mouse (8 μg/ml). To stain the actin fibers, permeabilized cells were incubated with phalloidin-FITC. Flow Cytometry—HUVEC were collected by trypsinization, washed, and incubated with anti-integrin or anti-ICAM-1 antibodies (1 μg/ml) for 1 h at 4 °C. After washing, cells were incubated with a FITC-labeled antiserum for 30 min at 4 °C. Samples were analyzed with a FACScan II® and Cell Quest® software (BD Biosciences). For annexin-V binding, HUVEC were harvested as above and sequentially incubated with FITC-conjugated annexin-V (1:40 dilutions) and propidium iodide (1 μg/ml) following the manufacturer's recommendations (Apotech). For detection of DNA degradation, control and treated adherent and non-adherent HUVEC were collected as above, resuspended in 70% ice-cold ethanol under vortex, and incubated for 2 h at –20 °C. Cells were recovered by centrifugation and resuspended in phosphate-buffered saline. 50 μg/ml RNase A (Roche Applied Science) was added, and samples were incubated at room temperature for 5 min before staining with propidium iodide (PI, 50 μg/ml) for 30 min at 37 °C. Stained cells were analyzed with a FACScan II® and Cell Quest® software. Western Blotting—Total cell lysates (40 μl/lane) were resolved by SDS-PAGE and blotted onto Immobilon-P membranes (Millipore, Volketswil, Switzerland). For caspase-3, PARP, phospho-PKB, phospho-FAK, phospho-c-Src, and phospho-IκB detection, membranes were sequentially incubated in 5% dry milk, primary antibodies (at recommended concentrations), and the appropriate horseradish peroxide-labeled secondary antibody (1 μg/ml). The ECL system was used for detection (Amersham Biosciences). To determine total FAK, PKB/Akt, and IκB, membranes were stripped 15 min at 50 °C in stripping buffer (62 mm Tris-HCl, 2% SDS, and 100 mm 2-β-mercaptoethanol) and re-probed with the appropriate antibodies. Caspase Activity Assay—106 cells were lysed in 70 μl of 0.2% Nonidet P-40, 20 mm Tris, pH 7.4, 150 mm NaCl, and 10% glycerol. 20 μl of cell lysate were transferred into a black microwell plate and mixed with 100 μl of substrate buffer (0.1% CHAPS, 2 mm MgCl2, 1 mm dithiothreitol, 5 mm EGTA, 150 mm NaCl, 10 mm Tris, pH 7.4) supplemented with 7 μl of a 5 mm Ac-DEVD-amidomethylcoumarin solution (Alexis Biochemicals, Basel, Switzerland). After a 30-min incubation at 30 °C in the dark, substrate conversion was determined in a multi-well fluorimeter (excitation 355 nm, emission 460 nm) (Packard Fluoro Count, Meriden, CT). Results are expressed as arbitrary units and represent the mean value of duplicate determinations ± S.D. Hoechst 33258 Staining—HUVEC were cultured for 16 h in complete medium alone or in medium supplemented with EDTA, clodronate, or zoledronate (100 μm each) with and without ZVAD (50 μm). Cells were fixed in 4% paraformaldehyde and 4% sucrose, permeabilized with 0.1% Triton X-100, phosphate-buffered saline, and then incubated with 2.5 μg/μl Hoechst 33258 in 0.1% Triton X-100, phosphate-buffered saline for 5 min. Stained cells were washed, and nuclei were viewed by epifluorescence microscopy (Axioskop, Carl Zeiss AG, Zürich) equipped with a CCD camera (Photonic Science Milham, UK) at 360 nm. Zoledronate Inhibits α V β 3-mediated HUVEC Adhesion— Bisphosphonates have been reported to decrease the adhesion of tumor cells to bone matrices (30Boissier S. Magnetto S. Frappart L. Cuzin B. Ebetino F.H. Delmas P.D. Clezardin P. Cancer Res. 1997; 57: 3890-3894PubMed Google Scholar, 31van der Pluijm G. Vloedgraven H. van Beek E. van der Wee-Pals L. Lowik C. Papapoulos S. J. Clin. Invest. 1996; 98: 698-705Crossref PubMed Scopus (298) Google Scholar) and to modulate endothelial cell adhesion to vitronectin (17Wood J. Bonjean K. Ruetz S. Bellahcene A. Devy L. Foidart J.M. Castronovo V. Green J.R. J. Pharmacol. Exp. Ther. 2002; 302: 1055-1061Crossref PubMed Scopus (722) Google Scholar). Whether bisphosphonates may differentially modulate adhesion mediated by different integrins was not reported. HUVEC adhesion to gelatin and vitronectin fully depends on integrin αVβ3, whereas HUVEC adhesion to fibronectin is predominantly mediated by integrin α5β1, with only a minor contribution from αVβ3 (Fig. 1A). To test whether bisphosphonates may selectively interfere with αVβ3-mediated adhesion, we cultured HUVEC for 24 h in medium alone or in medium supplemented with zoledronate, clodronate, or EDTA (100 μm each) and assessed their ability to attach to fibronectin, vitronectin, and gelatin in a short term adhesion assay. Because bisphosphonates chelate cations and integrin function is modulated by divalent cations (e.g. Ca2+, Mg2+, and Mn2+), EDTA treatment was included in this and the successive experiments to exclude nonspecific effects due to cation chelation. Zoledronate treatment inhibited adhesion to vitronectin and gelatin by 50 and 80%, respectively, but did not affect adhesion to fibronectin (Fig. 1B). Pretreatment with clodronate or EDTA (100 μm each) had no effect on HUVEC adhesion (Fig. 1B). Zoledronate Inhibits HUVEC Migration—To test for the effects of zelodronate and clodronate on cell migration, HUVEC were cultured until they reached confluence and then supplemented with EDTA, zelodronate, or clodronate for 16 h. A "scratch" wound was then made with a plastic tip, and 10 h later wells were fixed, and migration was quantified (32Mariotti A. Kedeshian P.A. Dans M. Curatola A.M. Gagnoux-Palacios L. Giancotti F.G. J. Cell Biol. 2001; 155: 447-458Crossref PubMed Scopus (36) Google Scholar). Zoledronate treatment suppressed cell migration by more than 90%, whereas treatment with EDTA or clodronate had no effect (Fig. 2). Zoledronate Does Not Affect Cell Surface Integrin Expression or Affinity—Decreased HUVEC adhesion and migration caused by zoledronate could be due to a reduced cell surface expression or a decreased affinity of integrins. To test for these possibilities, the total cell surface level of β1 and αVβ3 integrins and LIBS expression (33Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) on β1 and β3 subunits were determined. HUVEC cultured for 24 h in medium alone or in the presence of EDTA, clodronate, or zoledronate (100 μm each) were first stained with anti-αVβ3 or -β1 integrin mAbs and analyzed by flow cytometry. Exposure to zoledronate had no effect on β1 or αVβ3 integrin expression levels (Fig. 3A and data not shown). Thereafter, control or treated HUVEC were stained with mAb HUTS-21 and LIBS-1, which specifically bind to the high affinity conformation of β1 or β3 integrin subunits, respectively (34Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 35Frelinger A.d. Cohen I. Plow E.F. Smith M.A. Roberts J. Lam S.C. Ginsberg M.H. J. Biol. Chem. 1990; 265: 6346-6352Abstract Full Text PDF PubMed Google Scholar), to test whether zoledronate suppressed integrin affinity maturation. Zoledronate-treated HUVEC had β1 and β3 LIBS-staining profiles identical to control or clodronate-treated HUVEC (Fig. 3B and data not shown). MnCl2, which is used to promote integrin affinity maturation (36Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar), induced LIBS expression regardless of HUVEC pretreatment (Fig. 3B). MnCl2 did not modify total β1 and αVβ3 integrin levels (Fig. 3A). Taken together, these experiments demonstrated that zoledronate, but not clodronate, inhibit αVβ3-mediated HUVEC attachment to vitronectin and gelatin and block HUVEC migration without affecting αVβ3 and β1 integrin cell surface expression or affinity. Zoledronate Disrupts Focal Adhesions and Actin Stress Fibers—Stable integrin-dependent cell adhesion and efficient cell migration are critically dependent on integrin post-receptor events, including focal adhesion assembly and actin cytoskeleton rearrangement (37Webb D.J. Parsons J.T. Horwitz A.F. Nat. Cell Biol. 2002; 4: 97-100Crossref PubMed Scopus (610) Google Scholar). To test whether zoledronate interferes with these events, HUVEC were cultured for 24 h in medium alone or in the presence of EDTA, clodronate, or zoledronate and then stained for paxillin, to visualize focal adhesions, and for F-actin, to visualize the actin cytoskeleton. Untreated and EDTA- and clodronate-treated HUVEC showed the typical focal paxillin staining and the fibrillar actin cytoskeleton, consistent with focal adhesion assembly and actin stress fiber formation. In contrast, in zoledronate-treated HUVEC the focal paxillin staining was completely lost, and the F-actin cytoskeleton was nearly completely dissolved (Fig. 4A). Zoledronate Treatment Inhibits Phosphorylation of Focal Adhesion Kinase—FAK is a critical regulator of focal adhesion formation, maintenance, and turnover (38Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (566) Google Scholar). FAK is activated by auto-phosphorylation in response to integrin ligation and by c-Src-mediated phosphorylation (39Abbi S. Guan J.L. Histol. Histopathol. 2002; 17: 1163-1171PubMed Google Scholar). To analyze the effect of zoledronate on FAK phosphorylation, HUVEC were cultured for 24 h in the absence or presence of EDTA, clodronate, or zoledronate (100 μm each), and FAK phosphorylation was analyzed by Western blotting using a phospho-FAK-specific antibody. Zoledronate, but not clodronate or EDTA treatment, efficiently suppressed FAK phosphorylation without altering total FAK protein levels (Fig. 4B). Phosphorylation of c-Src was not diminished by zoledronate, suggesting that decreased FAK phosphorylation was not due to suppressed c-Src activation (Fig. 4B). These experiments demonstrated that zoledronate disrupted focal adhesions and actin stress fibers in stably adherent cells and inhibited FAK phosphorylation, three events downstream of integrin ligation. Zoledronate Reduces HUVEC Viability and Enhances TNF Cytotoxic Activity—FAK-dependent signaling events promote cell survival and protect cells against death induced by stress, cytotoxic drugs, and TNF (40Kasahara T. Koguchi E. Funakoshi M. Aizu-Yokota E. Sonoda Y. Antioxid. Redox Signal. 2002; 4: 491-499Crossref PubMed Scopus (74) Google Scholar, 41Fang Y. Wang L. Jin J. Zha X. Eur. J. Biochem. 2001; 268: 4513-4519Crossref PubMed Scopus (15) Google Scholar, 42Chan P.C. Lai J.F. Cheng C.H. Tang M.J. Chiu C.C. Chen H.C. J. Biol. Chem. 1999; 274: 26901-26906Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To test whether zoledronate treatment had an effect on cell viability, HUVEC were cultured in medium alone or in the presence of bisphosphonates, and cell viability was determined after 24 h. Zoledronate treatment caused a dose-dependent decrease in HUVEC viability, with a 30% cell loss at 100 μm, whereas EDTA and clodronate had no effect (Fig. 5A). Zoledronate-treated cells had a retracted cytoplasm, an elongated shape with long protrusions at the ends (Fig. 5B). Because HUVEC are normally resistant to TNF-induced death (29Yilmaz A. Bieler G. Spertini O. Lejeune F.J. Ruegg C. Int. J. Cancer. 1998; 77: 592-599Crossref PubMed Scopus (36) Google Scholar, 43Ruegg C. Yilmaz A. Bieler G. Bamat J. Chaubert P. Lejeune F.J. Nat. Med. 1998; 4: 408-414Crossref PubMed Scopus (428) Google Scholar), we decided to test whether zoledronate may sensitize HUVEC to TNF cytotoxicity. HUVEC were cultured for 8 h in the absence or presence of bisphosphonates and then exposed to TNF at doses ranging from 0.02 ng/ml up to 200 ng/ml, and viability was determined 24 h later. Treatment with TNF alone had a minimal effect on viability. Zoledronate pretreatment, however, induced a dose-dependent decrease in viability of cells exposed to TNF (Fig. 5C). Zoledronate/TNF-treated cells showed a severely altered morphology, with many cells rounding up and retractin
Referência(s)