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Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.
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[This corrects the article DOI: 10.3389/fimmu.2018.01896.].
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Targeted and triggered release of liposomal drug using ultrasound (US) induced cavitation represents a promising treatment modality to increase the therapeutic-toxicity ratio of encapsulated chemotherapy. OBJECTIVES: To study the feasibility and efficacy of a combination of focused US and liposomal doxorubicin (US-L-DOX) release in orthotopic murine models of pancreatic cancer. MATERIAL AND METHODS: A confocal US setup was developed to generate US inertial cavitation delivery in a controlled and reproducible manner and designed for two distinct murine orthotopic pancreatic cancer models. Controlled cavitation at 1 MHz was applied within the tumors after L-DOX injection according to a preliminary pharmacokinetic study. RESULTS: In vitro studies confirmed that L-DOX was cytostatic. In vivo pharmacokinetic study showed L-DOX peak tumor accumulation at 48h. Feasibility of L-DOX injection and US delivery was demonstrated in both murine models. In a nude mouse model, at W9 after implantation (W5 after treatment), US-L-DOX group (median [IQR] 51.43 mm3 [35.1-871.95]) exhibited significantly lower tumor volumes than the sham group (216.28 [96.12-1202.92]), the US group (359.44 [131.48-1649.25]), and the L-DOX group (255.94 [84.09-943.72]), and a trend, although not statistically significant, to a lower volume than Gemcitabine group (90.48 [42.14-367.78]). CONCLUSION: This study demonstrates that inertial cavitation can be generated to increase the therapeutic effect of drug-carrying liposomes accumulated in the tumor. This approach is potentially an important step towards a therapeutic application of cavitation-induced drug delivery in pancreatic cancer.
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Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Estudios de Factibilidad , Femenino , Liposomas , Masculino , Ratones , Ratones Desnudos , Polietilenglicoles/administración & dosificación , Ratas , Ratas Endogámicas Lew , UltrasonografíaRESUMEN
The endogenous cholinergic system plays a key role in neuronal cells, by suppressing neurite outgrowth and myelination and, in some cancer cells, favoring tumor growth. Platinum compounds are widely used as part of first line conventional cancer chemotherapy; their efficacy is however limited by peripheral neuropathy as a major side-effect. In a multiple sclerosis mouse model, benztropine, that also acts as an anti-histamine and a dopamine re-uptake inhibitor, induced the differentiation of oligodendrocytes through M1 and M3 muscarinic receptors and enhanced re-myelination. We have evaluated whether benztropine can increase anti-tumoral efficacy of oxaliplatin, while preventing its neurotoxicity.We showed that benztropine improves acute and chronic clinical symptoms of oxaliplatin-induced peripheral neuropathies in mice. Sensory alterations detected by electrophysiology in oxaliplatin-treated mice were consistent with a decreased nerve conduction velocity and membrane hyperexcitability due to alterations in the density and/or functioning of both sodium and potassium channels, confirmed by action potential analysis from ex-vivo cultures of mouse dorsal root ganglion sensory neurons using whole-cell patch-clamp. These alterations were all prevented by benztropine. In oxaliplatin-treated mice, MBP expression, confocal and electronic microscopy of the sciatic nerves revealed a demyelination and confirmed the alteration of the myelinated axons morphology when compared to animals injected with oxaliplatin plus benztropine. Benztropine also prevented the decrease in neuronal density in the paws of mice injected with oxaliplatin. The neuroprotection conferred by benztropine against chemotherapeutic drugs was associated with a lower expression of inflammatory cytokines and extended to diabetic-induced peripheral neuropathy in mice.Mice receiving benztropine alone presented a lower tumor growth when compared to untreated animals and synergized the anti-tumoral effect of oxaliplatin, a phenomenon explained at least in part by benztropine-induced ROS imbalance in tumor cells.This report shows that blocking muscarinic receptors with benztropine prevents peripheral neuropathies and increases the therapeutic index of oxaliplatin. These results can be rapidly transposable to patients as benztropine is currently indicated in Parkinson's disease in the United States.
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Antineoplásicos/administración & dosificación , Benzotropina/administración & dosificación , Oxaliplatino/administración & dosificación , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/prevención & control , Animales , Línea Celular Tumoral , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/fisiopatología , Nefropatías Diabéticas/prevención & control , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Hiperalgesia/inducido químicamente , Hiperalgesia/prevención & control , Masculino , Ratones Endogámicos BALB C , Oxaliplatino/efectos adversos , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiopatología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiologíaRESUMEN
Systemic sclerosis (SSc) is an autoimmune disease with fibrosis of the skin and internal organs and vascular alterations. Dysregulations in the oxidant/antioxidant balance are known to be a major factor in the pathogenesis of the disease. Indeed, reactive oxygen species (ROS) trigger neoepitopes leading to a breach of immune tolerance and autoimmune responses, activate fibroblasts to proliferate and to produce excess of type I collagen. ROS also alter endothelial cells leading to vascular dysfunction. Glutathione (GSH) is the most potent antioxidant system in eukaryotic cells. Numerous studies have reported a defect in GSH in SSc animal models and humans, but the origin of this defect remains unknown. The transcription factor NRF2 is a key player in the antioxidant defense, as it can induce the transcription of antioxidant and cytoprotective genes, including GSH, through its interaction with the antioxidant response elements. In this work, we investigated whether NRF2 could be implicated in the pathogenesis of SSc, and if this pathway could represent a new therapeutic target in this orphan disease with no curative medicine. Skin biopsies from 11 patients and 10 controls were harvested, and skin fibroblasts were extracted. Experimental SSc was induced both in BALB/c and in nrf2-/- mice by daily intradermal injections of hypochloric acid. In addition, diseased BALB/c mice were treated with an nrf2 agonist, dimethyl fumarate, or placebo. A drop in nrf2 and target genes mRNA levels was observed in skin fibroblasts of SSc patients compared to controls. Moreover, the nrf2 pathway is also downregulated in skins and lungs of SSc mice. In addition, we observed that nrf2-/- mice have a more severe form of SSc with increased fibrosis and inflammation compared to wild-type SSc mice. Diseased mice treated with the nrf2 agonist dimethyl fumarate (DMF) exhibited reduced fibrosis and immune activation compared to untreated mice. The ex vivo treatment of skin fibroblasts from SSc mice with DMF restores GSH intracellular content, decreases ROS production and cell proliferation. These results suggest that the nrf2 pathway is highly dysregulated in human and SSc mice with deleterious consequences on fibrosis and inflammation and that Nrf2 modulation represents a therapeutic target in SSc.
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Elementos de Respuesta Antioxidante , Autoinmunidad , Factor 2 Relacionado con NF-E2/metabolismo , Esclerodermia Sistémica/etiología , Esclerodermia Sistémica/metabolismo , Transducción de Señal , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Fibroblastos/metabolismo , Fibrosis , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/terapia , Adulto JovenRESUMEN
A disintegrin and metalloproteinase 9 (ADAM9) possesses potent metastasis-inducing capacities and is highly expressed in several cancer cells. Previous work has shown that ADAM9 participates in the adhesive-invasive phenotype in lung cancer cells in vitro. In this study, we evaluated whether ADAM9 expression plays a critical role in metastatic processes in vivo and in angiogenesis. We first found that high ADAM9 expression was correlated with poor lung adenocarcinoma patient prognosis on Prognoscan data base. In vivo model based on intravenous injection in nude mice showed that a stable downregulation of ADAM9 in A549 (TrA549 A9-) cells was associated with a lower number of nodules in the lung, suggesting lower potentials for extravasation and metastasis. On a subcutaneous xenograft we showed that TrA549 A9- produced significantly smaller tumours and exhibited fewer neovessels. In addition, in vitro human umbilical vein endothelial cells exposed to supernatant from TrA549 A9- could reduce the formation of more vessel-like structures. To further understand the mechanism, a human antibody array analysis confirmed that five cytokines were downregulated in TrA549 A9- cells. Interleukin 8 was the most significantly downregulated, and its interaction with CXCR2 was implicated in angiogenesis on an in vitro model. These results emphasize the critical influence of ADAM9 on lung cancer progression and aggressiveness. ADAM9 should at least be a marker of cancer aggressiveness and a potential therapeutic target for cancer treatment.
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Proteínas ADAM/genética , Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Neovascularización Patológica/genética , Células A549 , Proteínas ADAM/biosíntesis , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Biomarcadores de Tumor/biosíntesis , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Proteínas de la Membrana/biosíntesis , Ratones , Neovascularización Patológica/patología , Receptores de Interleucina-8B/genéticaRESUMEN
Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and inner organs, vasculopathy and immunological abnormalities. Recent insights into the polarization of macrophages in scleroderma and into the implication of STAT6 and KLF4 in this process have prompted us to investigate the effects of the inhibition of STAT6 signaling pathway by leflunomide in mice. SSc was induced in BALB/c mice by daily subcutaneous injections of hypochlorous acid (HOCl) or bleomycin. Mice were treated (or not) every other day, for 4 or 6 weeks, by leflunomide. Skin and lung fibrosis as well as immunological features were studied. Mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti-DNA topoisomerase 1 auto-antibodies. STAT6 pathway was hyperactivated and KLF4 was overexpressed in the skin and the lungs of diseased mice. Their inhibition by leflunomide prevented skin and lung fibrosis. Moreover, the hyperproliferative and pro-oxidative phenotype of skin and lung fibroblasts was reversed by leflunomide. Beneficial immunological effects of leflunomide were associated with decreased activation of CD4+ and CD8+ T cells, B cell activation, decreased auto-antibodies production and restored polarization of macrophages in the spleen. The improvement provided by leflunomide in both mouse models of SSc provides a rationale for the evaluation of this immunomodulating drug in the management of patients affected by this disease.
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Antirreumáticos/uso terapéutico , Isoxazoles/uso terapéutico , Macrófagos/inmunología , Fibrosis Pulmonar/tratamiento farmacológico , Piel/patología , Animales , Autoanticuerpos/sangre , Bleomicina/administración & dosificación , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibrosis , Humanos , Ácido Hipocloroso/administración & dosificación , Terapia de Inmunosupresión , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leflunamida , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Fibrosis Pulmonar/inducido químicamente , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Piel/metabolismoRESUMEN
The transcription factor NRF2 (NFE2L2), regulates important antioxidant and cytoprotective genes. It enhances cancer cell proliferation and promotes chemoresistance in several cancers. Dimethyl fumarate (DMF) is known to promote NRF2 activity in noncancer models. We combined in vitro and in vivo methods to examine the effect of DMF on cancer cell death and the activation of the NRF2 antioxidant pathway. We demonstrated that at lower concentrations (<25 µmol/L), DMF has a cytoprotective role through activation of the NRF2 antioxidant pathway. At higher concentrations, however (>25 µmol/L), DMF caused oxidative stress and subsequently cytotoxicity in several cancer cell lines. High DMF concentration decreases nuclear translocation of NRF2 and production of its downstream targets. The pro-oxidative and cytotoxic effects of high concentration of DMF were abrogated by overexpression of NRF2 in OVCAR3 cells, suggesting that DMF cytotoxicity is dependent of NRF2 depletion. High concentrations of DMF decreased the expression of DJ-1, a NRF2 protein stabilizer. Using DJ-1 siRNA and expression vector, we observed that the expression level of DJ-1 controls NRF2 activation, antioxidant defenses, and cell death in OVCAR3 cells. Finally, antitumoral effect of daily DMF (20 mg/kg) was also observed in vivo in two mice models of colon cancer. Taken together, these findings implicate the effect of DJ-1 on NRF2 in cancer development and identify DMF as a dose-dependent modulator of both NRF2 and DJ-1, which may be useful in exploiting the therapeutic potential of these endogenous antioxidants. Mol Cancer Ther; 16(3); 529-39. ©2017 AACR.
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Dimetilfumarato/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Glutatión/metabolismo , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuropathic pain is a limiting factor of platinum-based chemotherapies. We sought to investigate the neuroprotective potential of niclosamide in peripheral neuropathies induced by oxaliplatin. Normal neuron-like and cancer cells were treated in vitro with oxaliplatin associated or not with an inhibitor of STAT3 and NF-κB, niclosamide. Cell production of reactive oxygen species and viability were measured by 2',7'-dichlorodihydrofluorescein diacetate and crystal violet. Peripheral neuropathies were induced in mice by oxaliplatin with or without niclosamide. Neurologic functions were assessed by behavioral and electrophysiologic tests, intraepidermal innervation, and myelination by immunohistochemical, histologic, and morphologic studies using confocal microscopy. Efficacy on tumor growth was assessed in mice grafted with CT26 colon cancer cells. In neuron-like cells, niclosamide downregulated the production of oxaliplatin-mediated H2O2, thereby preventing cell death. In colon cancer cells, niclosamide enhanced oxaliplatin-mediated cell death through increased H2O2 production. These observations were explained by inherent lower basal levels of GSH in cancer cells compared with normal and neuron-like cells. In neuropathic mice, niclosamide prevented tactile hypoesthesia and thermal hyperalgesia and abrogated membrane hyperexcitability. The teniacide also prevented intraepidermal nerve fiber density reduction and demyelination in oxaliplatin mice in this mixed form of peripheral neuropathy. Niclosamide prevents oxaliplatin-induced increased levels of IL6, TNFα, and advanced oxidized protein products. Niclosamide displayed antitumor effects while not abrogating oxaliplatin efficacy. These results indicate that niclosamide exerts its neuroprotection both in vitro and in vivo by limiting oxaliplatin-induced oxidative stress and neuroinflammation. These findings identify niclosamide as a promising therapeutic adjunct to oxaliplatin chemotherapy. Mol Cancer Ther; 16(2); 300-11. ©2016 AACR.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Fármacos Neuroprotectores/farmacología , Niclosamida/farmacología , Compuestos Organoplatinos/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Glutatión/metabolismo , Humanos , Ratones , Neuronas Motoras/efectos de los fármacos , Compuestos Organoplatinos/toxicidad , Oxaliplatino , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Tacto/efectos de los fármacos , Carga Tumoral/efectos de los fármacosRESUMEN
Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and inner organs, vasculopathy, and immunological abnormalities. Recent insights on the implication of STAT3, AKT, and Wnt/ß-catenin in fibrosis have prompted us to investigate, in a mouse model of ROS-induced SSc, the effects of niclosamide, an antihelmintic drug that inhibits both of these signaling pathways. SSc was induced in BALB/c mice by daily s.c. injections of hypochlorous acid (HOCl). Mice were treated or not every other day, 5 d a week, for 6 wk, by niclosamide. Skin and lung fibrosis as well as immunological features were studied. Mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti-DNA topoisomerase 1 autoantibodies. STAT3, AKT, and Wnt/ß-catenin pathways were hyperactivated in the skin and the lungs of diseased mice. Niclosamide reversed fibrosis of the skin and the lungs. Beneficial immunological effects were also observed because niclosamide decreased the activation of CD4+ and CD8+ T cells, autoimmune B cell activation, as well as IL-4 and IL-13 production in the skin. The improvement permitted by niclosamide in the mouse model of HOCl-induced SSc as well as the well-documented safety profile of this drug provide a rationale for the evaluation of niclosamide in the management of patients affected by this disease.
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Antihelmínticos/uso terapéutico , Linfocitos B/efectos de los fármacos , Pulmón/efectos de los fármacos , Niclosamida/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Piel/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Autoanticuerpos , Linfocitos B/inmunología , Bleomicina , Células Cultivadas , Citocinas/metabolismo , ADN-Topoisomerasas de Tipo I/inmunología , Modelos Animales de Enfermedad , Femenino , Fibrosis , Humanos , Ácido Hipocloroso , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/metabolismo , Esclerodermia Sistémica/inmunología , Transducción de Señal , Piel/patología , Linfocitos T/inmunologíaRESUMEN
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and visceral organs and vascular alterations. SSc pathophysiology involves systemic inflammation and oxidative stress. Because the vanin-1 gene (vnn1) encodes an enzyme with pantetheinase activity that converts vasculoprotective pantethine into profibrotic pantothenic acid and pro-oxidant cystamine, we tested this pathway in the pathophysiology of SSc. Activation of the vanin-1/pantetheinase pathway was investigated in wild-type BALB/c mice with hypochlorous acid (HOCl)-induced SSc by ELISA and Western blotting. We then evaluated the effects of the inactivation of vnn1 on the development of fibrosis, endothelial alterations, and immunological activation in mice with HOCl- and bleomycin-induced SSc. We then explored the vanin-1/pantetheinase pathway in a cohort of patients with SSc and in controls. In wild-type mice with HOCl-induced SSc, the vanin-1/pantetheinase pathway was dysregulated, with elevation of vanin-1 activity in skin and high levels of serum pantothenic acid. Inactivation of the vnn1 gene in vnn1-/- mice with HOCl-induced SSc prevented the development of characteristic features of the disease, including fibrosis, immunologic abnormalities, and endothelial dysfunction. Remarkably, patients with diffuse SSc also had increased expression of vanin-1 in skin and blood and elevated levels of serum pantothenic acid that correlated with the severity of the disease. Our data demonstrate that vanin-1/pantetheinase controls fibrosis, vasculopathy, autoimmunity, and oxidative stress in SSc. The levels of vanin-1 expression and pantothenic acid determine SSc severity and can be used as markers of disease severity. More importantly, inhibition of vanin-1 can open new therapeutic approaches in SSc.
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Amidohidrolasas/metabolismo , Esclerodermia Sistémica/metabolismo , Animales , Femenino , Proteínas Ligadas a GPI/metabolismo , Ratones , Ratones Endogámicos BALB C , Ácido Pantoténico/metabolismoRESUMEN
Sclerodermatous graft-versus-host disease, a frequent complication of allogeneic hematopoietic stem cell graft, shares many features with systemic sclerosis, such as production of autoantibodies and fibrosis of skin and inner organs. Recent reports on the implication of signal transducer and activator of transcription 3 and of Wnt/ß-catenin in fibrosis have prompted us to investigate the effects of the inhibition of both signaling pathways in a mouse model of sclerodermatous graft-versus-host disease, using niclosamide, an anthelmintic drug, with a well-defined safety profile. Sclerodermatous graft-versus-host disease was induced in BALB/c mice by B10.D2 bone marrow and spleen cell transplantation. Mice were treated every other day, 5 days a week, for 5 weeks by niclosamide. Clinical and biological features were studied 42 days after transplantation. Niclosamide reversed clinical symptoms including alopecia, vasculitis, and diarrhea and prevented fibrosis of the skin and visceral organs. Beneficial immunological effects were also observed: niclosamide decreased the production of effector memory CD4 and CD8 T cells, T-cell infiltration of the skin and visceral organs, and decreased productions of IL-4 and IL-13, and autoimmune B-cell activation. The improvement provided by niclosamide in the mouse model of sclerodermatous graft-versus-host disease provides a rationale for the evaluation of niclosamide in the management of patients affected by systemic fibrotic disease.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Inmunidad Celular/efectos de los fármacos , Niclosamida/uso terapéutico , Esclerodermia Localizada/tratamiento farmacológico , Piel/patología , Animales , Linfocitos B/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Enfermedad Injerto contra Huésped/complicaciones , Enfermedad Injerto contra Huésped/inmunología , Activación de Linfocitos , Ratones Endogámicos BALB C , Esclerodermia Localizada/diagnóstico , Esclerodermia Localizada/etiología , Piel/metabolismo , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
OBJECTIVE: Mitogen-activated protein kinases (MAPKs) are involved in the proliferation and survival of endometriotic lesions. Vemurafenib (PLX4032) is a novel protein kinase inhibitor that targets BRAF, a member of the MAPK pathway. The present study tested the in vitro and in vivo effects of PLX4032 on endometriotic cells. RESEARCH DESIGN AND METHODS: We conducted a laboratory study in a tertiary-care university hospital from January 2013 to September 2013. We enrolled a cohort of 40 patients: 20 with histologically proven endometriosis and 20 unaffected women. A thorough surgical examination of the abdominopelvic cavity was performed on all of the study participants. Ex vivo stromal and epithelial cells were extracted from endometrial and endometriotic biopsies from both sets of patients. Proliferation, apoptosis, pERK/ERK ratio, cell cycle regulation (Cyclin D1 and CDK4) and inflammation (PTGS2) were explored with and without PLX4032 treatment. Human endometriotic lesions were implanted into 40 nude mice that were separated into two groups according to PLX4032 or vehicle treatment, which they received for four weeks, before sacrifice and histological examination. RESULTS: Treating endometriotic cells with PLX4032 abrogated the phosphorylation of ERK, significantly reducing the pERK/ERK ratio in both epithelial and stromal cells from endometriotic women compared to the controls (p < 0.05). In addition, treatment with PLX4032 significantly decreased proliferation in both stromal and epithelial cells with a concomitant decrease in Cyclin D1/CDK4 complex and PTGS2 levels. Using a murine model of endometriosis, we observed that PLX4032-treated mice displayed a significant decrease in implant volume compared to the initial size; a slight, but non-significant, increase in size was observed in the vehicle-treated mice. CONCLUSION: Our data suggest that MAPKs and BRAF are involved in the pathogenesis of endometriosis. PLX4032-induced inhibition of BRAF controlled endometriotic growth, both in vitro and in vivo, and could constitute a promising target for the treatment of endometriosis.
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Antineoplásicos/farmacología , Endometriosis/tratamiento farmacológico , Indoles/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sulfonamidas/farmacología , Adulto , Animales , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Endometriosis/patología , Células Epiteliales/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Células del Estroma/metabolismo , Vemurafenib , Adulto JovenRESUMEN
BACKGROUND: Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and multiple internal organs. Recent reports found that RhoA/Rho-kinase (ROCK) pathway is implicated in various fibrogenic diseases. Intradermal injection of hypochlorous acid (HOCl)-generating solution induced inflammation, autoimmune activation, and fibrosis, mimicking the cutaneous diffuse form of SSc in humans. Our study aimed firstly to describe pulmonary inflammation and fibrosis induced by HOCl in mice, and secondly to determine whether fasudil, a selective inhibitor of ROCK, could prevent lung and skin fibroses in HOCl-injected mice. METHODS: Female C57BL/6 mice received daily intradermal injection of hypochlorous acid (HOCl) for 6 weeks to induce SSc, with and without daily treatment with fasudil (30 mg·kg(-1)·day(-1)) by oral gavage. RESULTS: HOCl intoxication induced significant lung inflammation (macrophages and neutrophils infiltration), and fibrosis. These modifications were prevented by fasudil treatment. Simultaneously, HOCl enhanced ROCK activity in lung and skin tissues. Inhibition of ROCK reduced skin fibrosis, expression of α-smooth-muscle actin and 3-nitrotyrosine, as well as the activity of ROCK in the fibrotic skin of HOCl-treated mice, through inhibition of phosphorylation of Smad2/3 and ERK1/2. Fasudil significantly decreased the serum levels of anti-DNA-topoisomerase-1 antibodies in mice with HOCl-induced SSc. CONCLUSIONS: Our findings confirm HOCl-induced pulmonary inflammation and fibrosis in mice, and provide further evidence for a key role of RhoA/ROCK pathway in several pathological processes of experimental SSc. Fasudil could be a promising therapeutic approach for the treatment of SSc.
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Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Ácido Hipocloroso/farmacología , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neumonía/metabolismo , Esclerodermia Sistémica/inducido químicamente , Piel/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Exhaled nitric oxide (NO) is increased as a result of lung inflammation, which in turn causes subsequent interstitial lung disease in patients with systemic sclerosis (SSc). However, the exact time course of inflammatory and fibrotic changes in the SSc lung has not yet been described. Our objective was to assess the chronological evolution of lung inflammatory and fibrotic processes in mice pre-treated with hypochlorous acid (HOCl) or bleomycin. C57BL/6 mice were randomized into three groups receiving subcutaneous injections of HOCl, bleomycin, or PBS for 2, 4 or 6 weeks. Exhaled NO (eNO) was measured at the end of each injection period and after 2 resting weeks without injection (8 week group). Mice were then sacrificed to obtain skin and lung tissues to measure fibrotic changes and NO synthases (NOS) expression. Increased eNO, inducible NOS and nitrotyrosine expression in bronchial epithelium, lung neutrophils and macrophages were observed at early phases in both HOCl- and bleomycin-treated mice. Conversely, lung vascular endothelial NOS expression decreased significantly at 6th and 8th weeks. Skin fibrosis was significantly increased from the 4th week and lung fibrosis from 6th week. We conclude that lung inflammation occurs early after injury as reflected by increased exhaled NO and inducible NOS expression, and precedes fibrotic changes in skin and lungs of mice pre-treated with bleomycin and HOCl. Early detection and treatment of pulmonary inflammation might be useful in preventing subsequent occurrence of lung fibrosis in SSc patients.
Asunto(s)
Pruebas Respiratorias/métodos , Óxido Nítrico/metabolismo , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/metabolismo , Animales , Bleomicina , Modelos Animales de Enfermedad , Femenino , Ácido Hipocloroso , Ratones , Ratones Endogámicos C57BL , Piel/metabolismoRESUMEN
Chronic graft-versus-host disease (GVHD) follows allogeneic hematopoietic stem cell transplantation. It results from alloreactive processes induced by minor histocompatibility antigen incompatibilities leading to the activation of CD4 T cells and the development of fibrosis and inflammation of the skin and visceral organs and autoimmunity that resemble systemic sclerosis. EGFR is a ubiquitous cell receptor deeply involved in cell proliferation, differentiation, and motility. EGFR has recently been implicated in autoimmune and fibrotic diseases. Therefore, we tested whether Erlotinib, an EGFR tyrosine kinase inhibitor, can prevent sclerodermatous GVHD (Scl-GVHD). Scl-GVHD was induced in BALB/c mice by B10.D2 bone marrow and spleen cell transplantation. Transplanted mice displayed severe clinical symptoms including alopecia, fibrosis of the skin and visceral organs, vasculitis, and diarrhea. The symptoms were reversed in mice treated with Erlotinib. These beneficial effects were mediated by the decreased production of activated/memory CD4(+) T cells and the reduction in T-cell infiltration of the skin and visceral organs along with a decrease in IFN-γ and IL-13 production and autoimmune B-cell activation. The improvement provided by Erlotinib in the mouse model of Scl-GVHD supplies a rationale for the evaluation of Erlotinib in the management of patients affected by chronic GVHD.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Clorhidrato de Erlotinib/farmacología , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/prevención & control , Esclerodermia Localizada/tratamiento farmacológico , Esclerodermia Localizada/prevención & control , Aloinjertos , Análisis de Varianza , Animales , Biopsia con Aguja , Western Blotting , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunohistoquímica , Interferón gamma/metabolismo , Interleucina-13/metabolismo , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa/métodos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/farmacología , Distribución Aleatoria , Valores de Referencia , Esclerodermia Localizada/etiología , Esclerodermia Localizada/patologíaRESUMEN
OBJECTIVE: Endothelial cell (EC) damage in systemic sclerosis (SSc) is reflected by the shedding of microparticles (MPs). The aim of this study was to show that inhibiting MP release using pantethine or by inactivating ATP-binding cassette transporter A1 (ABCA1) ameliorates murine SSc. METHODS: First, the effects of pantethine on MP shedding and on basal oxidative and nitrosative stresses in ECs and fibroblasts were determined in vitro. The effects of pantethine were then tested in vivo. SSc was induced in BALB/c mice by daily intradermal injection of HOCl. Mice were simultaneously treated daily with pantethine by oral gavage. RESULTS: In vitro, pantethine inhibited MP shedding from tumor necrosis factor-stimulated ECs and abrogated MP-induced oxidative and nitrosative stresses in ECs and fibroblasts. Ex vivo, pantethine also restored redox homeostasis in fibroblasts from mice with SSc. In vivo, mice with SSc displayed skin and lung fibrosis associated with increased levels of circulating MPs and markers of oxidative and endothelial stress, which were normalized by administration of pantethine or inactivation of ABCA1. CONCLUSION: Pantethine is a well-tolerated molecule that represents a potential treatment of human SSc.
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Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/patología , Células Endoteliales/patología , Panteteína/análogos & derivados , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/prevención & control , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Administración Oral , Animales , Bleomicina/administración & dosificación , Bleomicina/efectos adversos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Homeostasis/efectos de los fármacos , Ácido Hipocloroso/administración & dosificación , Ácido Hipocloroso/efectos adversos , Técnicas In Vitro , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Panteteína/administración & dosificación , Panteteína/farmacología , Panteteína/uso terapéutico , Esclerodermia Sistémica/inducido químicamente , Resultado del TratamientoRESUMEN
Cancer cells display an overproduction of reactive oxygen species resulting from an exaggerated intrinsic oxidative stress. However, the concept of deleterious oxidants versus beneficial antioxidants has recently evolved. Indeed, molecules like natural coumarins have shown anti-oxidant or pro-oxidant properties depending on their intracellular concentration. Therefore, we have investigated the structure-activity relationship of a variety of coumarin derivatives in terms of cytotoxicity towards human and murine carcinoma cell lines (HT29, HepG2, A549, MCF7, OVCAR and CT26). Amongst those compounds, (E)-7-methoxy-4-(3-oxo-3- phenylprop-1-enyl)-2H-chromen-2-one and (E)-7-hydroxy-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2H-chromen-2-one displayed the most potent cytotoxic effect on colon cancer cells, CT26, (IC50=4.9µM) linked to their pro-oxidant properties. Those compounds triggered the in vitro production of reactive oxygen species by tumor cells, leading to their death through a necrotic process. In vivo, molecules also slowed down tumor growth by 65.7% and 35.4%, respectively, without inducing significant side effects.
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Antineoplásicos/farmacología , Chalconas/farmacología , Cumarinas/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chalconas/química , Cumarinas/química , Femenino , Xenoinjertos , Humanos , Concentración 50 Inhibidora , Riñón/patología , Hígado/patología , Ratones Endogámicos BALB C , Necrosis , Trasplante de Neoplasias , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: The majority of patients receiving the platinum-based chemotherapy drug oxaliplatin develop peripheral neurotoxicity. Because this neurotoxicity involves ROS production, we investigated the efficacy of mangafodipir, a molecule that has antioxidant properties and is approved for use as an MRI contrast enhancer. METHODS: The effects of mangafodipir were examined in mice following treatment with oxaliplatin. Neurotoxicity, axon myelination, and advanced oxidized protein products (AOPPs) were monitored. In addition, we enrolled 23 cancer patients with grade ≥ 2 oxaliplatin-induced neuropathy in a phase II study, with 22 patients receiving i.v. mangafodipir following oxaliplatin. Neuropathic effects were monitored for up to 8 cycles of oxaliplatin and mangafodipir. RESULTS: Mangafodipir prevented motor and sensory dysfunction and demyelinating lesion formation. In mice, serum AOPPs decreased after 4 weeks of mangafodipir treatment. In 77% of patients treated with oxaliplatin and mangafodipir, neuropathy improved or stabilized after 4 cycles. After 8 cycles, neurotoxicity was downgraded to grade ≥ 2 in 6 of 7 patients. Prior to enrollment, patients received an average of 880 ± 239 mg/m2 oxaliplatin. Patients treated with mangafodipir tolerated an additional dose of 458 ± 207 mg/m2 oxaliplatin despite preexisting neuropathy. Mangafodipir responders managed a cumulative dose of 1,426 ± 204 mg/m2 oxaliplatin. Serum AOPPs were lower in responders compared with those in nonresponders. CONCLUSION: Our study suggests that mangafodipir can prevent and/or relieve oxaliplatin-induced neuropathy in cancer patients. Trial registration. Clinicaltrials.gov NCT00727922. Funding. Université Paris Descartes, Ministère de la Recherche et de l'Enseignement Supérieur, and Assistance Publique-Hôpitaux de Paris.
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Antineoplásicos/efectos adversos , Antioxidantes/administración & dosificación , Ácido Edético/análogos & derivados , Compuestos Organoplatinos/efectos adversos , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Fosfato de Piridoxal/análogos & derivados , Potenciales de Acción/efectos de los fármacos , Administración Intravenosa , Anciano , Animales , Antineoplásicos/uso terapéutico , Antioxidantes/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/mortalidad , Ácido Edético/administración & dosificación , Ácido Edético/farmacología , Femenino , Humanos , Hipoestesia/inducido químicamente , Hipoestesia/prevención & control , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Actividad Motora/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Nocicepción/efectos de los fármacos , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino , Estrés Oxidativo , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Fosfato de Piridoxal/administración & dosificación , Fosfato de Piridoxal/farmacología , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Análisis de SupervivenciaRESUMEN
Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.