Additive effect of PTK787/ZK 222584, a potent inhibitor of VEGFR phosphorylation, with Idarubicin in the treatment of acute myeloid leukemia
2009; Elsevier BV; Volume: 37; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2009.03.001
ISSN1873-2399
AutoresNuria Barbarroja, Luis Arístides Torres, M. Luque, R.M. Carretero, Araceli Valverde-Estepa, Laura M. López‐Sánchez, Antonio Rodríguez‐Ariza, Francisco Velasco, António Torres, C. López-Pedrera,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoAcute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express vascular endothelial growth factor (VEGF) as well as Flt-1 and KDR, resulting in an autocrine pathway for cell survival. PTK787/ZK 222584 is a new oral antiangiogenic molecule that inhibits tyrosine kinase activity of all known VEGF receptors. The present study aimed to investigate the therapeutic efficacy of combining PTK787/ZK 222584 with a chemotherapeutic agent, such as Idarubicin, for treatment of AML. We have analyzed in four AML cell lines and seven AML patient samples, cell proliferation, apoptosis, angiogenesis. and activation of several related intracellular pathways after treatment with PTK787/ZK 222584 alone or combined with Idarubicin. PTK787/ZK 222584 decreased VEGF levels and VEGF receptor phosphorylation in the AML cells showing Fms-like tyrosine kinase 3/internal tandem duplication mutation (Flt3/ITD). Both drugs, given separately, inhibited cell proliferation and promoted apoptosis. Moreover, combined treatment promoted more apoptosis and inhibition of cell proliferation than each compound administered separately in all AML cells. In conclusion, PTK787/ZK 222584 combined with Idarubicin achieved a better therapeutic efficacy than chemotherapy alone in AML cells, especially in those with Flt3/ITD, in which the combination further prevented activation of the angiogenic process. Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express vascular endothelial growth factor (VEGF) as well as Flt-1 and KDR, resulting in an autocrine pathway for cell survival. PTK787/ZK 222584 is a new oral antiangiogenic molecule that inhibits tyrosine kinase activity of all known VEGF receptors. The present study aimed to investigate the therapeutic efficacy of combining PTK787/ZK 222584 with a chemotherapeutic agent, such as Idarubicin, for treatment of AML. We have analyzed in four AML cell lines and seven AML patient samples, cell proliferation, apoptosis, angiogenesis. and activation of several related intracellular pathways after treatment with PTK787/ZK 222584 alone or combined with Idarubicin. PTK787/ZK 222584 decreased VEGF levels and VEGF receptor phosphorylation in the AML cells showing Fms-like tyrosine kinase 3/internal tandem duplication mutation (Flt3/ITD). Both drugs, given separately, inhibited cell proliferation and promoted apoptosis. Moreover, combined treatment promoted more apoptosis and inhibition of cell proliferation than each compound administered separately in all AML cells. In conclusion, PTK787/ZK 222584 combined with Idarubicin achieved a better therapeutic efficacy than chemotherapy alone in AML cells, especially in those with Flt3/ITD, in which the combination further prevented activation of the angiogenic process. Angiogenesis plays a crucial role in facilitating tumor growth and the metastatic process. Although this process first seemed to be related to solid tumors, there is emerging evidence that angiogenesis also contributes to pathogenesis of hematological malignancies. Several reports confirmed that patients with acute myeloid leukemia (AML) have increased bone marrow vascularization. In most cases, leukemia progression acute phase appears to be accompanied by an increase in bone marrow microvessel density and elevated local and circulating proangiogenic growth factors [1Padro T. Ruiz S. Bieker R. et al.Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia.Blood. 2000; 96: 2637-2644Google Scholar, 2Hussong J.W. Rodgers G.M. Shami P.J. Evidence of increased angiogenesis in patients with acute myeloid leukemia.Blood. 2000; 95: 309-313Crossref PubMed Google Scholar, 3de Bont E.S. Rosati S. Jacobs S. Kamps W.A. Vellenga E. Increased bone marrow vascularization in patients with acute myeloid leukaemia: a possible role for vascular endothelial growth factor.Br J Haematol. 2001; 113: 296-304Crossref PubMed Scopus (100) Google Scholar]. Among the angiogenic regulators, vascular endothelial growth factor (VEGF) is one of the major proangiogenic molecules involved in the angiogenic process. Because of that, the VEGF/VEGF receptor (VEGFR) pathways have been intensively studied in basic and clinical cancer research [4Hicklin D.J. Ellis L.M. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.J Clin Oncol. 2005; 23: 1011-1027Crossref PubMed Scopus (2393) Google Scholar]. It has been demonstrated that circulating VEGF levels are significantly increased in approximately 90% of AML patients, and appear to accompany disease progression. Moreover, high levels of plasma/serum VEGF correlated with increased number of circulating blasts and the decrease of complete remission achievements and reduced survival in AML patients untreated [5Aguayo A. Estey E. Kantarjian H. et al.Cellular vascular endothelial growth factor is a predictor of outcome in patients with acute myeloid leukemia.Blood. 1999; 94: 3717-3721PubMed Google Scholar]. Leukemic blasts may promote their own development via both paracrine and autocrine interactions, through expression of VEGF and its tyrosine kinase receptors: VEGFR1/Flt-1 and VEGFR2/KDR [6Fragoso R. Elias A.P. Dias S. Autocrine VEGF loops, signaling pathways, and acute leukemia regulation.Leuk Lymphoma. 2007; 48: 481-488Crossref PubMed Scopus (24) Google Scholar]. Inhibition of both paracrine and autocrine VEGF/KDR pathways is necessary to induce long-term remission in mice bearing acute leukemia [7Dias S. Hattori K. Heissig B. et al.Inhibition of both paracrine and autocrine VEGF/VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias.Proc Natl Acad Sci U S A. 2001; 98: 10857-10862Crossref PubMed Scopus (251) Google Scholar]. Moreover, it has been shown that in bone marrow, expression of VEGFR2 is restored to normal expression levels in patients with complete remission after chemotherapy [8Padro T. Bieker R. Ruiz S. et al.Overexpression of vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia.Leukemia. 2002; 16: 1302-1310Crossref PubMed Scopus (166) Google Scholar]. In response to VEGF stimulation, KDR and Flt-1 have been shown to transmit intracellular signals leading to cell proliferation and survival. The signaling pathways activated by VEGF, downstream of its receptors and linked to these particular functions, include the mitogen-activated protein kinase, phosphatidylinositol 3 kinase, and signal transducer activator of transcription (STAT) cascades [9Weber-Nordt R.M. Mertelsmann R. Finke J. The JAK-STAT pathway: signal transduction involved in proliferation, differentiation and transformation.Leuk Lymphoma. 1998; 28: 459-467PubMed Google Scholar, 10Grandage V.L. Gale R.E. Linch D.C. Kchwaja A. PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-kappaB. Mapkinase and p53 pathways.Leukemia. 2005; 19: 586-594Crossref PubMed Scopus (243) Google Scholar, 11Lewis T.S. Shapiro P.S. Ahn N.G. Signal transduction through MAP kinase cascades.Adv Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar]. In addition, in AML cells, these pathways are also activated by overexpression and/or mutations of others tyrosine kinase receptors, such as Fms-like tyrosine kinase 3 (Flt3). Flt3 is involved in disease progression of AML and the presence of AML blasts with high expression of the wild-type Flt3 messenger RNA (mRNA) is associated with lower overall survival [12Kainz B. Fonatsch C. Schwarzinger I. Sperr W.R. Jäger U. Gaiger A. Limited value of FLT3 mRNA expression in the bone marrow for prognosis and monitoring of patients with acute myeloid leukemia.Haematologica. 2005; 90: 695-696PubMed Google Scholar]. Furthermore, internal tandem duplications (ITDs) in the juxtamembrane domain of Flt3 are the most common molecular defects identified in AML and have been shown to be associated with an even worse prognosis [13Kottaridis P.D. Gale R.E. Frew M.E. et al.The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials.Blood. 2001; 98: 1752-1759Crossref PubMed Scopus (1232) Google Scholar, 14Thiede C. Steudel C. Mohr B. et al.Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis.Blood. 2002; 99: 4326-4335Crossref PubMed Scopus (1378) Google Scholar, 15Schnittger S. Schoch C. Dugas M. et al.Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease.Blood. 2002; 100: 59-66Crossref PubMed Scopus (820) Google Scholar]. The well-established role of both VEGFR and Flt3 in promoting tumor growth and pathogenesis of hematological malignancies has led to the rational design and development of agents that selectively target this pathway. Several small molecule tyrosine kinase inhibitors that target the tyrosine kinase receptors are already being studied in clinical trials. These include ZD6474, a VEGFR2 inhibitor, SU11248, a small molecule that potently inhibits platelet-derived growth factor receptors (PDGFR), VEGFR1, VEGFR2, c-KIT, and Flt3 and PTK787/ZK 222584, which inhibits VEGFR1 and VEGFR2 [16O'Farrell A.M. Yuen H.A. Smolich B. et al.Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia.Leukemia Res. 2004; 28: 679-689Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. PTK787/ZK 222584 (PTK/ZK) is an oral angiogenesis inhibitor targeting VEGFR tyrosine kinases, including VEGFR1/Flt-1, VEGFR2/KDR, VEGFR3/Flt-4, PDGFR, and c-KIT [17Wood J.M. Bold G. Buchdunger E. et al.PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.Cancer Res. 2000; 60: 2178-2189PubMed Google Scholar, 18Drevs J. Muller-Driver R. Wittig C. et al.PTK787/ZK 222584, a specific vascular endothelial growth factor-receptor tyrosine kinase inhibitor, affects the anatomy of the tumor vascular bed and the functional vascular properties as detected by dynamic enhanced magnetic resonance imaging.Cancer Res. 2002; 62: 4015-4022PubMed Google Scholar]. It has no known effects against other tyrosine or serine/threonine kinases. PTK/ZK is currently being investigated in patients with different solid tumor types for its therapeutic utility [17Wood J.M. Bold G. Buchdunger E. et al.PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.Cancer Res. 2000; 60: 2178-2189PubMed Google Scholar, 18Drevs J. Muller-Driver R. Wittig C. et al.PTK787/ZK 222584, a specific vascular endothelial growth factor-receptor tyrosine kinase inhibitor, affects the anatomy of the tumor vascular bed and the functional vascular properties as detected by dynamic enhanced magnetic resonance imaging.Cancer Res. 2002; 62: 4015-4022PubMed Google Scholar, 19Hess-Stumpp H. Haberey M. Thierauch K.H. PTK 787/ZK 222584, a tyrosine kinase inhibitor of all known VEGF receptors, represses tumor growth with high efficacy.Chembiochem. 2005; 6: 550-557Crossref PubMed Scopus (65) Google Scholar]. In those preclinical models, PTK/ZK caused a significant decrease in vessel permeability, reduction in tumor vessel density and repression of tumor growth with high efficacy. Different clinical studies with inhibitors of VEGFR signaling, namely tyrosine kinase inhibitors, have also shown promising results in treatment of AML patients [20Giles F.J. Stopeck A.T. Silverman L.R. et al.SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplastic syndromes.Blood. 2003; 102: 795-801Crossref PubMed Scopus (180) Google Scholar, 21Fiedler W. Mesters R. Tinnefeld H. et al.A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia.Blood. 2003; 102: 2763-2767Crossref PubMed Scopus (239) Google Scholar]. However, to date, only two studies have analyzed the biological activity of PTK/ZK in AML [22Roboz G.J. Giles F.J. List A.F. et al.Phase I study of PTK787/ZK 222584, a small molecule tyrosine kinase receptor inhibitor, for the treatment of acute myeloid leukemia and myelodysplastic syndrome.Leukemia. 2006; 6: 952-957Crossref Scopus (75) Google Scholar, 23Weidenaar A.C. de Jonge H.J. Fidler V. et al.Addition of PTK787/ZK 222584 can lower the dosage of amsacrine to achieve equal amounts of acute myeloid leukemia cell death.Anticancer Drugs. 2008; 19: 45-54Crossref PubMed Scopus (10) Google Scholar]. Thus, the aim of the present study was to gain more insight into the effects of PTK/ZK on both growth and angiogenic process in AML cells with or without Flt3/ITD mutation. We and others have previously reported a constitutive and simultaneous activation of the main signaling pathways (extracellular signal-regulated kinase [ERK], Akt, and STAT) involved in survival and differentiation of AML [24Srinivasa S.P. Doshi P.D. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways cooperate in mediating cytokine-induced proliferation of a leukemic cell line.Leukemia. 2002; 16: 244-253Crossref PubMed Scopus (45) Google Scholar, 25Xu Q. Simpson S.E. Scialla T.J. Bagg A. Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation.Blood. 2003; 102: 972-980Crossref PubMed Scopus (421) Google Scholar, 26Kubota Y. Ohnishi H. Kitanaka A. Ishida T. Tanaka T. Constitutive activation of PI3K is involved in the spontaneous proliferation of primary acute myeloid leukemia cells: direct evidence of PI3K activation.Leukemia. 2004; 18: 1438-1440Crossref PubMed Scopus (64) Google Scholar, 27Xia Z. Baer M.R. Block A.W. Baumann H. Wetzler M. Expression of signal transducers and activators of transcription proteins in acute myeloid leukemia blasts.Cancer Res. 1998; 58: 3173-3180PubMed Google Scholar, 28Barbarroja N. Torres L.A. Hernandez V. et al.Coordinated deregulation of cellular receptors, proangiogenic factors and intracellular pathways in acute myeloid leukaemia.Leuk Lymphoma. 2007; 48: 1187-1199Crossref PubMed Scopus (13) Google Scholar, 29Siendones E. Barbarroja N. Torres L.A. et al.Inhibition of Flt3-activating mutations does not prevent constitutive activation of ERK/Akt/STAT pathways in some AML cells: a possible cause for the limited effectiveness of monotherapy with small-molecule inhibitors.Hematol Oncol. 2007; 25: 30-37Crossref PubMed Scopus (32) Google Scholar]. Thus, if multiple independent pathways are abnormally expressed in AML cells, reversion of leukemogenesis might be enhanced through the simultaneous inhibition of different cellular targets. Moreover, an increasing number of studies have suggested that tyrosine kinase inhibitors need to be combined with other antileukemic treatments to obtain the best clinical results. Theoretically, it would be advantageous to add chemotherapy agents that act synergistically with tyrosine kinase inhibitors for antileukemic effects. Thus, we also sought to profile the activity of PTK787/ZK in combination with a traditional antileukemic agent, Idarubicin. In this study, we show that PTK/ZK monotherapy has in vitro proapoptotic and antiangiogenic effects against four AML cell lines and primary AML blasts, causing greater effect in AML cell lines harboring the Flt3/ITD mutation. Moreover, the combined strategy with Idarubicin achieved a better therapeutic efficacy than the chemotherapeutic agent given alone in AML cells, especially in those showing Flt3/ITD mutations, in which the angiogenic process was further abrogated. The cell lines NB4 (Flt3 WT/WT), THP-1 (Flt3 WT/WT), MOLM-13 (Flt3 ITD/WT), and MV4-11 (Flt3 ITD/–) were obtained from DMSZ (Braunschweig, Germany). They were cultivated on fresh RPMI-1640 medium (Cambrex Bio Science, Verviers, Belgium) supplemented with 10% fetal bovine serum, 100 U penicillin/mL, and 100 μg streptomycin/mL, in a humidified atmosphere of 5% CO2. AML blasts cells were obtained from bone marrow aspirates after informed consent, according to ethical standards of the institutional guidelines. In all analyzed samples, the percentage of leukemic infiltration exceeded 80%. Leukemia cells were isolated on Ficoll-Hypaque density gradient centrifugation. Cell number was determined by trypan blue dye exclusion. AML blasts were cultured in complete RPMI-1640 medium. Details of patients examined in the study are indicated in Table 1. All AML samples presented Flt3 wild-type.Table 1Clinical details of acute myeloid leukemia patientsNo.GenderAge (y)FABBlasts (%)KaryotypeWBC (×109/l)Flt-1 (%)KDR (%)1M43M09245, XY, 766.12529.52F72M18847, XX, +618.339.16273M71M189NA27.125.523.64M46M29045, XY, del(5), -7, +815.524.934.55F69M39346, XX, t(15;17)3028.241.046F54M48546, XX, t(1;11)(p32;q23)10.72856.27M63M59547, XY, +11, del (12)/4620.522.163.7FAB=acute myeloid leukemia subtype, according to the classification of the French-American-British committee; WBC=white blood count. Open table in a new tab FAB=acute myeloid leukemia subtype, according to the classification of the French-American-British committee; WBC=white blood count. Cell lines and AML blast cells were seeded at 5×105/mL in RPMI-1640 complete medium for 24 and 48 hours and treated with different doses of PTK/ZK (a kind gift from the Novartis Pharmaceutic, Basel, Switzerland) or Idarubicin alone or combined. PTK/ZK was added 30 minutes prior to the addition of Idarubicin. Controls were performed with vehicles of PTK/ZK solvent. All experiments were carried out in duplicate and repeated at least three times. Cells washed twice in phosphate-buffered saline were lysated on ice for 10 minutes in NP-40 lysis buffer, containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM Na2−ethylene glycol tetraacetic acid (EGTA), 0.1 mM Na2−ethylenediamine tetraacetic acid, 1 mM sodium orthovanadate, 1% NP-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 μg/mL aprotinin. Samples were pelleted by centrifugation at 15,000g for 1 minute at 4°C. The supernatant (cytoplasmic lysates) was recovered and frozen at −80°C. Pellets were incubated on ice for 15 minutes in a lysis buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1 mM EGTA, 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 25 μg/mL aprotinin. Samples were centrifuged at 14,000g for 5 minutes at 4°C. Supernatant (nuclear proteins) was recovered and frozen at −80°C. Protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Whole-cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblottings were incubated with the following antibodies: human anti-pSTAT5 a/b, anti−phospho-ERK 1/2, anti−phospho-Akt. The immunoblots were reprobed with human anti-STAT5, anti−ERK 1/2, anti-Akt, and anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Nuclear cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis 7% and immunoblottings were incubated with anti−phospho-VEGFR2 monoclonal antibody, then the immunoblots were reprobed with human anti-TFII-B (Santa Cruz Biotechnology). Immunocomplexes were detected with appropriate horseradish peroxidase−conjugated secondary antibodies and detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK). Cytoplasmic cell lysates (1 mg) were incubated with 2 μg antibodies to human anti-VEGFR1 (Santa Cruz Biotechnology) for 4 hours at 4°C, followed by incubation with 20 μL protein A-Agarose overnight at 4°C with continuous mixing. After centrifugation at 12,000g during 5 minutes, the immunoprecipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis 7% and immunoblottings were incubated with human anti−phospho-Tyr antibody (Santa Cruz Biotechnology). Immunocomplexes were detected with the appropriate horseradish peroxidase−conjugated secondary antibody and detected by enhanced chemiluminescence (GE Healthcare). Viability was assessed by using an Annexin-V staining kit (Bender MedSystems, Vienna, Austria), according to manufacturer's recommendations. Briefly, cells were harvested, washed in PBS, and finally dissolved in 1×195 μL binding buffer and 5 μL Annexin-V, mixed and incubated in darkness for 10 minutes at room temperature. Cells were then washed with PBS and dissolved in 190 μL in 1× binding buffer and 10 μL (20 μg/mL) propidium iodide. Subsequently, binding of fluorescein-conjugated Annexin-V and propidium iodide was measured by fluorescein-activated cell sorting (BD, Franklin Lakes, NJ, USA). Total RNA was extracted by Tri-Reagent (Sigma, St Louis, MO, USA), according to manufacturer's recommendations. RNA concentration was determined spectrophotometrically at 260 and 280 nm and its integrity was verified following separation by electrophoresis into a 0.8% agarose gel containing ethidium bromide. RNA samples were stored at −80°C until use. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on one-step using the QuantiTect SYBR Green RT-PCR kit (QIAGEN GmbH, Hilden, Germany) following manufacturer's protocol. Expression levels of VEGF gene and glyceraldehyde phosphate dehydrogenase (GAPDH) as housekeeping gene, were measured by quantitative real-time RT-PCR using the LightCycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA). Sequence of primers used for study, theoretic size of PCR products, and efficiency were: VEGF: (470 bp), forward primer: 5′-CGAAGTGGTGAAGTTCATGGATG 3′; reverse primer: 5′-TTCTGTATCAGTCTTTCCTGGTGA-3′, efficiency, 1.89; GAPDH: (240 bp), forward primer: 5′-TGATGACATCAAGAAGGTGGTGAAG-3′; reverse primer: 5′-TCCTTGGAGGCCATGTAGGCCAT-3′, efficiency, 1.9. Quantification of relative expression was determined by standard curve method according to manufacturer's instructions. Target amount was normalized to GAPDH gene and relative expression was calculated by ratio of normalized target values and calibrator normalized target values. Cell viability was assessed using an XTT colorimetric assay (Roche Applied Science, Indianapolis, IN, USA). Cells were seeded on 96-well plates at a concentration of 25,000 cells/well. After treatment with PTK/ZK and Idarubicin at indicated times and doses, the XTT assay was performed following the protocol supplied by the manufacturer. In brief, 50 μL XTT reagent were added to each well and plates were incubated at 37°C for 4 hours. Plates were then analyzed at a wavelength of 450 nm using a GENios Reader (TECAN, Salzburg, Austria). VEGF enzyme-linked immunosorbent assay were performed on culture supernatants at 48 hours of culture. Cultures were centrifuged at 2000g for 5 minutes. Supernatant was separated, aliquoted, and stored at −80°C until assay. VEGF concentrations were determined using a commercially available enzyme-linked immunosorbent assay designed to measure VEGF levels (Quantikine; R&D Systems Europe, UK). The assay employs the quantitative sandwich enzyme immunoassay technique using Sf21-expressed recombinant human VEGF165 and antibodies raised against the recombinant protein. Optical density was measured at 450 nm, with the correction wavelength set at 540 nm, using a microtiter plate reader. All experiments were assayed in triplicate. Flt3 phosphorylation, VEGFR1/Flt-1, and VEGFR2/KDR expression were determined by flow cytometry. To analyze Flt3 phosphorylation, cells were centrifuged and resuspended in PBS containing 2% paraformaldehyde to fix for 10 minutes. Cells were permeabilized with 90% cold methanol for 30 minutes and resuspended in PBS containing 1% bovine serum albumin. The cells were incubated with specific monoclonal antibodies to human phospho-Flt3 (Tyr591) Alexa Fluor-conjugated (Cell Signalling Technology Inc, Beverly, MA, USA) for 1 hour at 4°C. To asses the VEGFR1/Flt-1 and VEGFR2/KDR expression, cells were incubated with specific monoclonal antibodies to human Flt-1 and KDR (R&D Systems) for 20 minutes at 4°C. After washing with PBS, the phosphorylation of Flt3 and VEGFR expression were analyzed on a FACScan (BD). All data are expressed as mean ± standard error of mean. Statistical analyses were carried out with the SigmaStat software package (Systat, Point Richmond, CA, USA). Before comparing two data groups, a normality test and an equal variance test were performed. If data groups passed both tests, a comparison was made by a parametric approach (paired Student's t-test). If the normality and/or equal variance test was violated, a comparison was made by a nonparametric method (Mann-Whitney rank sum test). Differences were considered statistically significant at p<0.05. We investigated the effects of inhibition of the VEGF kinase domain containing receptors by PTK/ZK in four AML cell lines in the induction of apoptosis and the inhibition of cell proliferation. PTK/ZK-induced apoptosis in a dose- and time-dependent manner and inhibited cell proliferation in a dose-dependent way in the four AML cell lines. Effects were significant at 48 hours and 40 μM concentration in all cell lines and stronger in MOLM-13 and MV4-11cell lines (Fig. 1). Cytometry analysis showed that the four cell lines expressed high levels of VEGFR: Flt-1: NB4 (85% ± 5%), THP-1 (92% ± 6.5%), MOLM-13 (25.5% ± 3.6%), MV4-11 (61.5% ± 4.5%). KDR: NB4 (70% ± 7.2%), THP-1 (60.5% ± 5.4%), MOLM-13 (45.8% ± 5.5%), MV4-11 (54.5% ± 3.5%). The treatment with different doses of PTK/ZK (2.5−40 μM) showed no change in VEGFR expression levels seen in AML cell lines (data not shown). PTK/ZK did not inhibit the levels of VEGF mRNA and protein in NB4 and THP-1 cells (data not shown). However, PTK/ZK decreased those levels in a dose-dependent way in MOLM-13 and MV4-11 cells (Fig. 2). When VEGF supernatant levels were normalized for viable cell number, the inhibitory effect was still significant, suggesting that the reduced VEGF levels are not a reflection of decreased cell number in the presence of PTK/ZK (Fig. 2A). On the other hand, complete inhibition of the expression of VEGF protein was seen in these two cell lines after 40 μM PTK/ZK treatment (Fig. 2B). We analyzed activation of the VEGF receptors after treatment with PTK/ZK (5−40 μM) in the four cell lines. Western blot results showed that PTK/ZK inhibited Flt-1 phosphorylation and the nuclear phosphorylated KDR form (KDR internalization requires phosphorylation of the receptor) from a dose of 5 μM in MOLM-13 and MV4-11 cells. The effects of PTK/ZK on inhibition of the VEGFR phosphorylation were slight at 40 μM in NB4 and THP-1 cells (Fig. 3A ). This fact might be correlated to the modest activity of this inhibitor on both inhibition of cell proliferation and induction of apoptosis in these cells. To evaluate the significance of the VEGFR signal blockade in the activation of several intracellular pathways, we studied the phosphorylation of Akt, ERK, and STAT5 kinases. Parallel downregulation of Akt, ERK, and STAT5 kinases in a dose-dependent manner was observed in MOLM-13 and MV4-11 cells after treatment with 20 to 40 μM PTK/ZK. The stronger effects were seen after treatment with 40 μM. A low inhibition of Akt Ser phosphorylation was seen in THP-1 cells. In contrast, no changes were found in the constitutive activation of these kinases in NB4 cells (Fig. 3B). On the other hand, inhibition of Bcl-2 expression after PTK/ZK 40 μM treatment, as measured by densitometric analysis of Western blot (NB4: control 4582±411, PTK/ZK treatment 3830±255; THP-1: control 3981±403, PTK/ZK treatment 3078±190; MOLM-13: control 4525±216, PTK/ZK treatment 3252±205; MV4-11: 4100±199, 2740±256 AU of IOD; all p<0.05) (Fig. 3B) showed a parallel behavior with the apoptosis levels induced by such treatment (Fig. 1). PTK/ZK reduced the Bcl-2 expression in a different grade in the cell lines studied according to the percentage of apoptosis induced by the inhibitor. The effects of PTK/ZK in cell death and inhibition of intracellular pathways activation were stronger in the MOLM-13 and MV4-11 cells, which carried the Flt3/ITD mutation. These data suggested that this mutation could be responsible for the sensitivity of these cells to treatment with PTK/ZK. Therefore, we examined the Flt3 phosphorylation levels by flow cytometry after treatment with different doses of PTK/ZK (20−40 μM). Results showed that this inhibitor did not affect the Flt3 activation status seen in MOLM-13 and MV4-11 cells (62.5% ± 3.6% and 92.7% ± 4.5%, respectively), indicating that the mutation in Flt3 was not a direct target for PTK787/ZK in these cell lines. Anthracyclines are a class of chemotherapeutic agents commonly used in the treatment of AML [30Lowenberg B. Downing J.R. Burnett A. Acute myeloid leukemia.N Engl J Med. 1999; 341: 1051-1062Crossref PubMed Scopus (1216) Google Scholar]. Therefore, the next step was to test the addition of Idarubicin to the VEGFR inhibitor PTK787/ZK treatment of the AML cells, and to determine the cell death and inhibition of the cell proliferation percentage induced by both drugs. We first treated the AML cel
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