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Renal ischemia-reperfusion is a common cause of acute kidney injury leading to significant morbidity and mortality. There are no effective treatments available in clinical practice. This meta-analysis aims to assess the effect of IL-10 immunotherapy on renal ischemia-reperfusion injury. Medline, Embase, Cochrane-library, Google Scholar and clinicaltrials.gov were searched up to 31 March 2023. Preclinical and clinical interventional studies investigating IL-10 immunotherapy for renal ischemia-reperfusion were eligible for inclusion. The primary endpoint was renal function (serum creatinine) following ischemia-reperfusion. The secondary endpoints included mitochondrial integrity, cellular proliferation, regulated cell death (TUNEL assay), expression of inflammatory cytokines (TNF-α, IL-6 and IL-1ß), M1/M2 macrophage polarization, tissue integrity (tubular injury score), long-term kidney fibrosis (fibrotic area %) and adverse events (pulmonary toxicity, cardiotoxicity hepatotoxicity). The search returned 861 records. From these, 16 full texts were screened and subsequently, seven animal studies, corresponding to a population of 268 mice/rats, were included. Compared to the control treatment, IL-10 immunotherapy reduced serum creatinine more effectively within 24 h of administration (95% CI: -9.177, -5.601, I2 = 22.42%). IL-10 immunotherapy promoted mitochondrial integrity and cellular proliferation and reduced regulated cell death (95% CI: -11.000, -4.184, I2 = 74.94%). It decreased the expression of TNF-α, IL-6 and IL-1ß, led to M2 polarization of the local macrophages, reduced tubular injury score (95% CI: -8.917, -5.755, I2 = 22.71%), and long-term kidney fibrosis (95% CI: -6.963, -3.438, I2 = 0%). No adverse outcomes were captured. In Conclusion, IL-10 immunotherapy safely improves outcomes in animal models of renal ischemia-reperfusion; the translational potential of IL-10 immunotherapy needs to be further investigated in clinical trials.
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Interleucina-10 , Traumatismo por Reperfusão , Traumatismo por Reperfusão/terapia , Animais , Interleucina-10/metabolismo , Humanos , Imunoterapia/métodos , Rim/patologia , Rim/metabolismo , Injúria Renal Aguda/terapia , CamundongosRESUMO
The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.
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Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , RNA de Cadeia Dupla/uso terapêutico , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo/cirurgia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/radioterapia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ablação por Radiofrequência , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance.
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Proteínas Estimuladoras de Ligação a CCAAT/genética , Inflamação/terapia , Monócitos/efeitos dos fármacos , RNA/genética , Animais , Anti-Inflamatórios/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Interleucina-10/genética , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Camundongos , Monócitos/metabolismo , RNA/farmacologia , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic.
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Neoplasias Hepáticas/genética , RNA de Cadeia Dupla/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , Biologia Computacional/métodos , Células Hep G2 , Humanos , Neoplasias Hepáticas/terapia , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) remains dismal despite current chemotherapeutic agents and inhibitors of molecular targets. As the incidence of PDAC constantly increases, more effective multidrug approaches must be made. Here, we report a novel method of delivering antitumorigenic therapy in PDAC by upregulating the transcriptional factor CCAAT/enhancer-binding protein-α (C/EBPα), recognized for its antiproliferative effects. Small activating RNA (saRNA) duplexes designed to increase C/EBPα expression were linked onto PDAC-specific 2'-Fluropyrimidine RNA aptamers (2'F-RNA) - P19 and P1 for construction of a cell type-specific delivery vehicle. Both P19- and P1-C/EBPα-saRNA conjugates increased expression of C/EBPα and significantly suppressed cell proliferation. Tail vein injection of the saRNA/aptamer conjugates in PANC-1 and in gemcitabine-resistant AsPC-1 mouse-xenografts led to reduced tumor size with no observed toxicity. To exploit the specificity of the P19/P1 aptamers for PDAC cells, we also assessed if conjugation with Cy3 would allow it to be used as a diagnostic tool on archival human pancreatic duodenectomy tissue sections. Scoring pattern from 72 patients suggested a positive correlation between high fluorescent signal in the high mortality patient groups. We propose a novel aptamer-based strategy for delivery of targeted molecular therapy in advanced PDAC where current modalities fail.
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Aptâmeros de Nucleotídeos/administração & dosagem , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Carcinoma Ductal Pancreático/terapia , Neoplasias Pancreáticas/terapia , RNA/administração & dosagem , Animais , Aptâmeros de Nucleotídeos/farmacologia , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Especificidade de Órgãos , Neoplasias Pancreáticas/genética , RNA/farmacologia , Resultado do Tratamento , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The prognosis for hepatocellular carcinoma (HCC) remains poor and has not improved in over two decades. Most patients with advanced HCC who are not eligible for surgery have limited treatment options due to poor liver function or large, unresectable tumors. Although sorafenib is the standard-of-care treatment for these patients, only a small number respond. For the remaining, the outlook remains bleak. A better approach to target "undruggable" molecular pathways that reverse HCC is therefore urgently needed. Small activating RNAs (saRNAs) may provide a novel strategy to activate expression of genes that become dysregulated in chronic disease. The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), a critical regulator of hepatocyte function, is suppressed in many advanced liver diseases. By using an saRNA to activate C/EBPα, we can exploit the cell's own transcription machinery to enhance gene expression without relying on exogenous vectors that have been the backbone of gene therapy. saRNAs do not integrate into the host genome and can be modified to avoid immune stimulation. In preclinical models of liver disease, treatment with C/EBPα saRNA has shown reduction in tumor volume and improvement in serum markers of essential liver function such as albumin, bilirubin, aspartate aminotransferase (AST), and alanine transaminase (ALT). This saRNA that activates C/EBPα for advanced HCC is the first saRNA therapy to have entered a human clinical trial. The hope is that this new tool will help break the dismal 20-year trend and provide a more positive prognosis for patients with severe liver disease.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Pequeno RNA não Traduzido/uso terapêutico , Humanos , FígadoRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. CONCLUSION: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.
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Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Terapia Genética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , RNA/uso terapêutico , Albuminas/metabolismo , Animais , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Injeções Intravenosas , Fígado/patologia , Cirrose Hepática/complicações , Testes de Função Hepática , Neoplasias Hepáticas Experimentais/complicações , Neoplasias Hepáticas Experimentais/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Wistar , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.
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Antígenos CD34/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura Livres de Soro , Citocinas/genética , Citocinas/metabolismo , Humanos , Regeneração Hepática/efeitos dos fármacos , Masculino , Cultura Primária de Células , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Ratos , TranscriptomaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic's such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.
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Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1ß), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.
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Síndrome Metabólica , Humanos , Síndrome Metabólica/genética , Síndrome Metabólica/terapia , RNA Mensageiro , Expressão Gênica , Lipídeos , Sirtuína 1/genéticaRESUMO
INTRODUCTION: Cannabinoids are a group of terpenophenolic compounds derived from the Cannabis sativa L. plant. There is a growing body of evidence from cell culture and animal studies in support of cannabinoids possessing anticancer properties. METHOD: A database search of peer reviewed articles published in English as full texts between January 1970 and April 2021 in Google Scholar, MEDLINE, PubMed and Web of Science was undertaken. References of relevant literature were searched to identify additional studies to construct a narrative literature review of oncological effects of cannabinoids in pre-clinical and clinical studies in various cancer types. RESULTS: Phyto-, endogenous and synthetic cannabinoids demonstrated antitumour effects both in vitro and in vivo. However, these effects are dependent on cancer type, the concentration and preparation of the cannabinoid and the abundance of receptor targets. The mechanism of action of synthetic cannabinoids, (-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) has mainly been described via the traditional cannabinoid receptors; CB1 and CB2, but reports have also indicated evidence of activity through GPR55, TRPM8 and other ion channels including TRPA1, TRPV1 and TRPV2. CONCLUSION: Cannabinoids have shown to be efficacious both as a single agent and in combination with antineoplastic drugs. These effects have occurred through various receptors and ligands and modulation of signalling pathways involved in hallmarks of cancer pathology. There is a need for further studies to characterise its mode of action at the molecular level and to delineate efficacious dosage and route of administration in addition to synergistic regimes.
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Canabinoides/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , HumanosRESUMO
PURPOSE: To evaluate the mechanisms of how therapeutic upregulation of the transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα), prevents tumor progression in patients with advanced hepatocellular carcinoma (HCC) and in different mouse tumor models. EXPERIMENTAL DESIGN: We conducted a phase I trial in 36 patients with HCC (NCT02716012) who received sorafenib as part of their standard care, and were given therapeutic C/EBPα small activating RNA (saRNA; MTL-CEBPA) as either neoadjuvant or adjuvant treatment. In the preclinical setting, the effects of MTL-CEBPA were assessed in several mouse models, including BNL-1ME liver cancer, Lewis lung carcinoma (LLC), and colon adenocarcinoma (MC38). RESULTS: MTL-CEBPA treatment caused radiologic regression of tumors in 26.7% of HCC patients with an underlying viral etiology with 3 complete responders. MTL-CEBPA treatment in those patients caused a marked decrease in peripheral blood monocytic myeloid-derived suppressor cell (M-MDSC) numbers and an overall reduction in the numbers of protumoral M2 tumor-associated macrophages (TAM). Gene and protein analysis of patient leukocytes following treatment showed CEBPA activation affected regulation of factors involved in immune-suppressive activity. To corroborate this observation, treatment of all the mouse tumor models with MTL-CEBPA led to a reversal in the suppressive activity of M-MDSCs and TAMs, but not polymorphonuclear MDSCs (PMN-MDSC). The antitumor effects of MTL-CEBPA in these tumor models showed dependency on T cells. This was accentuated when MTL-CEBPA was combined with checkpoint inhibitors or with PMN-MDSC-targeted immunotherapy. CONCLUSIONS: This report demonstrates that therapeutic upregulation of the transcription factor C/EBPα causes inactivation of immune-suppressive myeloid cells with potent antitumor responses across different tumor models and in cancer patients. MTL-CEBPA is currently being investigated in combination with pembrolizumab in a phase I/Ib multicenter clinical study (NCT04105335).
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Antineoplásicos/uso terapêutico , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Células Mieloides/fisiologia , Sorafenibe/uso terapêutico , Regulação para Cima , Animais , Humanos , Camundongos , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Non-alcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. Because there are no approved pharmacological treatment agents for non-alcoholic steatohepatitis (NASH) and NAFLD, different signaling pathways are under investigation for drug development with the focus on metabolic pathways. Hepatocyte nuclear factor 4-alpha (HNF4A) is at the center of a complex transcriptional network where its disruption is directly linked to glucose and lipid metabolism. Resetting HNF4A expression in NAFLD is therefore crucial for re-establishing normal liver function. Here, small activating RNA (saRNA) specific for upregulating HNF4A was injected into rats fed a high-fat diet for 16 weeks. Intravenous delivery was carried out using 5-(G5)-triethanolamine-core polyamidoamine (PAMAM) dendrimers. We observed a significant reduction in liver triglyceride, increased high-density lipoprotein/low-density lipoprotein (HDL/LDL) ratio, and decreased white adipose tissue/body weight ratio, all parameters to suggest that HNF4A-saRNA treatment induced a favorable metabolic profile. Proteomic analysis showed significant regulation of genes involved in sphingolipid metabolism, fatty acid ß-oxidation, ketogenesis, detoxification of reactive oxygen species, and lipid transport. We demonstrate that HNF4A activation by oligonucleotide therapy may represent a novel single agent for the treatment of NAFLD and insulin resistance.
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PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.
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Antineoplásicos/administração & dosagem , Proteínas Estimuladoras de Ligação a CCAAT/agonistas , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oligorribonucleotídeos/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Infusões Intravenosas , Lipossomos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Nanopartículas/administração & dosagem , Estadiamento de Neoplasias , Oligorribonucleotídeos/efeitos adversos , Oligorribonucleotídeos/farmacocinética , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPß occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.
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Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , RNA Interferente Pequeno/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica/genética , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Proteínas Nucleares/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies; it preferentially metastasizes to the liver and is the main cause of death from this disease. In previous studies, small activating RNA against CCAAT/enhancer-binding protein-α (C/EBPα-saRNA) demonstrated efficacy of PDAC in a local subcutaneous tumor model. In this study, we focused on the efficacy of C/EBPα-saRNA in advanced stage PDAC. For targeted delivery, we selected a new anti-transferrin receptor aptamer (TR14), which demonstrated a high binding affinity to target proteins. The TR14 aptamer was internalized with clathrin-mediated endocytosis, distributed in early endosome, late endosome, and lysosome subcellularly. To investigate its anti-tumor effects to advanced PDAC, we conjugated C/EBPα-saRNA to TR14. Treatment of pancreatic cancer cells with the conjugates upregulated expression of C/EBPα and its downstream target p21, and inhibited cell proliferation. For in vivo assays, we established an advanced PDAC mouse model by engrafting luciferase reporter-PANC-1 cells directly into the livers of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After treatment of aptamer-C/EBPα conjugates, we observed significant reduction of tumor growth in this advanced PDAC mouse model. Combinational treatment of the conjugates with gemcitabine also demonstrated enhanced anti-tumor effects in advanced PDAC. This suggests that aptamer-C/EBPα conjugates could be used as an adjuvant, along with other conventional anti-cancer drugs in advanced PDAC. In conclusion, targeted delivery of C/EBPα-saRNAs by aptamers might have potential therapeutic effects in advanced PDAC.
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Aspirin and clopidogrel are used therapeutically for their anti-platelet effects. We examined the effects of aspirin and clopidogrel on basal and beta-adrenoceptor (beta-AR)-mediated platelet nitric oxide (NO) synthesis in healthy subjects and patients with coronary heart disease (CHD). Healthy subjects (n=19) were randomized in a double-blind cross-over manner to receive aspirin or clopidogrel, each at 75 mg daily, for 14 days. Patients (n=17) of similar age with CHD, taking aspirin, were randomized double-blind to either continue on aspirin 75 mg daily or to receive clopidogrel 75 mg daily for 14 days. NO synthase (NOS) activity was measured from l-[(3)H]arginine to l-[(3)H]citrulline conversion, and cGMP was determined by radioimmunoassay, in platelets basally and following incubation with isoproterenol or albuterol (each at 10(-5) mol/L). In healthy subjects, aspirin did not affect basal NOS activity or cGMP in platelets, but suppressed the normal increase in both by isoproterenol and albuterol. Clopidogrel suppressed platelet NOS activity and cGMP both basally and in response to beta-AR agonists. In platelets from CHD patients, clopidogrel suppressed basal and beta-AR-stimulated NOS activity and cGMP as compared with aspirin. Platelet NOS activity and cGMP were lower in CHD subjects pre-randomization compared with healthy subjects both pre-randomization and post-aspirin. We conclude that chronic aspirin treatment suppresses beta-AR-stimulated but not basal platelet NO synthesis, as previously described, whereas chronic clopidogrel treatment suppresses both, with resultant functional consequences. Moreover, CHD may itself be associated with decreased platelet NO biosynthesis.
Assuntos
Aspirina/uso terapêutico , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Óxido Nítrico/biossíntese , Inibidores da Agregação Plaquetária/uso terapêutico , Ticlopidina/análogos & derivados , Antagonistas Adrenérgicos beta/efeitos adversos , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Plaquetas/enzimologia , Clopidogrel , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/metabolismo , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ticlopidina/uso terapêuticoRESUMO
Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL-CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Terapia Genética/métodos , Cirrose Hepática Experimental/terapia , Pequeno RNA não Traduzido/farmacologia , Ativação Transcricional , Animais , Dietilnitrosamina/toxicidade , Doença Hepática Terminal/induzido quimicamente , Doença Hepática Terminal/genética , Doença Hepática Terminal/terapia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Cirrose Hepática Experimental/genética , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/terapia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/terapia , Pequeno RNA não Traduzido/administração & dosagem , Ratos Sprague-Dawley , Ratos WistarRESUMO
Aptamer-drug conjugates (ApDCs) have the potential to improve the therapeutic index of traditional chemotherapeutic agents due to their ability to deliver cytotoxic drugs specifically to cancer cells while sparing normal cells. This study reports on the conjugation of cytotoxic drugs to an aptamer previously described by our group, the pancreatic cancer RNA aptamer P19. To this end, P19 was incorporated with gemcitabine and 5-fluorouracil (5-FU), or conjugated to monomethyl auristatin E (MMAE) and derivative of maytansine 1 (DM1). The ApDCs P19-dFdCMP and P19-5FdUMP were shown to induce the phosphorylation of histone H2AX on Ser139 (γ-H2AX) and significantly inhibited cell proliferation by 51%-53% in PANC-1 and by 54%-34% in the gemcitabine-resistant pancreatic cancer cell line AsPC-1 (p ≤ 0.0001). P19-MMAE and P19-DM1 caused mitotic G2/M phase arrest and inhibited cell proliferation by up to 56% in a dose-dependent manner when compared to the control group (p ≤ 0.001). In addition, the cytotoxicity of P19-MMAE and P19-DM1 in normal cells and the control human breast cancer cell line MCF7 was minimal. These results suggest that this approach may be useful in decreasing cytotoxic side effects in non-tumoral tissue.
RESUMO
OBJECTIVE: We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets. METHODS: NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration. RESULTS: Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P<0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation. CONCLUSIONS: Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.