RESUMO
BACKGROUND: Patients with borderline resectable colorectal liver metastases (CRLM) frequently receive neoadjuvant chemotherapy (NC) to reduce tumour burden, thus making surgical resection feasible. Even though NC can induce severe liver injury, most studies investigating tissue-based prognostic markers focus on tumour tissue. Here, we assessed the prognostic significance of pyruvate-dehydrogenase-kinase isoenzyme 4 (PDK4) within liver tissue of patients undergoing surgical resection due to CRLM. METHODS: Transcript levels of hypoxia-adaptive genes (such as PDK isoenzymes) were assessed in the tissue of healthy liver, corresponding CRLM, healthy colon mucosa and corresponding tumour. Uni- and multivariate analyses were performed. Responses to chemotherapy upon up- or down-regulation of PDK4 were studied in vitro. RESULTS: PDK4 expression within healthy liver tissue was associated with increased overall survival and liver function following surgical resection of CRLM. This association was enhanced in patients with NC. PDK4 expression in CRLM tissue did not correlate with overall survival. Up-regulation of PDK4 increased the resistance of hepatocytes and colon cancer cells against chemotherapy-induced toxicity, whereas knockdown of PDK4 enhanced chemotherapy-associated cell damage. CONCLUSION: Our findings suggest that up-regulated PDK4 expression reduces hepatic chemotherapy-induced oxidative stress and is associated with improved postoperative liver function in patients undergoing multimodal treatment and resection of CRLM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/terapia , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/terapia , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Idoso , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Terapia Combinada , Regulação para Baixo , Feminino , Fenofibrato/farmacologia , Fluoruracila/farmacologia , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatectomia , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Metastasectomia , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Oxaliplatina/farmacologia , Prognóstico , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Regulação para CimaRESUMO
Liver fibrosis, eventually progressing to cirrhosis necessitating liver transplantation, poses a significant clinical problem. Oxygen shortage (hypoxia) and hypoxia-inducible transcription factors (HIFs) have been acknowledged as important drivers of liver fibrosis. The significance of oxygen-sensing HIF prolyl-hydroxylase (PHD) enzymes in this context has, however, remained elusive. In this study, we demonstrate that loss of PHD1 (PHD1-/-) attenuates the development of liver fibrosis in mice subjected to chronic bile duct injury, induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. This effect was accompanied with reduced recruitment of inflammatory leukocytes and attenuated occurrence of profibrotic myofibroblasts in PHD1-/- livers. Further analyses focused on the significance of PHD1 in the activation of hepatic stellate cells (HSCs), which represent the driving force in liver fibrosis. Primary HSCs isolated from PHD1-/- mice displayed significantly attenuated myofibroblast differentiation and profibrogenic properties compared with HSCs isolated from wild-type mice. Consistently, the expression of various profibrogenic and promitogenic factors was reduced in PHD1-/- HSCs, without alterations in HIF-1α protein levels. Of importance, PHD1 protein was expressed in HSCs within human livers, and PHD1 transcript expression was significantly increased with disease severity in hepatic tissue from patients with liver fibrosis. Collectively, these findings indicate that PHD1 deficiency protects against liver fibrosis and that these effects are partly due to attenuated activation of HSCs. PHD1 may represent a therapeutic target to alleviate liver fibrosis.
Assuntos
Ductos Biliares/patologia , Fibrose/patologia , Células Estreladas do Fígado/patologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Cirrose Hepática/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Índice de Gravidade de Doença , Animais , Ductos Biliares/metabolismo , Células Cultivadas , Fibrose/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.
Assuntos
Aminoácidos Dicarboxílicos/uso terapêutico , Dermatite Alérgica de Contato/prevenção & controle , Oxigenases de Função Mista/antagonistas & inibidores , Aminoácidos Dicarboxílicos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Eosinófilos/citologia , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamação/tratamento farmacológico , Camundongos , Neutrófilos/citologiaRESUMO
PURPOSE: DLG7 (disc large homolog 7) is a microtubule-associated protein encoded by DLGAP5 (DLG associated protein 5) gene and has an important role during spindle assembly. Spindle assembly deregulation is a well-known cause of genomic instability. The aim of this study was to investigate the influence of DLGAP5 expression on survival and to evaluate its potential use as a biomarker in colorectal cancer (CRC). METHODS: DLGAP5 expression was measured in the primary tumor and corresponding normal mucosa samples from 109 patients with CRC and correlated to clinical and pathological data. The results were validated in a second, publically available patient cohort. Molecular effects of DLG7/DLGAP5 in CRC were analyzed via functional assays in knockdown cell lines. RESULTS: DLGAP5 downregulation led to a significant reduction of the invasion and migration potential in CRC. In addition, DLGAP5 expression correlates with nodal status and advanced UICC stage (III-IV).Subgroup analyses revealed a correlation between DLGAP5 overexpression and poor survival in patients with non-metastatic disease (M0). Furthermore, overexpression of DLGAP5 is associated with worse overall survival in distinct molecular CRC subtypes. CONCLUSIONS: The results of this study suggest the importance of DLGAP5 in defining a more aggressive CRC phenotype. DLG7/DLGAP5 represents a potential biomarker for CRC in molecular subgroups of CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Instabilidade Cromossômica , Neoplasias Colorretais/patologia , Feminino , Humanos , Intestinos/patologia , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/patologia , Prognóstico , Células-Tronco/metabolismo , Análise de SobrevidaRESUMO
Ulceration in the bronchial mucosa is noted rarely in bronchoscopy. In the past, it was frequently encountered in endobronchial tuberculosis. Deep necrotic bronchial ulcers are seen very rarely in clinical practice. Here we are reporting a first-ever case report of malignant bronchial ulcer presenting as necrotic deep bronchial ulcer, in a 70-year-old male, chronic smoker, who complained of breathlessness for 3 months, cough for 3 months, loss of weight and of appetite for 1 month. Bronchoscopy showed a large necrotic ulcer with dense anthracotic pigmentation which bleeds in touch with forceps. Bronchial washings, brushings, endobronchial biopsy were taken from the ulcer which was suggestive of poorly differentiated bronchogenic carcinoma. TBNA from the mediastinal nodes showed the features of caseous necrosis with granulomatous inflammation. Consequently, with the diagnosis of poorly differentiated carcinoma with pulmonary tuberculosis and COPD, the patient was started on anti-tuberculosis drugs, inhaled bronchodilators and referred to an oncologist for chemotherapy.
Assuntos
Broncopatias/complicações , Carcinoma Broncogênico/complicações , Neoplasias Pulmonares/complicações , Tuberculose Pulmonar/complicações , Úlcera/complicações , Idoso , Broncopatias/diagnóstico , Broncopatias/etiologia , Broncoscopia , Carcinoma Broncogênico/diagnóstico , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Mucosa/patologia , Necrose , Tuberculose Pulmonar/diagnóstico , Úlcera/diagnóstico , Úlcera/etiologiaRESUMO
OBJECTIVE: We sought to assess whether pharmacological inhibition of hypoxia-inducible transcription factor (HIF)-prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) is a suitable strategy to stimulate liver regeneration after partial hepatectomy for colorectal liver metastases (CRLM). BACKGROUND: Liver regeneration occurs in a hypoxic environment. PHD1 to PHD3 are molecular oxygen sensors and increasingly considered as putative therapeutic targets. However, little is known about the effect of pharmacological PHD inhibition on tumor expansion, and on liver regeneration after surgical resection. METHODS: Various mouse models of liver regeneration after extended partial hepatectomy and portal vein ligation for multiple bilobar CRLM were applied to assess the effect of the small molecule pan-PHD inhibitor ethyl-3,4-dihydroxybenzoate (EDHB) on liver regeneration and metastatic tumor growth. Metabolism and biodistribution of EDHB were analyzed using liquid chromatography coupled to tandem mass spectrometry. RESULTS: EDHB selectively augmented liver regeneration after partial hepatectomy and portal vein ligation, and increased the expression of cell cycle-promoting cyclin proteins, without enhancing metastatic tumor growth. Systemically administered EDHB and its active metabolite 3,4-dihydroxybenzoic acid accumulated in the liver to selectively induce hepatoprotective effects in the liver, but not in tumor tissue, without humoral adverse effects. CONCLUSIONS: Pharmacological inhibition of PHDs using EDHB might represent a novel and safe strategy in the treatment of multiple bilobar CRLM.
Assuntos
Neoplasias Colorretais/patologia , Hepatectomia/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/secundário , Regeneração Hepática/efeitos dos fármacos , Inibidores de Prolil-Hidrolase/farmacologia , Análise de Variância , Animais , Western Blotting , Neoplasias Colorretais/cirurgia , Modelos Animais de Doenças , Feminino , Neoplasias Hepáticas/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
BACKGROUND: The Empty Spiracles Homeobox (EMX-) 2 gene has been associated with regulation of growth and differentiation in neuronal development. While recent studies provide evidence that EMX2 regulates tumorigenesis of various solid tumors, its role in colorectal cancer remains unknown. We aimed to assess the prognostic significance of EMX2 expression in stage III colorectal adenocarcinoma. METHODS: Expression levels of EMX2 in human colorectal cancer and adjacent mucosa were assessed by qRT-PCR technology, and results were correlated with clinical and survival data. siRNA-mediated knockdown and adenoviral delivery-mediated overexpression of EMX2 were performed in order to investigate its effects on the migration of colorectal cancer cells in vitro. RESULTS: Compared to corresponding healthy mucosa, colorectal tumor samples had decreased EMX2 expression levels. Furthermore, EMX2 down-regulation in colorectal cancer tissue was associated with distant metastasis (M1) and impaired overall patient survival. In vitro knockdown of EMX2 resulted in increased tumor cell migration. Conversely, overexpression of EMX2 led to an inhibition of tumor cell migration. CONCLUSIONS: EMX2 is frequently down-regulated in human colorectal cancer, and down-regulation of EMX2 is a prognostic marker for disease-free and overall survival. EMX2 might thus represent a promising therapeutic target in colorectal cancer.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Fatores de Transcrição/genética , Adenoviridae/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Estudos de Coortes , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Feminino , Seguimentos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/terapia , Masculino , Estadiamento de Neoplasias , Prognóstico , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease, a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases, which confer oxygen sensitivity upon the hypoxia-inducible factor pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate, followed by a period of recovery, developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine ameliorated fibrosis. TGF-ß1 is a key regulator of fibrosis that acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-ß1-induced expression of fibrotic markers in cultured fibroblasts, suggesting a direct role for hydroxylases in TGF-ß1 signaling. This was at least in part due to inhibition of noncanonical activation of extracellular signal-regulated kinase (ERK) signaling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis through suppression of TGF-ß1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress profibrotic pathways.
Assuntos
Colágeno/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Intestinos/patologia , Oxigenases de Função Mista/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista/antagonistas & inibidores , Transdução de SinaisRESUMO
BACKGROUND: Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials. METHODS: Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux. RESULTS: This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12. CONCLUSIONS: The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Pirróis/farmacologia , Compostos de Bifenilo/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Células HT29 , Humanos , Indóis , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3(-/-)) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1(-/-)) or PHD2 (PHD2(+/-)). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α- and NF-κB-mediated enhancement of the innate immune response.
Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Pró-Colágeno-Prolina Dioxigenase/imunologia , Sepse/imunologia , Transdução de Sinais/imunologia , Animais , Western Blotting , Quimiotaxia de Leucócito/imunologia , Citocinas/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , NF-kappa B/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sepse/metabolismoRESUMO
The COVID-19 pandemic caused lifestyle changes and has led to the new electricity demand patterns in the presence of non-pharmaceutical interventions such as work-from-home policy and lockdown. Quantifying the effect on electricity demand is critical for future electricity market planning yet challenging in the context of limited smart metered buildings, which leads to limited understanding of the temporal and spatial variations in building energy use. This study uses a large scale private smart meter electricity demand data from the City of Austin, combined with publicly available environmental data, and develops an ensemble regression model for long term daily electricity demand prediction. Using 15-min resolution data from over 400,000 smart meters from 2018 to 2020 aggregated by building type and zip code, our proposed model precisely formalizes the counterfactual universe in the without COVID-19 scenario. The model is used to understand building electricity demand changes during the pandemic and to identify relationships between such changes and socioeconomic patterns. Results indicate the increase in residential usage , demonstrating the spatial redistribution of energy consumption during the work-from-home period. Our experiments demonstrate the effectiveness of our proposed framework by assessing multiple socioeconomic impacts with the comparison between the counterfactual universe and observations.
RESUMO
PURPOSE: Liver regeneration after partial hepatectomy (PH) occurs in conditions of reduced oxygen supply. HIF prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) are oxygen sensors involved in adaptive response to hypoxia. Specific functions of these PHD enzymes in liver regeneration have, however, remained enigmatic. Here, we investigated the significance of PHD1 in liver regeneration following hepatectomy. METHODS: Liver regeneration was studied in PHD1-deficient (PHD1(-/-)) and wild type (WT) mice subjected to 80% hepatectomy. For in vitro analyses, hepatocytes were isolated from PHD1(-/-) and WT livers. Cell cycle progression was studied via FACS-based analysis of nuclear DNA profile. Transcription factor binding assays, qRT-PCR, and immunoblotting were applied to study the relevance of PHD1 downstream effectors during liver regeneration. RESULTS: Liver regeneration was significantly enhanced in PHD1(-/-) mice compared to WT littermates. This effect was due to enhanced proliferation rather than to hypertrophy of liver cells. Cell cycle progression was significantly enhanced, and transcriptional activity of the cell cycle regulator c-Myc was increased in PHD1-deficient hepatocytes. These changes coincided with increased expression of cyclin D2, a cell cycle-promoting c-Myc target, and decreased expression of the cell cycle-delaying c-Myc target p21. CONCLUSIONS: Loss of PHD1 enhances liver regeneration by boosting hepatocyte proliferation in a c-Myc-dependent fashion. PHD1 might, therefore, represent a potential target to facilitate liver regeneration after surgical resection.
Assuntos
Hepatectomia , Hepatócitos/metabolismo , Regeneração Hepática/fisiologia , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Ciclo Celular , Proliferação de Células , Células Cultivadas , Hepatócitos/citologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Ischemia and reperfusion injury (IRI) determines primary allograft function after liver transplantation (LT). Primary graft dysfunction (PGD) is associated with increased morbidity and impaired graft survival and can eventually progress to graft failure requiring retransplantation. Hypoxia-inducible transcription factor-prolyl hydroxylase containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors, which control the adaptive hypoxia response through the hypoxia-inducible factor (HIF). In this study, we have investigated pharmacological activation of the HIF pathway through inhibition of PHDs as a strategy to reduce PGD after LT. METHODS: Primary rat hepatocytes were isolated and the impact of the pan-PHD small-molecule inhibitor ethyl-3,4-dihydroxybenzoate (EDHB) on HIF-1 and its downstream target gene expression assessed. Subsequently, various rodent models of segmental warm liver ischemia and reperfusion and orthotopic LT were applied to study the impact of EDHB on normothermic or combined cold and warm liver IRI. Liver enzyme levels and histology were analyzed to quantify hepatic IRI. RESULTS: In vitro, EDHB induced HIF-1 signaling and significantly upregulated its downstream target heme-oxygenase 1 in primary rat hepatocytes. In vivo, after establishment of the optimal EDHB pretreatment conditions in a murine IRI model, EDHB pretreatment significantly mitigated hepatic IRI after warm segmental liver ischemia and reperfusion and allograft injury after orthotopic LT in rats. Mechanistically, EDHB stabilized HIF-1 in the liver and subsequently increased hepatoprotective heme-oxygenase 1 levels, which correlated with reduced hepatic IRI in these models. CONCLUSIONS: This proof-of-concept study establishes a strong therapeutic rationale for targeting PHDs with small-molecule inhibitors to mitigate PGD after LT.
Assuntos
Transplante de Fígado , Traumatismo por Reperfusão , Aloenxertos/metabolismo , Animais , Heme , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Transplante de Fígado/efeitos adversos , Camundongos , Oxigênio , Pró-Colágeno-Prolina Dioxigenase/genética , Prolil Hidroxilases/metabolismo , Ratos , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle , Fatores de TranscriçãoRESUMO
Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD). HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) control cellular adaptation to hypoxia and are considered promising therapeutic targets in IBD. However, their relevance in the pathogenesis of CAC remains elusive. We induced CAC in Phd1-/-, Phd2+/-, Phd3-/-, and WT mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Phd1-/- mice were protected against chronic colitis and displayed diminished CAC growth compared with WT mice. In Phd3-/- mice, colitis activity and CAC growth remained unaltered. In Phd2+/- mice, colitis activity was unaffected, but CAC growth was aggravated. Mechanistically, Phd2 deficiency (i) increased the number of tumor-associated macrophages in AOM/DSS-induced tumors, (ii) promoted the expression of EGFR ligand epiregulin in macrophages, and (iii) augmented the signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 signaling, which at least in part contributed to aggravated tumor cell proliferation in colitis-associated tumors. Consistently, Phd2 deficiency in hematopoietic (Vav:Cre-Phd2fl/fl) but not in intestinal epithelial cells (Villin:Cre-Phd2fl/fl) increased CAC growth. In conclusion, the 3 different PHD isoenzymes have distinct and nonredundant effects, promoting (PHD1), diminishing (PHD2), or neutral (PHD3), on CAC growth.
Assuntos
Neoplasias Associadas a Colite , Colite , Animais , Camundongos , Azoximetano , Colite/induzido quimicamente , Colite/complicações , Colite/metabolismo , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/metabolismo , Células Epiteliais/metabolismo , Prolil Hidroxilases/metabolismoRESUMO
Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2 haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immunomodulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favorable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.
Assuntos
Anastomose Cirúrgica , Colo/metabolismo , Macrófagos/metabolismo , Prolil Hidroxilases/metabolismo , Abdome/cirurgia , Aminoácidos Dicarboxílicos , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica , Animais , Células CACO-2 , Colágeno/metabolismo , Colo/patologia , Colo/cirurgia , Feminino , Humanos , Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Isquemia , Masculino , Camundongos , RNA Mensageiro/metabolismo , Sepse , CicatrizaçãoRESUMO
The characteristic desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a key contributor to its lethality. This stromal microenvironment is populated by cancer-associated fibroblasts (CAFs) that interact with cancer cells to drive progression and chemo-resistance. Research has focused on CAFs in the primary tumour but not in metastases, calling into question the role of analogous metastasis-associated fibroblasts (MAFs). We infer a role of MAFs in murine hepatic metastases following untargeted treatment with the anti-angiogenic drug sunitinib in vivo. Treated metastases were smaller and had fewer stromal cells, but were able to maintain angiogenesis and metastasis formation in the liver. Furthermore, sunitinib was ineffective at reducing MAFs alongside other stromal cells. We speculate that cancer cells interact with MAFs to maintain angiogenesis and tumour progression. Thus, we tested interactions between metastatic pancreatic cancer cells and fibroblasts using in vitro co-culture systems. Co-cultures enhanced fibroblast proliferation and induced angiogenesis. We identify carcinoma-educated fibroblasts as the source of angiogenesis via secretions of CXCL8 (aka IL-8) and CCL2 (aka MCP-1). Overall, we demonstrate that metastasis-associated fibroblasts have potential as a therapeutic target and highlight the CXCL8 and CCL2 axes for further investigation.
Assuntos
Quimiocina CCL2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Interleucina-8/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/metabolismo , Pâncreas/patologia , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente Tumoral/fisiologia , Neoplasias PancreáticasRESUMO
BACKGROUND: Pouchitis is the most common long-term complication after restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) or familial adenomatous polyposis (FAP), which can eventually progress to pouch failure, necessitating permanent stoma construction. Hypoxia-inducible transcription factor prolyl hydroxylase-containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors that control adaptive gene expression through hypoxia-inducible factor (HIF). Emerging evidence supports PHDs as being therapeutic targets in intestinal inflammation. However, pharmacological inhibition of PHDs has not been validated as a treatment strategy in pouchitis. METHODS: PHD1-3 mRNA and protein expression were analyzed in mucosal pouch and prepouch ileal patient biopsies. After establishment of a preclinical IPAA model in rats, the impact of the pan-PHD small-molecule inhibitor dimethyloxalylglycine (DMOG) on dextran sulfate sodium (DSS)-induced pouchitis was studied. Clinical and molecular parameters were investigated. RESULTS: PHD1, but not PHD2 or PHD3, was overexpressed in pouchitis in biopsies of patients with IPAA for UC but not FAP. In addition, PHD1 expression correlated with disease activity. DMOG treatment profoundly mitigated DSS-induced pouchitis in a rodent IPAA model. Mechanistically, DMOG restored intestinal epithelial barrier function by induction of tight junction proteins zona occludens-1 and claudin-1 and alleviation of intestinal epithelial cell apoptosis, thus attenuating pouch inflammation. CONCLUSIONS: Together, these results establish a strong therapeutic rationale for targeting PHD1 with small-molecule inhibitors in pouchitis after IPAA for UC.
Assuntos
Pouchite/prevenção & controle , Prolil Hidroxilases/química , Inibidores de Prolil-Hidrolase/uso terapêutico , Animais , Humanos , Pouchite/enzimologia , Pouchite/patologiaRESUMO
Tumors are influenced by the mechanical properties of their microenvironment. Using patient samples and atomic force microscopy, we found that tissue stiffness is higher in liver metastases than in primary colorectal tumors. Highly activated metastasis-associated fibroblasts increase tissue stiffness, which enhances angiogenesis and anti-angiogenic therapy resistance. Drugs targeting the renin-angiotensin system, normally prescribed to treat hypertension, inhibit fibroblast contraction and extracellular matrix deposition, thereby reducing liver metastases stiffening and increasing the anti-angiogenic effects of bevacizumab. Patients treated with bevacizumab showed prolonged survival when concomitantly treated with renin-angiotensin inhibitors, highlighting the importance of modulating the mechanical microenvironment for therapeutic regimens.
Assuntos
Bevacizumab/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Sinergismo Farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Sistema Renina-Angiotensina/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Fibroblastos Associados a Câncer/patologia , Captopril/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Losartan/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Microambiente Tumoral/efeitos dos fármacosRESUMO
INTRODUCTION: Pleural biopsy is the commonest mode of obtaining thoracoscopic pleural specimens from suspected pleural lesions. However, this may be associated with arisk of bleeding in certain cases. The decision to perform biopsy could be difficult, especially when the lesions are close to vascular structures and the visceral pleura. So, pleural brushing can be used to get safely thoracoscopic specimens in addition to biopsy samples. AIM: To determine the sensitivity and specificity of thoracoscopic pleural brushing in exudative pleural effusions. MATERIAL AND METHODS: This prospective study was done in the Department of Pulmonary Medicine, Sri Manakula Vinayagar Medical College, Pondicherry, India on 80 patients with exudative pleural effusion in whom pleural fluid analysis and closed pleu-ral biopsy results were inconclusive. All these patients were subjected to medical thoracoscopy after getting informed consent. Pleural biopsy and pleural brushings were taken and sent for analysis. RESULTS: Thoracoscopic pleural biopsy was diagnostic in 76 of 80 patients (95%). Thoracoscopic pleural brushing was diagnostic in 74 patients (92.5%). Histopathology revealed malignancy (82.7%), granulomatous inflammation (11.5%) and nonspecific inflam-mation (5.7%). The sensitivity and specificity of pleural brushing were 96% and 75%, respectively. Interestingly, pleural brushing was the only diagnostic modality in one patient that was reported to be adenocarcinoma. CONCLUSIONS: Thoracoscopic pleural brushing is an easy, convenient and safe procedure as it can augment the diagnostic yield of thoracoscopy. It is of significant value, especially in sampling pleural lesions close to vessels and the visceral pleura compared to pleural biopsy.
Assuntos
Pleura/patologia , Doenças Pleurais/diagnóstico , Manejo de Espécimes/métodos , Toracoscopia/métodos , Adulto , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Doenças Pleurais/patologia , Derrame Pleural/diagnóstico , Estudos Prospectivos , Tuberculose Pleural/diagnósticoRESUMO
OBJECTIVES: Salinomycin is a polyether antibiotic with selective activity against human cancer stem cells. The impact of salinomycin on patient-derived primary human colorectal cancer cells has not been investigated so far. Thus, here we aimed to investigate the activity of salinomycin against tumor initiating cells isolated from patients with colorectal cancer. METHODS: Primary tumor-initiating cells (TIC) isolated from human patients with colorectal liver metastases or from human primary colon carcinoma were exposed to salinomycin and compared to treatment with 5-FU and oxaliplatin. TICs were injected subcutaneously into NOD/SCID mice to induce a patient-derived mouse xenograft model of colorectal cancer. Animals were treated either with salinomycin, FOLFOX regimen, or salinomycin and FOLFOX. Human colorectal cancer cells were used to delineate an underlying molecular mechanism of salinomycin in this tumor entity. RESULTS: Applying TICs isolated from human patients with colorectal liver metastases or from human primary colon carcinoma, we demonstrated that salinomycin exerts increased antiproliferative activity compared to 5-fluorouracil and oxaliplatin treatment. Consistently, salinomycin alone or in combination with FOLFOX exerts superior antitumor activity compared to FOLFOX therapy in a patient-derived mouse xenograft model of colorectal cancer. Salinomycin induces apoptosis of human colorectal cancer cells, accompanied by accumulation of dysfunctional mitochondria and reactive oxygen species. These effects are associated with expressional down-regulation of superoxide dismutase-1 (SOD1) in response to salinomycin treatment. CONCLUSION: Collectively, the results of this pre-clinical study indicate that salinomycin alone or in combination with 5-fluorouracil and oxaliplatin exerts increased antitumoral activity compared to common chemotherapy.