RESUMO
BACKGROUND: Curcumin is the main active ingredient of the spice turmeric, investigated extensively for putative anticancer properties. OBJECTIVES: This phase IIa open-labelled randomized controlled trial aimed to assess safety, efficacy, quality of life, neurotoxicity, curcuminoids, and C-X-C-motif chemokine ligand 1 (CXCL1) in patients receiving folinic acid/5-fluorouracil/oxaliplatin chemotherapy (FOLFOX) compared with FOLFOX + 2 g oral curcumin/d (CUFOX). METHODS: Twenty-eight patients aged >18 y with a histological diagnosis of metastatic colorectal cancer were randomly assigned (1:2) to receive either FOLFOX or CUFOX. Safety was assessed by Common Toxicity Criteria-Adverse Event reporting, and efficacy via progression-free survival (PFS) and overall survival (OS). Quality of life and neurotoxicity were assessed using questionnaires (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 and Functional Assessment of Cancer Treatment-Gynecologic Oncology Group-Neurotoxicity). Plasma curcuminoids were determined with liquid chromatography (LC) electrospray ionization tandem mass spectrometry and CXCL1 by ELISA. RESULTS: Addition of daily oral curcumin to FOLFOX chemotherapy was safe and tolerable (primary outcome). Similar adverse event profiles were observed for both arms. In the intention-to-treat population, the HR for PFS was 0.57 (95% CI: 0.24, 1.36; P = 0.2) (median of 171 and 291 d for FOLFOX and CUFOX, respectively) and for OS was 0.34 (95% CI: 0.14, 0.82; P = 0.02) (median of 200 and 502 d for FOLFOX and CUFOX, respectively). There was no significant difference between arms for quality of life (P = 0.248) or neurotoxicity (P = 0.223). Curcumin glucuronide was detectable at concentrations >1.00 pmol/mL in 15 of 18 patients receiving CUFOX. Curcumin did not significantly alter CXCL1 over time (P = 0.712). CONCLUSION: Curcumin is a safe and tolerable adjunct to FOLFOX chemotherapy in patients with metastatic colorectal cancer. This trial was registered at clinicaltrials.gov as NCT01490996 and at www.clinicaltrialsregister.eu as EudraCT 2011-002289-19.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Curcumina/uso terapêutico , Administração Oral , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Colorretais/patologia , Curcumina/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/uso terapêutico , Humanos , Leucovorina/administração & dosagem , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/uso terapêutico , Resultado do TratamentoRESUMO
IL-12 is a pro-inflammatory cytokine that induces the production of interferon-γ (IFNγ) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjögren's syndrome (pSS)(autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (nâ¯=â¯17) and pSS monocytes (nâ¯=â¯6) resulted in significant induction of IL12A mRNA (pâ¯<â¯0.05, pâ¯<â¯0.01, respectively) compared to PSC monocytes (nâ¯=â¯13) and at similar levels to HC monocytes (nâ¯=â¯8). PSC monocytes expressed significantly less IL-12p70 (108â¯pg/ml, mean) and IL-23 (358â¯pg/ml) compared to HC (458â¯pg/ml and 951â¯pg/ml, respectively) (pâ¯<â¯0.01, pâ¯<â¯0.05). Treg cells from patients with PBC (nâ¯=â¯16) and pSS (nâ¯=â¯3) but not PSC (nâ¯=â¯10) and HC (nâ¯=â¯8) responded to low dose (10â¯ng/ml) IL-12 stimulation by significant upregulation of IFNγ (mean 277 and 254â¯pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77â¯pg/ml, respectively)(pâ¯<â¯0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (pâ¯<â¯0.05) but not PSC and HC. In the liver of patients with PBC (nâ¯=â¯7) a significantly higher proportion of IL-12Rß2+Tregs (16% on average) was detected (pâ¯<â¯0.05) compared to other liver disease controls (5%)(nâ¯=â¯18) which also showed ex vivo high IFNG and TBET expression. CONCLUSION: Our data show an increased sensitivity of PBC and pSS Treg cells to low dose IL-12 stimulation, providing ongoing support for the importance of the IL12-IL-12Rß2-STAT4 pathway on Treg cells in disease pathogenesis and potentially treatment.
Assuntos
Colangite Esclerosante/imunologia , Interferon gama/imunologia , Subunidade p35 da Interleucina-12/imunologia , Cirrose Hepática Biliar/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Autoimunidade , Estudos de Casos e Controles , Diferenciação Celular , Colangite Esclerosante/genética , Colangite Esclerosante/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interleucina-12/genética , Interleucina-12/imunologia , Subunidade p35 da Interleucina-12/genética , Interleucina-23/genética , Interleucina-23/imunologia , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Fenótipo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/patologia , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity.
Assuntos
Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Puromicina Aminonucleosídeo/farmacologia , Células Cultivadas , Antagonismo de Drogas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Podócitos/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Albuminuria and elevated C-reactive protein (CRP) levels are common manifestations of many inflammatory diseases. Cardiovascular-based drugs, with secondary anti-inflammatory actions, such as angiotensin-converting enzyme-inhibitors are able to reduce both proteinuria and CRP levels, raising the question of whether CRP directly influences the processes that result in proteinuria. As proteinuria is thought to be induced as a result of podocyte dysfunction, we investigated whether there is a pathomechanistic link with CRP. METHODS: Podocytes were analysed for evidence of endogenous CRP production in response to inflammatory agents. In addition, they were incubated in the presence of various concentrations of exogenous CRP and analysed for evidence of a response to treatment. RESULTS: Our results demonstrated that inflammatory agents such as macrophage-conditioned medium and interleukin-1ß induced the expression of CRP messenger RNA in podocytes. However, they were unable to induce CRP protein. Stimulation of podocytes with exogenous CRP demonstrated that 10 µg/mL CRP induced a low but significant level of interleukin-6 secretion. Tumour necrosis factor α, however, was not detected. CRP did up-regulate the expression of the slit diaphragm proteins nephrin and CD2AP, as well as the structural proteins ezrin and podocalyxin-like protein-1, proteins known to be involved in signalling via the phosphotidylinositol-3 (PI-3) kinase pathway. CRP exposure reduced caspase-3 enzyme activity and up-regulated the expression of the anti-apoptotic protein Bcl-2. In the presence of the PI-3 kinase inhibitor LY294002, the ability of CRP to suppress caspase-3 activity was significantly reduced. CONCLUSIONS: Taken together, these data suggest that rather than inducing podocyte damage, CRP may be a survival factor for podocytes by maintaining their structural integrity and initiating a survival cascade, which may facilitate podocyte recovery from injury.
Assuntos
Proteína C-Reativa/metabolismo , Citoproteção , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Western Blotting , Proteína C-Reativa/antagonistas & inibidores , Proteína C-Reativa/genética , Caspase 3/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Inflamação , Interleucina-1beta/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Podócitos/patologia , Proteinúria/etiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDH(high)/CD133(-)). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Curcumina/administração & dosagem , Curcumina/efeitos adversos , Relação Dose-Resposta a Droga , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Xenoenxertos , Humanos , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Oxaliplatina , Esferoides CelularesRESUMO
Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including the turnover of proteases and the degradation of extracellular matrix proteins. As such, it can play a key role in the control of fibrosis. The aim of this investigation was to ascertain whether the anti-fibrotic effects exerted by the angiotensin-converting enzyme inhibitor (ACE-I) perindoprilat on macrophage-conditioned medium (MPCM)-injured human mesangial cells can be modulated by this receptor. Addition of receptor-associated protein to MPCM-injured mesangial cells with and without ACE-I increased the amount of tissue plasminogen activator protein detected in mesangial cell culture supernatants without affecting the protein levels of plasminogen activator inhibitor-1. The ability of ACE-I to reduce fibronectin was diminished in the presence of receptor-associated protein. ACE-I induced an increase in mesangial cell MMP9 mRNA, but reduced the MMP9 enzyme activity detected in mesangial cell supernatants. Mesangial cell lysates from ACE-I-treated cells were able to bind immobilized fibronectin at higher dilutions than cell lysates from untreated cells. Flow cytometry showed that MPCM induced an increase in LRP surface expression in mesangial cells over that in control cells and that this expression was further increased by ACE-I treatment. The increase in LRP expression in response to ACE-I was also observed by Western blotting. Northern blot analysis of RNA extracted from cells following a 24-h exposure to MPCM with and without ACE-I demonstrated that there was no change in LRP mRNA expression upon ACE-I treatment. In conclusion, we show that ACE-I treatment is able to modulate mesangial cell-surface expression of LRP, providing an additional mechanism whereby ACE-Is can mediate anti-fibrotic actions independent of their hemodynamic actions.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Células Mesangiais/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Meios de Cultivo Condicionados/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Regulação da Expressão Gênica/fisiologia , Humanos , Células Mesangiais/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Células U937RESUMO
The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.
Assuntos
Sistema Calicreína-Cinina/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Tiazepinas/farmacologia , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina , Meios de Cultivo Condicionados/toxicidade , Fibronectinas/metabolismo , Fibrose , Humanos , Sistema Calicreína-Cinina/fisiologia , Calicreínas/análise , Calicreínas/genética , Calicreínas/metabolismo , Cininogênios/análise , Cininogênios/genética , Cininogênios/metabolismo , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/genética , Ativador de Plasminogênio Tecidual/análiseRESUMO
BACKGROUND: The sympathetic nervous system is frequently activated in hypertension and may modify various aspects of renal function. Whether modulation of the sympathetic nervous system directly influences the development of renal fibrosis is yet to be established. The current study investigates the role of the alpha-1 adrenoceptor on human mesangial cell scarring. METHODS: Human mesangial cells were injured with macrophage-conditioned medium (MPCM) and treated with doxazosin for 1 or 3 days. RESULTS: alpha-1 Adrenoceptor antagonist doxazosin of 2 micromol/l reduced fibronectin protein in MPCM-injured female mesangial cells by 31 +/- 1.03% (P < 0.001) and by 9.5 +/- 0.3% (P = 0.01) in male mesangial cells. The differential response between sexes was significant (P = 0.004). alpha-1B Adrenoceptors were detected in human mesangial cells by reverse transcription-polymerase chain reaction with expression in female cells being 87% higher than in males (P = 0.04). Injury with MPCM reduced alpha-1B adrenoceptor mRNA expression in both cell types. Doxazosin had no effect on the protein levels of transforming growth factor-beta (TGF-beta) or interleukin-1beta (IL-1beta), however, a small reduction in tumour necrosis factor-alpha (TNF-alpha) levels was observed. Doxazosin had no effect on the modulators of matrix turnover matrix metalloproteinases MMP3, MMP9 and tissue inhibitor of matrix metalloproteinases (TIMP-1), although a significant reduction in tissue plasminogen activator (tPA); (36.5 +/- 2.6%, P < 0.001) was observed. Doxazosin caused an up-regulation of kallikrein expression, both at mRNA and protein levels. Co-treatment with the bradykinin B2 receptor antagonist HOE140 was able to attenuate the effects of doxazosin treatment on fibronectin levels. CONCLUSION: These data suggest that inhibition of alpha-1B adrenoceptors in mesangial cells exerts an anti-fibrotic effect in a sex-specific manner via modulation of the kallikrein-kinin/plasminogen activator system.
Assuntos
Fibronectinas/metabolismo , Mesângio Glomerular/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Northern Blotting , Células Cultivadas , Doxazossina/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Fenilefrina/farmacologia , RNA Mensageiro/genética , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Angiotensin-converting enzyme inhibitors (ACE-I) protect against the development of glomerulosclerosis using mechanisms partly dissociated from their systemic antihypertensive action. The aim of the current study was to delineate the mechanism of action underlying the antifibrotic effects of the ACE-I perindoprilat in the context of macrophage-mediated scarring in human mesangial cells. METHODS: Mesangial cells were treated with macrophage-conditioned medium (MPCM) in the presence or absence of the ACE-I perindoprilat. RESULTS: Forty micromol/L perindoprilat reduced MPCM-induced mesangial cell fibronectin levels by 19.4 +/- 0.6% (P < 0.001). Immunoprecipitation of 35S-methionine biosynthetically labeled fibronectin and Northern analysis suggested that the decrease in fibronectin levels was not caused by reduced synthesis. MPCM stimulated the production of matrix metalloproteinases (MMP) 2, 3, and 9 in mesangial cells; however, these were not significantly altered by ACE-I treatment, and neither was production of their tissue inhibitor of metalloproteinases (TIMP-1). Addition of exogenous bradykinin to MPCM-treated mesangial cells resulted in a 22.5 +/- 1.4% (P < 0.02) reduction in secreted fibronectin levels, while semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting demonstrated that bradykinin B2 receptor expression was up regulated by 71 +/- 30% in MPCM-stimulated mesangial cells in response to ACE-I treatment (P= 0.032). Moreover, the bradykinin B2 receptor antagonist HOE 140 attenuated the beneficial effects of perindoprilat. MPCM-stimulated mesangial cell protein expression levels of plasminogen activator system components tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were altered after treatment with ACE-I. CONCLUSION: These results suggest that ACE-I-induced renoprotection, in the context of macrophage-stimulated mesangial cell scarring, is mediated, at least in part, via the actions of bradykinin.