RESUMO
Introduction and aim. Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor. It is primarily caused by hepatic cirrhosis or chronic viral hepatitis. Hepatic carcinogenesis is associated with increased oxidative stress. Thus, the aim of our study was to assess expression of the genes involved in the homeostasis of oxidative stress in patients with HCC. MATERIAL AND METHODS: The study was performed on 32 patients with primary HCC (verified by liver histology in 29 patients) and 27 control subjects (in 11 subjects, liver histology was available either with no or minimal changes in the liver tissue). Gene expressions of heme oxygenase 1 (HMOX1), biliverdin reductase A/B (BLVRA/B), NADPH oxidase 2 (NOX2) and p22phox were analyzed in the liver and peripheral blood leukocytes (PBL) in the subjects. RESULTS: Compared to controls, almost a 3 times higher mRNA level of BLVRA was detected in livers of HCC patients (p = 0.002); while those of BLVRB as well as HMOX1 were unchanged (p > 0.05). In accord with these results in the liver tissue, BLVRA mRNA levels in PBL were also significantly increased in HCC patients (p = 0.012). mRNA levels of NOX2 and p22phox in the liver tissue, although higher in HCC patients, did not differ significantly compared to control subjects (p > 0.05). Nevertheless, NOX2 mRNA level in PBL was significantly higher in HCC patients (p = 0.003). CONCLUSIONS: BLVRA mRNA levels in the liver as well as in PBL are significantly higher in HCC patients most likely as a feedback mechanism to control increased oxidative stress associated with HCC progression.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , RNA Mensageiro/genética , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , NADPH Oxidase 2 , NADPH Oxidases/genética , Estresse Oxidativo/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/sangue , Transdução de Sinais , Regulação para CimaRESUMO
Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 gâ¢L-1 [S. platensis], 60 µM [PCB], and 125 µM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.
Assuntos
Antineoplásicos/farmacologia , Bilirrubina/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Extratos Vegetais/farmacologia , Spirulina , Tetrapirróis/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Nus , Oxirredução , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Heme oxygenase-1 (HMOX1) and bilirubin UDP-glucuronosyltransferase (UGT1A1), both enzymes involved in bilirubin homeostasis, play an important role in oxidative stress defense. OBJECTIVE: To assess the effect of promoter variations of HMOX1 and UGT1A1 genes on the progression of fibrosis in patients chronically infected with the hepatitis C virus (HCV). MATERIAL AND METHODS: The study was performed on146 chronic HCV infection patients, plus 146 age- and sex-matched healthy subjects. The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis in all subjects. RESULTS: No differences were found in the frequencies of each particular allele of both genes, between HCV patients and a control group (p > 0.05). Furthermore, no association was detected (p > 0.05) between either the HMOX1 or the UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. Finally, no differences in the HMOX1 and UGT1A1 genotype prevalence rates were found between pre-cirrhotic and cirrhotic patients (p > 0.05). CONCLUSION: Based on our data, microsatellite variations in the HMOX1 and UGT1A1 genes are not likely to protect from progression of liver disease in patients with chronic hepatitis C.
Assuntos
Variação Genética , Glucuronosiltransferase/genética , Heme Oxigenase-1/genética , Hepatite C Crônica/genética , Cirrose Hepática/genética , Fígado/enzimologia , Regiões Promotoras Genéticas , Biópsia , Estudos de Casos e Controles , República Tcheca , Frequência do Gene , Predisposição Genética para Doença , Hepacivirus/genética , Hepatite C Crônica/enzimologia , Hepatite C Crônica/patologia , Humanos , Fígado/patologia , Fígado/virologia , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Repetições de Microssatélites , Fenótipo , RNA Viral/sangue , Carga ViralRESUMO
The patatin-like phospholipase domain containing 3 (PNPLA3) gene (viz. its I148M variant) is one of the key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We have identified a novel insertion/deletion variant of 1114 bp, localized in the second intron of the PNPLA3 gene, which corresponds to the 3' terminal sequence of the long-interspersed element (LINE-1). DNA analysis of 122 NAFLD patients and 167 control subjects as well as RNA analysis of 19 liver biopsies revealed that the novel variant is very common (frequency = 0.41), fully linked to the clinically important I148M variant, and clinically silent. Although the LINE-1 insertion does not seem to have any biological effect, it can impede genotyping of the I148M variant. If insertion prevents the attachment of the diagnostic primer, then the non-insertion allele will be selectively amplified; and thus the frequency of the 148M "risk" allele will be significantly overestimated due to the complete linkage of the LINE-1 insertion and the 148I allele of the PNPLA3 gene. Therefore, our findings underline the importance of careful design and consistent documentation of the methodology, including primer sequences. Critical revisions of the results of some studies that have already been reported may therefore be needed.
Assuntos
Aciltransferases/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fosfolipases A2 Independentes de Cálcio/genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Predisposição Genética para Doença/genética , Genótipo , Humanos , Fígado/patologiaRESUMO
INTRODUCTION: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare autosomal recessive cholestatic liver disorder caused by genetic deficiency of ATP-binding cassette subfamily B member 4 (ABCB4), a hepatocanalicular floppase translocating phospholipids from the inner to the outer leaflet of the canalicular membrane lipid bilayer. PFIC3 is characterised by production of hydrophilic bile with lithogenic properties which is harmful to the hepatobiliary epithelia. Chronic cholestasis in some patients may be accompanied by excessive accumulation of copper in the liver and by increased urinary copper excretion, the findings mimicking Wilson disease (WD). METHODS AND RESULTS: We report an 11 y/o male patient with growth retardation, mild craniofacial dysmorphic features and chronic liver disease, initially diagnosed and treated as WD. Whereas genetic testing for WD was negative, further molecular and histopathological analysis revealed two novel mutations (c.833+1G>T and c.1798T>A) in ABCB4 and complete absence of the ABCB4/MDR3 protein in the liver, determining PFIC3 as the correct diagnosis. CONCLUSION: PFIC3 and WD display pleomorphic and sometimes overlapping clinical and laboratory features, which may pose a differential diagnostic problem. Since the patient management in WD and PFIC3 differs significantly, an early and accurate diagnosis is crucial for optimising of therapeutic approach and prevention of possible complications.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Colestase Intra-Hepática/diagnóstico , Degeneração Hepatolenticular/diagnóstico , Criança , Diagnóstico Diferencial , Humanos , MasculinoRESUMO
Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10-125 µmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables.
Assuntos
Antineoplásicos/farmacologia , Clorofila/farmacologia , Neoplasias Pancreáticas/metabolismo , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Feofitinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Superóxidos/metabolismo , Synechocystis/químicaAssuntos
Acetilcisteína/uso terapêutico , Falência Hepática/etiologia , Falência Hepática/cirurgia , Protoporfiria Eritropoética/diagnóstico , Adulto , Éxons , Ferroquelatase/genética , Humanos , Falência Hepática/tratamento farmacológico , Transplante de Fígado , Masculino , Mutação , Polimorfismo de Nucleotídeo Único , Protoporfiria Eritropoética/tratamento farmacológicoRESUMO
BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is associated with systemic oxidative stress. Since the heme catabolic pathway plays an important role in antioxidant protection, we attempted to assess the gene expression of key enzymes of heme catabolism, heme oxygenase 1 (HMOX1), heme oxygenase 2 (HMOX2), and biliverdin reductase A (BLVRA) in the liver and peripheral blood leukocytes (PBL) of patients chronically infected with HCV. METHODS: Gene expressions (HMOX1, HMOX2, BLVRA) and HCV RNA were analyzed in PBL of HCV treatment naïve patients (nâ=â58) and controls (nâ=â55), with a subset of HCV patients having data on hepatic gene expression (nâ=â35). Based upon the therapeutic outcome, HCV patients were classified as either responders (nâ=â38) or treatment-failure patients (nâ=â20). Blood samples in HCV patients were collected at day 0, and week 12, 24, 36, and 48 after the initiation of standard antiviral therapy. RESULTS: Compared to the controls, substantially increased BLVRA expression was detected in PBL (p<0.001) of therapeutically naïve HCV patients. mRNA levels of BLVRA in PBL closely correlated with those in liver tissue (r2â=â0.347,pâ=â0.03). A marked difference in BLVRA expression in PBL between the sustained responders and patients with treatment failure was detected at week 0 and during the follow-up (p<0.001). Multivariate analysis revealed that BLVRA basal expression in PBL was an independent predictor for sustained virological response (OR 15; 95% CI 1.05-214.2; Pâ=â0.046). HMOX1/2 expression did not have any effect on the treatment outcome. CONCLUSION: Our results suggest that patients with chronic HCV infection significantly upregulate BLVRA expression in PBL. The lack of BLVRA overexpression is associated with non-responsiveness to standard antiviral therapy; whereas, HMOX1/2 does not seem to have any predictive potential.
Assuntos
Expressão Gênica , Hepacivirus , Hepatite C/genética , Leucócitos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Adulto , Antivirais/uso terapêutico , Feminino , Heme/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Leucócitos/virologia , Fígado/metabolismo , Fígado/virologia , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Curva ROC , Resultado do TratamentoRESUMO
Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.
Assuntos
Aterosclerose/prevenção & controle , Heme Oxigenase-1/genética , Ficobilinas/administração & dosagem , Ficocianina/administração & dosagem , Spirulina/química , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Feminino , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: The cause of Rotor syndrome (RS), a rare-familial conjugated hyperbilirubinaemia with normal liver histology, is unclear. We hypothesized that RS can be an allelic variant of Dubin-Johnson syndrome, caused by mutation in ABCC2, and investigated ABCC2 (gene) and ABCC2 (protein) in two patients with RS. METHODS: A 57-year-old male presented with a 5-year history of predominantly conjugated hyperbilirubinaemia (170 micromol/l). Urinary porphyrin excretion was increased; cholescintigraphy revealed no chromoexcretion. A 68-year-old male presented with lifelong conjugated hyperbilirubinaemia (85 micromol/l). Bromosulfophthalein elimination was typical for RS. Both patients had histologically normal liver, without pigment. ABCC2 expression was investigated by confocal fluorescence microscopy. ABCC2 was sequenced from genomic DNA and cDNA, and exon deletions/duplications were sought by comparative genomic hybridization on a custom micro-array. RESULTS: In both patients, ABCC2 was expressed unremarkably at the apical membrane of hepatocytes and no sequence alterations were found in 32 exons, adjacent intronic regions and the promoter region of ABCC2. CONCLUSIONS: Rotor-type hyperbilirubinaemia is not an allelic variant of ABCC2 deficiency.
Assuntos
Hiperbilirrubinemia Hereditária/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Idoso , Análise Mutacional de DNA , Saúde da Família , Humanos , Hiperbilirrubinemia Hereditária/diagnóstico , Hiperbilirrubinemia Hereditária/etiologia , Icterícia Idiopática Crônica , Fígado/patologia , Masculino , Proteínas de Membrana Transportadoras/análise , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Sulfobromoftaleína/análiseRESUMO
BACKGROUND & AIMS: Dubin-Johnson syndrome is recessively inherited, conjugated hyperbilirubinemia induced by mutations in the ABCC2/MRP2 gene encoding the canalicular transporter for conjugated bilirubin. Gilbert's syndrome is recessively inherited, unconjugated hyperbilirubinemia caused by decreased conjugation rate of bilirubin associated mostly with homozygous A(TA) 7 TAA variant of the TATAA-box in the UGT1A1 gene promoter. Our aim was to establish the molecular diagnosis in a 3-year-old male with atypical, intermittent, predominantly unconjugated, hyperbilirubinemia. METHODS: 99m Tc-HIDA cholescintigraphy was used for imaging the biliary tree. Expression of ABCC2/MRP2 protein in hepatocytes was investigated immunohistochemically. UGT1A1 and ABCC2/MRP2 genes were sequenced from genomic DNA, and the mutations were verified by fragment analysis, sequencing the cloned exons, and restriction fragment length polymorphism. RESULTS: Cholescintigraphy revealed delayed visualization of the gallbladder. A brown granular lipopigment differing from melanin-like pigment reported in Dubin-Johnson syndrome was present in hepatocytes, but, otherwise, liver histology was normal. ABCC2/MRP2 protein was not detected on the canalicular membrane of hepatocytes, and 2 novel mutations were found in the ABCC2/MRP2 gene: a heterozygous in-frame insertion-deletion mutation 1256insCT/delAAACAGTGAACCTGATG in exon 10 inherited from the father and a heterozygous deletion 4292delCA in exon 30 inherited from the mother. In addition, the patient was homozygous for -3279T>G and A(TA) 7 TAA mutations in the UGT1A1 gene promoter. CONCLUSIONS: Our patient represents a case of digenic mixed hyperbilirubinemia-a distinct type of constitutive jaundice resulting from coinherited defects in ABCC2/MRP2 and UGT1A1 genes.