Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1+Mac-1+ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca2+ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines

Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (K-iapp = 43 mu M) and was more potent against Factor Xa (K-iapp = 8.6 mu M), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the beta-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (similar to 645 angstrom(2)). The structure differs from those of the most closely related proteins by the lack of the N-terminal beta-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 mM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and −4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and −4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-016-2203-7) contains supplementary material, which is available to authorized users

Problems caused by high concentration of ATP on activation of the P2X(7) receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2

Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+](i)). in mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+](i); from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPgammaS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. the physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. the P2X(7) inhibitors, Mg2+ (5 mM), OxATP (300 muM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. the J774 cell line and mononuclear cells with a higher expression of P2X(7) receptors show the same decrease in R340/380 as that induced by ATP. in the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of [Ca2+](i) in these cells in the presence of a higher concentration of ATP which activated the P2X(7) receptor.Universidade Federal de São Paulo, Dept Biophys, BR-04034060 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04034060 São Paulo, BrazilWeb of Scienc

A heparin mimetic isolated from a marine shrimp suppresses neovascularization

Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin-like compound in angiogenic processes. Methods and Results: the anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. the compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. the decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. the results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. the effect on CNV area correlates with decreases in the levels of VEGF and TGF-beta 1 in the choroidal tissue. the low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. Conclusions: the properties of the shrimp heparinoid, such as potent anti-angiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Mol Biol Grad Program, Disciplina Biol Mol, UNIFESP,Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Oftalmol, BR-04044020 São Paulo, BrazilUniv Fed Rio Grande do Norte, UFRN, Dept Bioquim, BR-59072970 Natal, RN, BrazilUniversidade Federal de São Paulo, Mol Biol Grad Program, Disciplina Biol Mol, UNIFESP,Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Oftalmol, BR-04044020 São Paulo, BrazilWeb of Scienc

Cell-Permeable Gomesin Peptide Promotes Cell Death by Intracellular Ca2+ Overload

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca2+ signaling during Gm-induced cell death. the results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 mu M Gm increases the cytosolic Ca2+ and induces membrane permeabilization after 30 min of treatment. Direct Ca2+ measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca2+ depletion, which in turn resulted in oscillatory mitochondrial Ca2+ signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca2+-dependent pathway involving Gm translocation into the cell, ER Ca2+ depletion and disruption, mitochondrial Ca2+ overload and oxidative stress.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Consolodacion de grupos UAM-CAMFPU from Ministerio de Educacion, SpainUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv Fed Parana, Dept Biol Celular, BR-81531990 Curitiba, Parana, BrazilUniv Autonoma Madrid, Inst Teofilo Hernando, Inst Invest Sanitaria, Hosp Univ Princesa,Serv Farmacol Clin, Madrid, SpainUniversidade Federal de São Paulo, Dept Ciencias Biol, Diadema, SP, BrazilUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, Diadema, SP, BrazilFAPESP: 2009/54869-2Consolodacion de grupos UAM-CAM: 1004040047FPU from Ministerio de Educacion, Spain: AP2009-0343Web of Scienc