AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells. Interference with the Akt/NF-kB survival pathway

8 páginas, 5 figuras -- PAGS nros. 572-579Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 M. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and BisI inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-B activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-B-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancyThis work was supported by grants 08.3/0030.1/2003 from the Comunidad Autónoma de Madrid, SAF2003-00824 from the Ministerio de Ciencia y Tecnología (MCyT), and 01/1183 from Fondo de Investigación Sanitaria (to AGP); and CIDEM Grant 301888 (Generalitat de Catalunya)/Fundació Bosch i Gimpera, to RPT). E Escobar and E López-Martín were supported by fellowships from MCyTPeer reviewe

A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4β1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells

13 páginas, 7 figuras, 2 tablas -- PAGS nros. 27601-27613We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC50 values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesisThis work was supported by Grants SAF2009–07035 and RTICC RD06/0020/0011 (to A. G.-P.) and RTICC RD06/0020/0080 (to M. J. T.) from the Ministerio de Ciencia e Innovación, Spain, and by a grant from the Fundación Puerta de Hierro (to J. A. G. M.)Peer reviewe

Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration

4 páginas, 2 figuras -- PAGS nros. 383-386B-cell chronic lymphocytic leukemia (B-CLL) progression is frequently accompanied by clinical lymphadenopathy, and the CCL21 chemokine may play an important role in this process. Indeed, CCR7 (the CCL21 receptor), as well as matrix metalloproteinase-9 (MMP-9), are overexpressed in infiltrating B-CLL cells. We have studied whether MMP-9 is regulated by CCL21 and participates in CCL21-dependent migration. CCL21 significantly increased B-CLL MMP-9 production, measured by gelatin zymography. This was inhibited by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) activity or by cell transfection with CCR7-siRNA. Accordingly, CCL21/CCR7 interaction activated the ERK1/2/c-Fos pathway and increased MMP-9 mRNA. CCL21-driven B-CLL cell migration through Matrigel or human umbilical vein endothelial cells (HUVEC) was blocked by anti-CCR7 antibodies, CCR7-siRNA transfection, or the ERK1/2 inhibitor U0126, as well as by anti-MMP-9 antibodies or tissue inhibitor of metalloproteinase 1 (TIMP-1). These results strongly suggest that MMP-9 is involved in B-CLL nodal infiltration and expand the roles of MMP-9 and CCR7 in B-CLL progression. Both molecules could thus constitute therapeutic targets for this diseaseThis work was supported by grants PI060400 (to A.G.P.) and PI061637 (to M.J.T.) from the Ministerio de Sanidad y Consumo, and by the Fundación de Investigación Médica Mutua Madrileña (FMM; to A.G.P.). J.R.M. was supported by FMM and the Fundación Ramón ArecesPeer reviewe

The heparin III-binding domain of fibronectin (III4-5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly

10 páginas, 8 figuras -- PAGS nros. 642-651Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4–5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4–5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4–5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4–5. These interactions were specific since FNIII4–5 did not bind to the FNIII7–10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4–5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4–5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated α4β1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135–142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4–5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimerThis work was supported by Grants SAF2003-00824, from the Ministerio de Educación y Ciencia; PI060400, from the Instituto de Salud Carlos III, Madrid, Spain; and by a grant from the Fundación de Investigación Médica Mutua Madrileña (Madrid, Spain) (to A. García-Pardo), as well as by the National Institutes of Health grant EY012515 (to D.M. Peters). A. Maqueda was supported by an I3P predoctoral fellowship from CSIC/European Union Social Fund; J.V. Moyano was supported by a fellowship from GlaxoSmithKlinePeer reviewe

VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase–9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration

Brief Report. Artículo breve.B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase–9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.Ministerio de Ciencia e Innovación, Spain (grants PI060400, SAF2009-07035, and RTICC RD06/0020/0011, A.G.-P.; and PI061637 and RTICC RD06/0020/0080, M.J.T.) and by the Fundación de Investigación Médica Mutua Madrileña (A.G.-P.). J.R.-M. was supported by the Fundación Ramón Areces. P.E. was supported by the Ministerio de Ciencia e Innovación.Peer reviewe

AT514, a cyclic depsipeptide from Serratia marcescens, induces apoptosis of B-chronic lymphocytic leukemia cells. Interference with the Akt/NF-kB survival pathway

8 páginas, 5 figuras -- PAGS nros. 572-579Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 M. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and BisI inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-B activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-B-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancyThis work was supported by grants 08.3/0030.1/2003 from the Comunidad Autónoma de Madrid, SAF2003-00824 from the Ministerio de Ciencia y Tecnología (MCyT), and 01/1183 from Fondo de Investigación Sanitaria (to AGP); and CIDEM Grant 301888 (Generalitat de Catalunya)/Fundació Bosch i Gimpera, to RPT). E Escobar and E López-Martín were supported by fellowships from MCyTPeer reviewe

Induction of B-chronic lymphocytic leukemia cell apoptosis by arsenic trioxide involves suppression of the phosphoinositide 3-kinase/Akt survival pathway via c-jun-N-terminal-kinase activation and PTEN upregulation

10 páginas, 5 figuras -- PAGS nros. 4382-4391Purpose: Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors. Experimental Design: B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses. Results: After 48 hours, 3 μmol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-κB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased the apoptotic effect of either agent alone. ATO also induced c-jun-NH2 terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-κB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production. Conclusions: We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapiesGrants PI060400, RD06/0020/0011, and SAF2009-07035 (A. García-Pardo), RD06/0020/0041 (A. Pandiella), and PI061637 and RD06/0020/0080 (M.J. Terol) from the Ministerio de Ciencia e Innovación, and grant from the Fundación de Investigación Médica Mutua Madrileña (A. García-Pardo). JRM was supported by a fellowship from the Fundación Ramón Areces, Madrid, SpainPeer reviewe

Matrix metalloproteinase-9 promotes chronic lymphocytic leukemia b cell survival through its hemopexin domain

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors alpha4beta1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesis.status: publishe

Matrix Metalloproteinase-9 Promotes Chronic Lymphocytic Leukemia B Cell Survival through Its Hemopexin Domain

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors α4β1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesisThis work was supported by grants PI060400, SAF2009-07035, and RTICC RD06/0020/0011 (to A.G.P.), by grants PI061637 and RTICC RD06/0020/0080 (to M.J.T.) from the Ministerio de Ciencia e Innovacio´ n, and by the Fundacio´ n de Investigacio´nMe´ dica Mutua Madrilen ˜ a (to A.G.P.). Research on the human proMMP-9 mutants was supported by the Geconcerteerde OnderzoeksActies (GOA 2007-2011) and the Fund for Scientific Research-Flanders (FWO-Vlaanderen). Research on the murine proMMP-9-PEX domain was supported by grants Ro 957/6-1 and Ro 957/7-1 from the Deutsche Forschungsgemeinschaft. J.R.M. was supported by the Fundacio´ n Ramo´ n Areces. P.E.V.D.S. is a postdoctoral fellow of the FWOVlaanderen.Peer reviewe