RESUMO
Spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease affecting children and young adults, caused by mutations of the survival motor neuron 1 gene (SMN1). SMA is characterized by the degeneration of spinal alpha motor neurons (αMNs), associated with muscle paralysis and atrophy, as well as other peripheral alterations. Both growth hormone-releasing hormone (GHRH) and its potent agonistic analog, MR-409, exert protective effects on muscle atrophy, cardiomyopathies, ischemic stroke, and inflammation. In this study, we aimed to assess the protective role of MR-409 in SMNΔ7 mice, a widely used model of SMA. Daily subcutaneous treatment with MR-409 (1 or 2 mg/kg), from postnatal day 2 (P2) to euthanization (P12), increased body weight and improved motor behavior in SMA mice, particularly at the highest dose tested. In addition, MR-409 reduced atrophy and ameliorated trophism in quadriceps and gastrocnemius muscles, as determined by an increase in fiber size, as well as upregulation of myogenic genes and inhibition of proteolytic pathways. MR-409 also promoted the maturation of neuromuscular junctions, by reducing multi-innervated endplates and increasing those mono-innervated. Finally, treatment with MR-409 delayed αMN death and blunted neuroinflammation in the spinal cord of SMA mice. In conclusion, the present study demonstrates that MR-409 has protective effects in SMNΔ7 mice, suggesting that GHRH agonists are promising agents for the treatment of SMA, possibly in combination with SMN-dependent strategies.
Assuntos
Hormônio Liberador de Hormônio do Crescimento , Atrofia Muscular Espinal , Animais , Camundongos , Atrofia/metabolismo , Modelos Animais de Doenças , Hormônio Liberador de Hormônio do Crescimento/agonistas , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Patients with type 1 diabetes (T1D) suffer from insufficient functional ß-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated ß-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and in vivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on ß-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in ß-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in ß-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of ß-cell mass. Increased IRS2 expression in ß-cells in the group treated with MR-409 corroborated the in vitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 in vivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of ß-cells death in T1D.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Estreptozocina , Citocinas , InsulinaRESUMO
Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.
Assuntos
Apoptose , Insulina , Humanos , Peptídeo C , Necrose , Morte CelularRESUMO
COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.
Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Camundongos , Masculino , Feminino , Animais , SARS-CoV-2 , COVID-19/patologia , Pulmão/patologia , Inflamação/patologia , Síndrome do Desconforto Respiratório/patologia , Redução de Peso , Camundongos Transgênicos , Modelos Animais de DoençasRESUMO
Ischemic stroke can induce neurogenesis. However, most stroke-generated newborn neurons cannot survive. It has been shown that MR-409, a potent synthetic agonistic analog of growth hormone-releasing hormone (GHRH), can protect against some life-threatening pathological conditions by promoting cell proliferation and survival. The present study shows that long-term treatment with MR-409 (5 or 10 µg/mouse/d) by subcutaneous (s.c.) injection significantly reduces the mortality, ischemic insult, and hippocampal atrophy, and improves neurological functional recovery in mice operated on for transient middle cerebral artery occlusion (tMCAO). Besides, MR-409 can stimulate endogenous neurogenesis and improve the tMCAO-induced loss of neuroplasticity. MR-409 also enhances the proliferation and inhibits apoptosis of neural stem cells treated with oxygen and glucose deprivation-reperfusion. The neuroprotective effects of MR-409 are closely related to the activation of AKT/CREB and BDNF/TrkB pathways. In conclusion, the present study demonstrates that GHRH agonist MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/agonistas , Hormônio Liberador de Hormônio do Crescimento/metabolismo , AVC Isquêmico/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Plasticidade Neuronal , Fármacos Neuroprotetores , Proteínas Tirosina Quinases/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/agonistas , Macrófagos/patologia , Neuroproteção , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Inflamação/genética , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neuroproteção/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Endogâmicos F344 , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fator de Transcrição STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismo , Zimosan/farmacologiaRESUMO
Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.
Assuntos
Cardiotônicos/farmacologia , Hormônio Liberador de Hormônio do Crescimento/agonistas , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Renal Crônica/tratamento farmacológico , Sermorelina/análogos & derivados , Volume Sistólico/fisiologia , Animais , Nitrogênio da Ureia Sanguínea , Cálcio/metabolismo , Conectina/genética , Conectina/metabolismo , Creatinina/sangue , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/sangue , Peptídeo Natriurético Encefálico/genética , Nefrectomia/métodos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Sermorelina/farmacologia , SuínosRESUMO
Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.
Assuntos
Hormônio Liberador de Hormônio do Crescimento , Neoplasias , Receptores de Neuropeptídeos , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Humanos , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Criança , Masculino , Feminino , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Projetos Piloto , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Pré-Escolar , Adolescente , Neoplasias/genética , Neoplasias/metabolismo , Hungria , Lactente , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Doenças Hematológicas/genética , Doenças Hematológicas/metabolismo , Estudos de CoortesRESUMO
Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need owing to its diverse pathophysiology and lack of effective therapies. Potent synthetic, agonists (MR-356 and MR-409) of growth hormone-releasing hormone (GHRH) improve the phenotype of models of HF with reduced ejection fraction (HFrEF) and in cardiorenal models of HFpEF. Endogenous GHRH exhibits a broad range of regulatory influences in the cardiovascular (CV) system and aging and plays a role in several cardiometabolic conditions including obesity and diabetes. Whether agonists of GHRH can improve the phenotype of cardiometabolic HFpEF remains untested and unknown. Here we tested the hypothesis that MR-356 can mitigate/reverse the cardiometabolic HFpEF phenotype. C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (l-NAME) for 9 wk. After 5 wk of HFD + l-NAME regimen, animals were randomized to receive daily injections of MR-356 or placebo during a 4-wk period. Control animals received no HFD + l-NAME or agonist treatment. Our results showed the unique potential of MR-356 to treat several HFpEF-like features including cardiac hypertrophy, fibrosis, capillary rarefaction, and pulmonary congestion. MR-356 improved cardiac performance by improving diastolic function, global longitudinal strain (GLS), and exercise capacity. Importantly, the increased expression of cardiac pro-brain natriuretic peptide (pro-BNP), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor-A (VEGF-A) was restored to normal levels suggesting that MR-356 reduced myocardial stress associated with metabolic inflammation in HFpEF. Thus, agonists of GHRH may be an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype.NEW & NOTEWORTHY This randomized study used rigorous hemodynamic tools to test the efficacy of a synthetic GHRH agonist to improve cardiac performance in a cardiometabolic HFpEF. Daily injection of the GHRH agonist, MR-356, reduced the HFpEF-like effects as evidenced by improved diastolic dysfunction, reduced cardiac hypertrophy, fibrosis, and pulmonary congestion. Notably, end-diastolic pressure and end-diastolic pressure-volume relationship were reset to control levels. Moreover, treatment with MR-356 increased exercise capacity and reduced myocardial stress associated with metabolic inflammation in HFpEF.
Assuntos
Insuficiência Cardíaca , Animais , Camundongos , Cardiomegalia , Modelos Animais de Doenças , Fibrose , Hormônio Liberador de Hormônio do Crescimento , Inflamação , NG-Nitroarginina Metil Éster , Volume Sistólico/fisiologia , Fator A de Crescimento do Endotélio Vascular , Função Ventricular EsquerdaRESUMO
Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-ß and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-ß, TNF-α, TGFß-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.
Assuntos
Pneumonia , Fibrose Pulmonar , Humanos , Células Epiteliais Alveolares/metabolismo , Fator de Necrose Tumoral alfa , Lipopolissacarídeos/farmacologia , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Inflamação , CitocinasRESUMO
The extrahypothalamic growth hormone-releasing hormone (GHRH) and its cognate receptors (GHRH-Rs) and splice variants are expressed in a variety of cancers. It has been shown that the pituitary type of GHRH-R (pGHRH-R) mediates the inhibition of tumor growth induced by GHRH-R antagonists. However, GHRH-R antagonists can also suppress some cancers that do not express pGHRH-R, yet the underlying mechanisms have not been determined. Here, using human esophageal squamous cell carcinoma (ESCC) as a model, we were able to reveal that SV1, a known splice variant of GHRH-R, is responsible for the inhibition induced by GHRH-R antagonist MIA-602. We demonstrated that GHRH-R splice variant 1 (SV1) is a hypoxia-driven promoter of tumor progression. Hypoxia-elevated SV1 activates a key glycolytic enzyme, muscle-type phosphofructokinase (PFKM), through the nuclear factor kappa B (NF-κB) pathway, which enhances glycolytic metabolism and promotes progression of ESCC. The malignant actions induced by the SV1-NF-κB-PFKM pathway could be reversed by MIA-602. Altogether, our studies demonstrate a mechanism by which GHRH-R antagonists target SV1. Our findings suggest that SV1 is a hypoxia-induced oncogenic promoter which can be an alternative target of GHRH-R antagonists.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Receptores LHRH/genética , Sermorelina/análogos & derivados , Processamento Alternativo , Animais , Apoptose , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Glicólise , Humanos , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Receptores LHRH/antagonistas & inibidores , Sermorelina/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.
Assuntos
Células Epiteliais/patologia , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Transdução de Sinais/imunologia , Uveíte/patologia , Animais , Linhagem Celular , Corpo Ciliar/citologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Janus Quinase 2/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Nitrilas , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas , Ratos , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/imunologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/antagonistas & inibidores , Receptores de Hormônios Reguladores de Hormônio Hipofisário/imunologia , Fator de Transcrição STAT3/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Sermorelina/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Uveíte/tratamento farmacológico , Uveíte/imunologiaRESUMO
BACKGROUND: Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE-1) as well as its transforming effect in a xenograft model. METHODS: We performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)-2 activity by means gelatin zymography, GHRH-R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE-1 cells. RESULTS: In cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP-2, expression of VEGF, as well as E-cadherin-mediated cell-cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE-1 tumors developed by subcutaneous injection of GHRH-treated cells in athymic nude mice, 49 days after inoculation. CONCLUSIONS: Thus, GHRH appears to act as a cytokine in the transformation of RWPE-1 cells by mechanisms that likely involve epithelial-mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.
Assuntos
Neoplasias da Próstata , Sermorelina , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células Epiteliais/metabolismo , Hormônio Liberador de Hormônio do Crescimento , Humanos , Masculino , Camundongos , Camundongos Nus , Próstata/patologia , Neoplasias da Próstata/patologia , Sermorelina/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.
Assuntos
Apoptose/efeitos dos fármacos , Decídua/citologia , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Células Estromais/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Decídua/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Gravidez , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Células Estromais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
Anxiety and depression have been suggested to increase the risk for post-traumatic stress disorders (PTSD). A link between all these mental illnesses, inflammation and oxidative stress is also well established. Recent behavior studies by our group clearly demonstrate a powerful anxiolytic and antidepressant-like effects of a novel growth hormone releasing hormone (GHRH) antagonist of MIAMI class, MIA-690, probably related to modulatory effects on the inflammatory and oxidative status. In the present work we investigated the potential beneficial effects of MIA-602, another recently developed GHRH antagonist, in mood disorders, as anxiety and depression, and the possible brain pathways involved in its protective activity, in adult mice. MIA-602 exhibited antinflammatory and antioxidant effects in ex vivo and in vivo experimental models, inducing anxiolytic and antidepressant-like behavior in mice subcutaneously treated for 4 weeks. The beneficial effect of MIA-602 on inflammatory and oxidative status and synaptogenesis resulting in anxiolytic and antidepressant-like effects could be related by increases of nuclear factor erythroid 2-related factor 2 (Nrf2) and of brain-derived neurotrophic factor (BDNF) signaling pathways in the hippocampus and prefrontal cortex. These results strongly suggest that GHRH analogs should be tried clinically for the treatment of mood disorders including PTSD.
Assuntos
Transtornos de Estresse Pós-Traumáticos , Animais , Fator Neurotrófico Derivado do Encéfalo , Camundongos , Transtornos do Humor/tratamento farmacológico , Receptores de Neuropeptídeos , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológicoRESUMO
Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Ferroptose/efeitos dos fármacos , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Células HEK293 , Células HT29 , Humanos , Insulina/metabolismo , Ferro/metabolismo , Ácido Linoleico/metabolismo , Camundongos , Mitotano/toxicidade , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Selênio/metabolismo , Sermorelina/análogos & derivados , Sermorelina/farmacologia , Transferrina/metabolismoRESUMO
Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 µg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.
Assuntos
Antineoplásicos/farmacologia , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Mesotelioma Maligno , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Pleurais/tratamento farmacológico , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pleural mesothelioma (PM) is an aggressive cancer with poor prognosis and no effective therapies, mainly caused by exposure to asbestos. Antagonists of growth hormone-releasing hormone (GHRH) display strong antitumor effects in many experimental cancers, including lung cancer and mesothelioma. Here, we aimed to determine whether GHRH antagonist MIA-690 potentiates the antitumor effect of cisplatin and pemetrexed in PM. In vitro, MIA-690, in combination with cisplatin and pemetrexed, synergistically reduced cell viability, restrained cell proliferation and enhanced apoptosis, compared with drugs alone. In vivo, the same combination resulted in a strong growth inhibition of MSTO-211H xenografts, decreased tumor cell proliferation and increased apoptosis. Mechanistically, MIA-690, particularly with chemotherapeutic drugs, inhibited proliferative and oncogenic pathways, such as MAPK ERK1/2 and cMyc, and downregulated cyclin D1 and B1 mRNAs. Inflammatory pathways such as NF-kB and STAT3 were also reduced, as well as oxidative, angiogenic and tumorigenic markers (iNOS, COX-2, MMP2, MMP9 and HMGB1) and growth factors (VEGF and IGF-1). Overall, these findings strongly suggest that GHRH antagonists of MIA class, such as MIA-690, could increase the efficacy of standard therapy in PM.
Assuntos
Proteína HMGB1 , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Ciclina D1 , Ciclo-Oxigenase 2 , Hormônio Liberador de Hormônio do Crescimento , Humanos , Fator de Crescimento Insulin-Like I/uso terapêutico , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz/genética , Mesotelioma/tratamento farmacológico , Mesotelioma/patologia , NF-kappa B/metabolismo , Pemetrexede/farmacologia , Pemetrexede/uso terapêutico , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.
Assuntos
Processamento Alternativo , Neoplasias do Endométrio , Hormônio Liberador de Hormônio do Crescimento/genética , Receptores de Neuropeptídeos/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Primers do DNA , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Feminino , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Age-related diseases such as cardiovascular diseases portend disability, increase health expenditures, and cause late-life mortality. Synthetic agonists of growth hormone-releasing hormone (GHRH) exhibit several favorable effects on heart function and remodeling. Here we assessed whether GHRH agonist MR409 can modulate heart function and systemic parameters in old mice. Starting at the age of 15 months, mice were injected subcutaneously with MR409 (10 µg/day, n = 8) or vehicle (n = 7) daily for 6 months. Mice treated with MR409 showed improvements in exercise activity, cardiac function, survival rate, immune function, and hair growth in comparison with the controls. More stem cell colonies were grown out of the bone marrow recovered from the MR409-treated mice. Mitochondrial functions of cardiomyocytes (CMs) from the MR409-treated mice were also significantly improved with more mitochondrial fusion. Fewer ß-gal positive cells were observed in endothelial cells after 10 passages with MR409. In Doxorubicin-treated H9C2 cardiomyocytes, cell senescence marker p21 and reactive oxygen species were significantly reduced after cultured with MR409. MR409 also improved cellular ATP production and oxygen consumption rate in Doxorubicin-treated H9C2 cells. Mitochondrial protein OPA1 long isoform was significantly increased after treatment with MR409. The effects of MR409 were mediated by GHRH receptor and protein kinase A (PKA). In short, GHRH agonist MR409 reversed the aging-associated changes with respect of heart function, mobility, hair growth, cellular energy production, and senescence biomarkers. The improvement of heart function may be related to a better mitochondrial functions through GHRH receptor/cAMP/PKA/OPA1 signaling pathway and relieved cardiac inflammation.