ABSTRACT
Defective apoptosis is a hallmark of the progression of B chronic lymphocytic leukaemia (B-CLL). Smac-mimetics have been shown to induce apoptosis in several tumours. We describe the in vitro pro-apoptotic activity and regulation of the molecular pathway induced by new Smac-mimetics in B-CLL. The cytotoxic effect was significantly higher in B-CLL samples than in healthy controls. No significant synergistic effect was observed in combined treatment. In conclusion one of our compounds (Smac66), used as monotherapy and not in combination, is highly active against B-CLL cells thus suggesting a promising therapeutic potential as a new class of antileukemic drugs in haematology.
Subject(s)
Apoptosis/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mitochondrial Proteins/metabolism , Oligopeptides/pharmacology , Peptidomimetics/pharmacology , Adult , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Case-Control Studies , Cells, Cultured , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , PrognosisABSTRACT
Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.
Subject(s)
Biomimetic Materials/chemical synthesis , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Oligopeptides/chemistry , Biomimetic Materials/chemistry , Dimerization , Inhibitor of Apoptosis Proteins/metabolismABSTRACT
Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.
Subject(s)
Biomimetic Materials/chemistry , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Oligopeptides/chemistry , Animals , Binding Sites , Biomimetic Materials/therapeutic use , Biomimetic Materials/toxicity , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Dimerization , Female , HL-60 Cells , Half-Life , Humans , Inhibitor of Apoptosis Proteins/metabolism , Mice , Mice, Nude , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Ovarian Neoplasms/drug therapy , Protein Structure, Tertiary , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
OBJECTIVE: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). METHODS: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. RESULTS: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. INTERPRETATION: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.
Subject(s)
Biomarkers/cerebrospinal fluid , Central Nervous System Diseases/cerebrospinal fluid , Central Nervous System Diseases/drug therapy , Inflammation/cerebrospinal fluid , Inflammation/drug therapy , Spinal Cord/metabolism , Animals , Blotting, Western , Calcium Signaling/physiology , Cell Communication , Cells, Cultured , Encephalitis/cerebrospinal fluid , Encephalitis/pathology , Flow Cytometry , Lentivirus/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Microscopy, Fluorescence , Multiple Sclerosis/pathology , Nervous System Autoimmune Disease, Experimental/cerebrospinal fluid , Nervous System Autoimmune Disease, Experimental/drug therapy , Neuroglia/metabolism , Neuroglia/physiology , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/physiologyABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34+ cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34+ cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.
Subject(s)
Antigens, CD34/biosynthesis , Environmental Pollutants/pharmacology , Gene Expression Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Hematopoietic System/drug effects , Polychlorinated Dibenzodioxins/pharmacology , Gene Expression Profiling/methods , Hematopoietic Stem Cells/physiology , Hematopoietic System/physiology , Humans , Oligonucleotide Array Sequence Analysis/methods , RNA/chemistry , RNA/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The Inhibitor of Apoptosis Proteins (IAPs) are important regulators of programmed cell death. XIAP is the most potent among them and is over-expressed in several hematological malignancies. Its activity is endogenously antagonized by SMAC/DIABLO, and also by small molecules mimicking Smac that can induce apoptosis in tumor cells. Here we describe the activity of 56 newly synthesized Smac-mimetics in human leukemic cell lines and normal CD34(+) progenitor cells. Our compounds bind to XIAP with high affinity, reduce the levels of cIAP1 and are cytotoxic at nanomolar or low micromolar concentrations. Furthermore, the Smac-mimetics synergize with Cytarabine, Etoposide and especially with TRAIL in combination treatments. Apoptosis activation was clearly detectable by the occurrence of sub G(1) apoptotic peak and the accumulation of cleaved PARP, caspase 8 and caspase 3. Interestingly, the down-regulation of XIAP sensitized Jurkat cells to drugs too, confirming the role of this protein in drug-resistance. In conclusion, while being very active in leukemic cells, our Smac-mimetics have modest effects on normal hematopoietic progenitors, suggesting their promising therapeutic potential as a new class of anticancer drugs in onco-hematology, particularly when combined with TRAIL, to overcome the resistance of cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia/drug therapy , Mitochondrial Proteins/metabolism , Antigens, CD34/metabolism , Antineoplastic Agents/chemistry , Apoptosis Regulatory Proteins , Cell Line, Tumor , Cytarabine/pharmacology , Drug Synergism , Etoposide/pharmacology , Humans , Inhibitor of Apoptosis Proteins/metabolism , Leukemia/pathology , Protein Binding , Stem Cells/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, nowadays considered as suitable candidate for autologous stem therapy with bone marrow (BM). A careful characterization of BM stem cell (SC) compartment is mandatory before its extensive application to clinic. Indeed, widespread systemic involvement has been recently advocated given that non-neuronal neighboring cells actively influence the pathological neuronal loss. We therefore investigated BM samples from 21 ALS patients and reported normal hematopoietic biological properties while an atypical behavior and impaired SC capabilities affected only the mesenchymal compartment. Moreover, by quantitative real-time approach, we observed altered Collagen IV and Metalloproteinase-9 levels in patients' derived mesenchymal stem cells (MSCs). Widespread metalloproteinase (MMPs) and their tissue inhibitor (TIMPs) alterations were established by multiplex ELISA analysis, demonstrating diffuse enzymatic variations in MSC compartment. Since MMPs act as fundamental effectors of extra-cellular matrix remodeling and stem cell mobilization, their modifications in ALS may influence reparative mechanisms effective in counteracting the pathology. In conclusion, ALS is further confirmed to be a systemic disease, not restricted to the nervous system, but affecting also the BM stromal compartment, even in sporadic cases. Therefore, therapeutic implantation of autologous BM derived SC in ALS patients needs to be carefully reevaluated.
Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Bone Marrow/enzymology , Matrix Metalloproteinases/metabolism , Mesenchymal Stem Cells/enzymology , Adult , Aged , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Cells, Cultured , Collagen Type IV/metabolism , Disease Progression , Female , Humans , Male , Matrix Metalloproteinases/genetics , Mesenchymal Stem Cells/cytology , Mice , Middle Aged , Tissue Inhibitor of Metalloproteinases/metabolismABSTRACT
The clinical and biochemical features of an acute and initial Wegener's granulomatosis case were analysed in a young woman. A multifactorial aspects are evident. A chronic inflammation of the superior respiratory tract has been observed. Staphylococcus aureus has been isolated. An oligoclonal component constituted of high levels of anti-PR3 autoantibodies was detected: initial autoreactive B cell clone activation is probable. The chronological link with postpartum is present: our study excluded foetal microchimerism; the hormonal state can be a trigger factor. Serical IL-17 was negative.
Subject(s)
Granulomatosis with Polyangiitis/blood , Granulomatosis with Polyangiitis/diagnosis , Female , Humans , Serologic TestsABSTRACT
Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.
Subject(s)
Antineoplastic Agents/chemical synthesis , Bridged Bicyclo Compounds/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Mitochondrial Proteins/chemistry , Neoplasms/drug therapy , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Apoptosis Regulatory Proteins , Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/toxicity , Cell Line, Tumor , Computer Simulation , Crystallography, X-Ray , Drug Design , Drug Screening Assays, Antitumor , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , X-Linked Inhibitor of Apoptosis Protein/chemistry , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.
Subject(s)
Inhibitor of Apoptosis Proteins , Intracellular Signaling Peptides and Proteins , Mitochondrial Proteins , Molecular Mimicry , X-Linked Inhibitor of Apoptosis Protein , Amino Acid Sequence , Apoptosis Regulatory Proteins , Cell Line , Crystallography, X-Ray , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Molecular Structure , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , X-Linked Inhibitor of Apoptosis Protein/chemistry , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Compelling evidence indicates the role of sphingosine kinase 1 (SPHK1) deregulation in the processes of carcinogenesis and acquisition of drug resistance, providing the rationale for an effective anti-cancer therapy. However, no highly selective inhibitors of SPHK1 are available for in vitro and in vivo studies, except for the newly discovered 'SK inhibitor' (SKI). The present study showed that, in a panel of myeloid leukaemia cell lines, basal level of SPHK1 correlated with the degree of kinase inhibition by SKI. Exposure to SKI caused variable anti-proliferative, cytotoxic effects in all cell lines. In particular, SKI induced an early, significant inhibition of SPHK1 activity, impaired cell cycle progression and triggered apoptosis in K562 cells. Moreover, SKI acted synergistically with imatinib mesylate (IM) to inhibit cell growth and survival. Finally, the inhibitor affected the clonogenic potential and viability of primary cells from chronic myeloid leukaemia (CML) patients, including one harbouring the IM-insensitive Abl kinase domain mutation T315I. Due to the fact that the phenomenon of resistance to IM remains a major issue in the treatment of patients with CML, the identification of alternative targets and new drugs may be of clinical relevance.
Subject(s)
Blast Crisis/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Thiazoles/therapeutic use , Apoptosis/drug effects , Benzamides , Blast Crisis/enzymology , Cell Cycle/drug effects , Colony-Forming Units Assay , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression , Humans , Imatinib Mesylate , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction/methods , Thiazoles/pharmacology , Time FactorsABSTRACT
OBJECTIVE: Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into multiple mesodermal tissues. We previously reported that monoclonal antibodies to the low-affinity nerve growth factor receptor (alpha-LNGFR) stain bone marrow (BM) mesenchymal cells. We now show that LNGFR antibodies label primitive MSCs with high specificity and purity in adult BM, and compare these cells to those isolated by plastic adherence (PA) and CD45(-)anti-glycophorin A(-) selection. MATERIALS AND METHODS: Low-density mononuclear cells (LD-MNCs) from normal BM were separated by PA or immunomagnetic selection for NGFR(+) or CD45(-)alpha-glycophorin A(-) cells. The three fractions were grown in Iscove's modified Dulbecco medium + 20% fetal bovine serum +/- basic fibroblast growth factor (bFGF) in order to assess their proliferative capacity and evaluate their phenotype during culture. The clonogenic potential of the MSCs was assessed using a colony-forming unit fibroblast (CFU-F) assay, whereas multipotential differentiation was determined after culture in adipocytic and osteoblastic conditioned media. RESULTS: The NGFR(+) mesenchymal cells grown without growth factors showed persistent NGFR expression (rapidly down-regulated after the addition of bFGF) and persistent CFU-F activity. The NGFR(+) fractions were rich in clonogenic precursors: CFU-F median frequency was 1584/1 x 10(6) cells (range 325-13,793) in the NGFR(+) cells and 35/1 x 10(6) cells (range 27-112) in the LD-MNCs. The NGFR(-) fraction never showed any residual CFU-F activity. Compared with the other two fractions, the NGFR(+) cells (+/- bFGF) showed a 1 to 3 log greater expansion in the number of fibroblastic cells and a greater capacity to give rise to adipocyte colonies and induce osteoblastic differentiation, and they had similar effects in supporting the growth of hematopoietic precursors. CONCLUSION: The data suggest that positive selection using low-affinity NGFR antibodies makes it possible to obtain homogeneous multipotent MSCs.
Subject(s)
Bone Marrow Cells , Immunomagnetic Separation , Mesoderm/cytology , Receptor, Nerve Growth Factor/immunology , Stem Cells , Adipocytes/cytology , Adipocytes/metabolism , Adult , Bone Marrow Cells/chemistry , Bone Marrow Cells/immunology , Cell Differentiation , Colony-Forming Units Assay , Culture Media/pharmacology , Culture Media, Conditioned/pharmacology , Fibroblasts/cytology , Glycophorins/analysis , Humans , Immunophenotyping , Leukocyte Common Antigens/analysis , Osteoblasts/cytology , Osteoblasts/metabolism , Stem Cells/chemistry , Stem Cells/immunologyABSTRACT
Inositol hexaphosphate (IP(6)), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP(6) action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP(6) had a dose-dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP(6) exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP(6) treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte-macrophage colony-forming unit (CFU-GM) formation (P = 0.0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP(6) and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.