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1.
J Cell Sci ; 131(1)2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29158223

RESUMO

Gene splicing profiles are frequently altered in cancer, and the splice variants of fibronectin (FN) that contain the extra-domains A (EDA) or B (EDB), referred to as EDA+FN or EDB+FN, are highly upregulated in tumor vasculature. Transforming growth factor ß (TGF-ß) signaling has been attributed a pivotal role in glioblastoma, with TGF-ß promoting angiogenesis and vessel remodeling. By using immunohistochemistry staining, we observed that the oncofetal FN isoforms EDA+FN and EDB+FN are expressed in glioblastoma vasculature. Ex vivo single-cell gene expression profiling of tumors by using CD31 and α-smooth muscle actin (αSMA) as markers for endothelial cells, and pericytes and vascular smooth muscle cells (VSMCs), respectively, confirmed the predominant expression of FN, EDA+FN and EDB+FN in the vascular compartment of glioblastoma. Specifically, within the CD31-positive cell population, we identified a positive correlation between the expression of EDA+FN and EDB+FN, and of molecules associated with TGF-ß signaling. Further, TGF-ß induced EDA+FN and EDB+FN in human cerebral microvascular endothelial cells and glioblastoma-derived endothelial cells in a SMAD3- and SMAD4-dependent manner. In turn, we found that FN modulated TGF-ß superfamily signaling in endothelial cells via the EDA and EDB, pointing towards a bidirectional influence of oncofetal FN and TGF-ß superfamily signaling.


Assuntos
Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Processamento Alternativo , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Neovascularização Patológica , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética
2.
Ann Rheum Dis ; 76(4): 756-764, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27793816

RESUMO

OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-ß (TGF-ß) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-ß receptor I. RESULTS: TGF-ß upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-ß signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.


Assuntos
Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fibrose Pulmonar/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Pele/patologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Anilidas/farmacologia , Animais , Células Cultivadas , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Técnicas de Inativação de Genes , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Pteridinas/farmacologia , Fibrose Pulmonar/induzido quimicamente , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Proteína Smad3/metabolismo , Receptor Smoothened/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologia , Adulto Jovem , Proteína Gli2 com Dedos de Zinco
3.
Biomater Sci ; 4(11): 1691-1703, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27709133

RESUMO

Three different heterologous substitutes for bone regeneration, manufactured with equine-derived cortical powder (CP), cancellous chips (CC) and demineralized bone matrix granules (DBM), were compared in in vitro and in vivo settings. We tested: a commercially available bone paste (Osteoplant-Activagen™, consisting of aqueous collagenous carrier, CP, DBM; named A); a second-generation injectable paste (20 kDa polyethylene glycol/hydroxypropyl-methyl cellulose-based hydrogel, CP, DBM; B); a pre-formed bone filler (400 kDa polyethylene oxide/hydroxypropyl-methyl cellulose-based hydrogel, CP, CC, DBM; C). Vitamin C acted as a visco-modulator during C and B ß-rays sterilization, modifying graft injectability. For each filler, we examined dissolution in culture medium, gene expression of the substitute-exposed osteogenically-induced human bone marrow stromal cells (hBMSC), and performance in a rabbit bone defect model. A dissolved after 1 h, while fragmentation of B peaked after 8 h. C remained unaltered for 2 days, but affected the microenvironmental pH, slowing the proliferation of exposed cells. B-exposed hBMSC overexpressed bone sialoprotein, osteocalcin and RUNX2. For all fillers histological results evidenced bridged lesion margins, marrow replenishment and bone-remodeling. However, B-treated lesions displayed a metachromatic type II collagen-rich matrix with prehypertrophic-like cells, matching the in vitro expression of cartilage-specific markers, and suggesting a possible application of B/C double-layer monolithic osteochondral plugs for full-thickness articular defects.


Assuntos
Materiais Biocompatíveis/química , Substitutos Ósseos/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Animais , Osso e Ossos/lesões , Linhagem Celular , Humanos , Células-Tronco Mesenquimais , Coelhos , Reologia
4.
Biochim Biophys Acta ; 1860(6): 1354-61, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27015759

RESUMO

BACKGROUND: Obesity is linked to increased thrombotic risk. Circulating leptin concentration correlates with body mass index. Microparticles are small (.05-1 µm) vesicles shed by activated and apoptotic cells, involved in numerous pathophysiologically relevant phenomena including blood coagulation and thrombosis. We tested the hypothesis that leptin induces the shedding of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells, and investigated the intracellular mechanisms leading to microparticle release upon incubation with leptin. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors. Cells were incubated with leptin in the presence or in the absence of a phospholipase C inhibitor, U73122, a calmodulin inhibitor, W-7, and three inhibitors of mitogen activated protein kinases. Microparticle generation was assessed as phosphatidylserine concentration with a prothrombinase assay and by cytofluorimetric analysis. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Intracellular calcium concentration was assessed by a fluorescent probe. RESULTS: Leptin increased intracellular calcium mobilization and stimulated the generation of tissue factor-bearing MP by peripheral blood mononuclear cells, as assessed by phosphatidylserine quantification, clotting tests and flow-cytometry. U73122, PD98059 (an extracellular signal-regulated kinase1/2 inhibitor), and W-7, significantly inhibited leptin-induced MP release. SB203580 (a p38 inhibitor), and SP600125 (a c-Jun N-terminal kinase inhibitor) had no effect. CONCLUSION: Leptin-induced generation of procoagulant microparticles might represent a link between obesity and atherothrombotic risk. Inhibition of leptin-induced microparticle generation might prove a viable strategy for the reduction of such risk in obese individuals.


Assuntos
Fatores de Coagulação Sanguínea/biossíntese , Micropartículas Derivadas de Células/efeitos dos fármacos , Leptina/farmacologia , Leucócitos Mononucleares/metabolismo , Tromboplastina/biossíntese , Cálcio/metabolismo , Calmodulina/fisiologia , Micropartículas Derivadas de Células/fisiologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fosfolipases Tipo C/fisiologia
5.
PLoS One ; 11(2): e0148103, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26867013

RESUMO

BACKGROUND: Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the "extra-domain-B" (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B. METHODOLOGY: We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6. CONCLUSION: The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity.


Assuntos
Anticorpos Monoclonais/química , Epitopos , Fibronectinas/química , Animais , Anticorpos/química , Simulação por Computador , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Humanos , Camundongos , Mutação , Neovascularização Patológica , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Precursores de RNA , Proteínas Recombinantes/química
6.
Inflamm Res ; 63(7): 539-47, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24599284

RESUMO

OBJECTIVES: Microparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism. MATERIALS AND METHODS: Human mononuclear cells were exposed to cigarette smoke extract. [Ca(2+)]i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA. RESULTS: Exposure to cigarette smoke extract induced a rapid increase in [Ca(2+)]i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators. CONCLUSIONS: Exposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.


Assuntos
Cálcio/metabolismo , Micropartículas Derivadas de Células/efeitos dos fármacos , Misturas Complexas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Nicotiana , Fumaça , Apoptose/efeitos dos fármacos , Brônquios/citologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Células Epiteliais , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/metabolismo
7.
PLoS One ; 8(12): e82878, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367567

RESUMO

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.


Assuntos
Anticorpos/imunologia , Escherichia coli/metabolismo , Fibronectinas/imunologia , Uteroglobina/metabolismo , Animais , Anticorpos/genética , Anticorpos/metabolismo , Escherichia coli/genética , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Camundongos , Camundongos SCID , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Uteroglobina/química
8.
Thromb Res ; 131(4): e168-74, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23414567

RESUMO

INTRODUCTION: Microparticles are small vesicles shed by cells upon activation and during apoptosis which participate in physiologically relevant phenomena, including blood coagulation. Intracellular calcium mobilization is one of the mechanisms of microparticle generation during cell activation. Because the renin-angiotensin system has been proposed as a link between hypertension and increased thrombotic risk, we investigated whether angiotensin II upregulates the generation of procoagulant microparticles by human mononuclear cells. MATERIALS AND METHODS: Human mononuclear cells were exposed to angiotensin II for 15min. Intracellular calcium concentration was assessed by a Fluo 4 based kit. The supernatants were analyzed for both microparticle content, with a commercially available kit based on phosphatidylserine analysis, and microparticle-associated tissue factor, with a one-stage clotting assay. RESULTS: Intracellular calcium concentration is increased upon exposure of mononuclear cells to angiotensin II. Incubation with angiotensin II stimulates microparticles release; microparticle-associated tissue factor is also upregulated. The effect is inhibited by an angiotensin receptor type 2 antagonist (PD123319) and not by two angiotensin type 1 antagonists (Losartan and Olmesartan). CONCLUSIONS: Angiotensin receptor 2-mediated upregulation of tissue factor-bearing, procoagulant microparticle generation represents a novel mechanism linking the renin-angiotensin system to thrombosis.


Assuntos
Angiotensina II/farmacologia , Micropartículas Derivadas de Células/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/sangue , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Micropartículas Derivadas de Células/metabolismo , Humanos , Imidazóis/farmacologia , Leucócitos Mononucleares/metabolismo , Piridinas/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais , Tromboplastina/metabolismo
9.
Thromb Res ; 130(3): 552-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22817879

RESUMO

BACKGROUND: Intimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (ANG) II, the effector arm of the Renin Angiotensin System (RAS). The RAS, in turn, plays a pathophysiological role in diabetes, a proinflammatory state to which elevated glucose, the disease hallmark, contributes by activating key signalling pathways and increasing the cellular content of RAS components. AIMS: To evaluate the effect of high glucose concentrations on TF antigen (Ag) expression and procoagulant activity (PCA) in lipopolysaccharide(LPS)-primed human mononuclear cell(MNC)s and to test whether pharmacological RAS blockade modifies that pattern. METHODS: LPS-activated MNCs exposed to increasing D-glucose (from 5.5 to 50mM) in absence or presence of aliskiren, a renin inhibitor, zofenopril, an ANG converting enzyme inhibitor, and olmesartan, an ANGII type I receptor blocker. PCA was assessed by one-stage clotting assay and TF antigen expression by ELISA. RESULTS: Increasing ambient glucose (range 5.5-50mM) potentiated LPS-induced PCA and TF Ag expression. Aliskiren, zofenopril and olmesartan downregulated those responses but the efficacy of the former decreased by ascending drug concentration while both zofenopril and olmesartan showed an opposite behaviour. TF Ag expression modulation by RAS blockade was stronger in 50 than 5mM ambient glucose. CONCLUSIONS: High glucose potentiates the procoagulant action of LPS in human MNCs and RAS blockers downregulate that response possibly as a reflection of the underlying involvement of the system in that mechanism.


Assuntos
Glucose/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Tromboplastina/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos
10.
Ann Rheum Dis ; 71(5): 785-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22402139

RESUMO

OBJECTIVES: Tissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach. METHODS: The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice. RESULTS: Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis. CONCLUSIONS: Inhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.


Assuntos
Fibrose , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais , Dermatopatias/patologia , Pele/patologia , Animais , Compostos de Bifenilo/farmacologia , Bleomicina/toxicidade , Modelos Animais de Doenças , Quimioterapia Combinada , Fibrose/metabolismo , Fibrose/patologia , Fibrose/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Dermatopatias/tratamento farmacológico , Dermatopatias/metabolismo , Receptor Smoothened
11.
Cardiovasc Res ; 94(3): 537-44, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22425902

RESUMO

AIMS: Microparticles are membrane vesicles shed by cells upon activation and/or apoptosis. Microparticles are involved in several processes, including blood coagulation and thrombosis. In addition to their role in the regulation of lipid metabolism, peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists exert other effects, both dependent on and independent of PPAR-γ activation. Some PPAR-γ agonists have been linked to an increased risk of thrombotic diseases. We aimed to investigate the potential role of PPAR-γ agonists on the generation of procoagulant microparticles by human monocytes/macrophages. METHODS AND RESULTS: Monocytes/macrophages were isolated from the buffy coats of normal donors. Cells were incubated with three structurally unrelated PPAR-γ agonists, namely, rosiglitazone, pioglitazone, and 15-deoxy-Δ(12,14)-prostaglandin J(2). Microparticle generation was assessed as phosphatidylserine concentration by a prothrombinase assay, after capturing the microparticles onto annexin V-coated wells. Intracellular calcium concentration was assessed by a fluorescent probe. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by western blot. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused a dose-dependent, significant increase in intracellular calcium mobilization and tissue factor-bearing microparticle generation. EGTA inhibited microparticle generation. The specific PPAR-γ inhibitor, GW9662, also inhibited microparticle generation.  Finally, rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2) caused phosphorylation of ERK; inhibition of ERK by PD98059 inhibited microparticle generation. CONCLUSION: The PPAR-γ agonists rosiglitazone and 15-deoxy-Δ(12,14)-prostaglandin J(2), but not pioglitazone, caused an increase in procoagulant, tissue factor-bearing microparticle generation by human monocytes/macrophages. The effect was dependent on ERK phosphorylation and partly mediated through intracellular calcium mobilization; however, direct activation of the PPAR-γ ligand was also involved.


Assuntos
Micropartículas Derivadas de Células/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , PPAR gama/agonistas , PPAR gama/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Pioglitazona , Rosiglitazona , Transdução de Sinais/fisiologia , Tiazolidinedionas/farmacologia
12.
Arthritis Rheum ; 64(8): 2724-33, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22354771

RESUMO

OBJECTIVE: Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc). METHODS: Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of α-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch(+/-) mice). RESULTS: SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch(+/-) mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis. CONCLUSION: We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.


Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Escleroderma Sistêmico/metabolismo , Transdução de Sinais/fisiologia , Pele/metabolismo , Adulto , Idoso , Animais , Bleomicina/efeitos adversos , Estudos de Casos e Controles , Células Cultivadas , Colágeno/metabolismo , Feminino , Fibroblastos/patologia , Fibrose/induzido quimicamente , Humanos , Masculino , Camundongos , Camundongos Mutantes , Pessoa de Meia-Idade , Modelos Animais , Proteínas Oncogênicas/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Escleroderma Sistêmico/patologia , Pele/patologia , Transativadores/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína GLI1 em Dedos de Zinco
13.
Invest New Drugs ; 29(1): 98-109, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19876599

RESUMO

We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4',6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC(50) reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Diltiazem/análogos & derivados , Diltiazem/farmacologia , Nifedipino/análogos & derivados , Nifedipino/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Anexina A5/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Diltiazem/química , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Indóis/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Nifedipino/química , Propídio/metabolismo , Coloração e Rotulagem
14.
J Med Chem ; 52(2): 259-66, 2009 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-19093883

RESUMO

The reversal of multidrug resistance by 22 molecules [8-aryl-8-hydroxy-5-R'-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (1a-i) and 8-aryl-8-alkoxy-5-methyl-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (2a-m)] related to myocardial-calcium-channel-modulator diltiazem was studied in multidrug resistant A2780/DX3 and their sensitive counterpart A2780 cells. MTT, cytofluorimetry assays, and fluorescence microscopy analyses were used to define activity and accumulation of doxorubicin with or without the diltiazem-like modulators. Of the 22 molecules, 1a, 2f, 2g, and 2m were able to overcome the established criteria for the selection in A2780/DX3 cells (IC(50) reduction > or = 25%), but only 2f, 2g, and 2m caused a significant increase of intracellular accumulation of doxorubicin. In conclusion, experiments lead to the identification of three diltiazem-like molecules able to increase the intracellular accumulation of doxorubicin by inhibiting the MDR1 function, thus potentiating its antiproliferative activity in multidrug resistant A2780/DX3 cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Diltiazem/análogos & derivados , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diltiazem/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas In Vitro , Microscopia de Fluorescência
15.
Eur J Pharmacol ; 588(1): 47-51, 2008 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-18495109

RESUMO

The inhibition of cell proliferation by methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) and (1E,3E)-1,4-bis(2-naphthyl)-2,3-dinitro-1,3-butadiene (2-Naph-DNB) has been studied in vitro against four cell lines selected for their resistance to doxorubicin, cisplatin, taxol and 5-fluorouracil. In previous experiments both compounds showed good in vitro antiproliferative, cytotoxic and pro-apoptotic activities against cell lines of different histologic origin. The results of the experiments presented here suggest that 1-Naph-NMCB is able to overcome all of the different mechanisms of resistance showed by the resistant cell lines used for our experiments. On the contrary, when we used the taxol-resistant A549-T12 cell line, characterized by a mechanism of resistance due to a mutation of the target site of taxol on microtubules, it displayed a partial but significant cross-resistance to 2-Naph-DNB. Although the actual mechanism of this cross-resistance has not yet been definitively elucidated, our results from immunostaining of microtubules suggest that it may be linked to the presence of a shared target site for taxol and 2-Naph-DNB on microtubules.


Assuntos
Antineoplásicos/farmacologia , Butadienos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Naftalenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Imunofluorescência , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Sais de Tetrazólio , Tiazóis
16.
Bioorg Med Chem ; 16(1): 240-7, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17936630

RESUMO

On the grounds of previous encouraging results on the antitumor activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1), we have designed and synthesized two new molecules [(1E,3E)-1,4-bis(4-carboxy-1-naphthyl)-2,3-dinitro-1,3-butadiene (2) and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (3)] characterized by a common naphthylnitrobutadiene array but with different structural properties, with the aim of approaching to some structure-activity correlation. When 2 and 3 were analyzed in vitro for their inhibition of cell proliferation and pro-apoptotic properties, the carboxyderivative 2 did not furnish appreciable results. In contrast, 3 (which contains only one of the two naphthylnitroethenyl moieties of the original compound 1) showed remarkable activities in the range of micromolar concentrations (in six over eight cell lines its IC(50)s are in the 1-3 microM range), with a significant improvement compared to 1. In particular, 3 proved able to bind to DNA, to upregulate p53, to block cells in the G2/M phase of their cycle, and to induce apoptosis. Thus, very interestingly, the performance of 3 with respect to 1 shows that a single 1-(1-naphthyl)-2-nitroethene moiety is able to ensure better (on four out of eight of the cell lines tested) or comparable levels of activity. This result suggests that the 'molecular-simplification strategy' could furnish a useful instrument for future design in our antitumor research.


Assuntos
Antineoplásicos/síntese química , Butadienos/síntese química , Butadienos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Interfase/efeitos dos fármacos , Naftalenos , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
17.
Pharmacol Res ; 56(4): 318-28, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17827031

RESUMO

Our interesting results on the antiproliferative (in vitro) and antitumour (in vivo) activities of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) have more recently induced us to design and synthesize some new 1,4-diaryl-2,3-dinitro-1,3-butadienes characterized by a common arylnitrobutadiene array but with different geometric and/or functional properties. This task was undertaken with the aim to obtain new compounds with an enhanced antiproliferative activity and, possibly, a different specificity with respect to the original (lead) compound. (1E,3E)-1,4-Bis(2-naphthyl)-2,3-dinitro-1,3-butadiene (2-Naph-DNB) is one of the molecules so obtained, a structural isomer of 1-Naph-DNB provided with a different spatial arrangement. When analyzed in vitro for its inhibition of cell proliferation 2-Naph-DNB showed a remarkable activity in the range of micromolar concentrations, with significant differences, with respect to 1-Naph-DNB, against some cell lines. Furthermore, it was able to significantly trigger apoptosis, to up-regulate p53, to block cells in the G2/M phase of the cell cycle and, finally, to slightly bind to DNA forming interstrand cross-links (ISCL). 2-Naph-DNB was then analyzed for its toxic activity in vivo in CD1 mice. This allowed the determination of toxicity parameters such as the lethal doses (LD) and the maximal tolerated dose (MTD) together with the definition of the spectrum of tissue alterations due to its administration i.v. Altogether our data suggest that the idea of modifying the geometry of the lead compound 1-Naph-DNB deserves further investigation aimed at synthesizing new molecules with similar chemical functionalities but with different spatial requirements, hopefully characterized by still enhanced activities in terms of inhibition of cell proliferation and apoptosis.


Assuntos
Antineoplásicos/síntese química , Butadienos/síntese química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Apoptose , Western Blotting , Butadienos/farmacologia , Butadienos/toxicidade , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/farmacologia , Reagentes de Ligações Cruzadas/toxicidade , DNA/química , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Naftalenos/síntese química , Naftalenos/farmacologia , Naftalenos/toxicidade , Baço/efeitos dos fármacos , Baço/patologia , Estereoisomerismo , Proteína Supressora de Tumor p53/biossíntese , Regulação para Cima
18.
Invest New Drugs ; 25(6): 535-44, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17572851

RESUMO

On the basis of our previous interesting results in vitro on the antiproliferative activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) we have designed and synthesized the new molecule methyl (2Z,4E)-2-methylsulphanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) characterized by the same naphthylnitrobutadiene array but with a different functional group at one end of the diene system. This new molecule showed an in vitro antiproliferative activity more significant than that found for the original 1-Naph-DNB. In order to verify in vivo our in vitro results we have tested the antitumour activity of 1-Naph-DNB and 1-Naph-NMCB in several murine tumour models, namely the myelomonocytic P388 and the Lewis lung carcinoma 3LL in BDF1 mice, the melanoma B16 in C57Bl mice, the fibrosarcoma WEHI 164 in nude mice and, finally, the C51 colon cancer in Balb/c mice. In the case of 1-Naph-NMCB the analysis of the antitumour activity has been preceded by toxicological experiments on CD-1 mice, in order to determine the lethal (LD) and the maximal tolerated (MTD) doses together with the spectrum of histological alterations caused by its iv administration. The results obtained show that the modification of the original structure of 1-Naph-DNB according to the molecular-simplification strategy has led to an asymmetric nitrobutadiene array, i.e. that of 1-Naph-NMCB, endowed with an antitumour activity which is in some cases even better than that showed by the parental compound itself, together with differences in tumour selectivity and negligible histological toxic effects.A promising, versatile route to new, more active and/or safe nitrobutadiene derivatives has thus been positively tested.


Assuntos
Antineoplásicos/farmacologia , Butadienos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Naftalenos/farmacologia , Animais , Antineoplásicos/toxicidade , Butadienos/toxicidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Testes de Toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Pharmacol Res ; 52(3): 271-82, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15921920

RESUMO

Our preliminary data suggested that 1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene [Viale M, Ottone M, Chiavarina B, Mariggiò MA, Prevosto C, Dell'Erba C, et al. Preliminary evaluation in vitro of the inhibition of cell proliferation, cytotoxicity and induction of apoptosis by 1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene. Invest New Drug 2004;22:359-67] (Naph-DNB), possesses good characteristics in terms of inhibition of cell proliferation in two cell lines derived from colon and gastric cancers. On this basis and to confirm the specificity of our compound towards gastrointestinal malignancies, we have analyzed the inhibition of cell proliferation, the cytotoxicity and the induction of apoptosis by Naph-DNB in seven cell lines derived from human colon (DLD-1, Lovo, HCT-8 and Colo 741), stomach (HGC-27) and pancreas (Panc-1 and Hup-T4) tumours. For the sake of comparison, cells have also been exposed to four anticancer drugs utilized for the treatment of gastrointestinal malignancies (oxaliplatin, irinotecan, gemcitabine and 5-fluorouracil). Moreover, toxicological data have been obtained in order to define the lethal dose (LD) and maximal tolerated dose (MTD) values and the spectrum of tissue alterations caused by the intraperitoneal (i.p.) and intravenous (i.v.) administration of Naph-DNB. IC50 data obtained by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay suggest that Naph-DNB is generally more active than two or more of the anticancer drugs above in most cell lines: it displayed the lowest activity only in HGC-27 cells, although data concerning the IC75 parameter enlighten a significantly better activity than irinotecan and 5-fluorouracil. Using the equitoxic concentrations IC50 and IC75, we have also evaluated the ability of Naph-DNB and of the other anticancer drugs to kill cells and to induce apoptosis. Our data show that at these concentrations Naph-DNB has a cytotoxic activity comparable or even better than that of some anticancer drugs. Similarly, Naph-DNB induces apoptosis better than the other anticancer drugs in HCT-8 and HGC-27 cells, while in Lovo and Panc-1 cells the induction is comparable. On the basis of toxicological data we defined the LD10, LD50, LD90 (i.p., 17.6, 36.1 and 54.1 mg kg(-1), respectively; i.v., 6.1, 14.1 and 22.0 mg kg(-1), respectively) and the MTD (i.p., 15 mg kg(-1); i.v., 5 mg kg(-1)) parameters. Histochemical analysis has shown that, in general, the administration of even toxic doses of Naph-DNB does not cause great structural injuries, although it can have some effects on the metabolism of glicogen and iron in organs as liver and spleen. In conclusion, our preclinical studies in vitro suggest that Naph-DNB may represent a good anticancer compound for the treatment of generally unresponsive tumours such as those of pancreas, stomach and colon. Moreover, the analysis of its toxic effects has allowed the definition of LD and MTD parameters, which will be used in further experiments in vivo for the definition of its antitumour activity.


Assuntos
Apoptose , Butadienos/farmacologia , Butadienos/toxicidade , Proliferação de Células/efeitos dos fármacos , Animais , Carcinoma , Linhagem Celular Tumoral , Feminino , Neoplasias Gastrointestinais , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Rim/efeitos dos fármacos , Rim/patologia , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos , Baço/efeitos dos fármacos , Baço/patologia
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