RESUMO
Hantavirus, a rodent-borne zoonotic pathogen, has a global distribution with 200,000 human infections diagnosed annually. In recent decades, repeated outbreaks of hantavirus infections have been reported in Eurasia and America. These outbreaks have led to public concern and an interest in understanding the underlying biological mechanisms. Here, we propose a climate-animal-Hantaan virus (HTNV) infection model to address this issue, using a unique dataset spanning a 54-y period (1960-2013). This dataset comes from Central China, a focal point for natural HTNV infection, and includes both field surveillance and an epidemiological record. We reveal that the 8-y cycle of HTNV outbreaks is driven by the confluence of the cyclic dynamics of striped field mouse (Apodemus agrarius) populations and climate variability, at both seasonal and interannual cycles. Two climatic variables play key roles in the ecology of the HTNV system: temperature and rainfall. These variables account for the dynamics in the host reservoir system and markedly affect both the rate of transmission and the potential risk of outbreaks. Our results suggest that outbreaks of HTNV infection occur only when climatic conditions are favorable for both rodent population growth and virus transmission. These findings improve our understanding of how climate drives the periodic reemergence of zoonotic disease outbreaks over long timescales.
Assuntos
Clima , Infecções por Hantavirus/epidemiologia , Interações Hospedeiro-Patógeno , Modelos Teóricos , Orthohantavírus/fisiologia , Roedores/virologia , Animais , China/epidemiologia , Reservatórios de Doenças , Vetores de Doenças , Humanos , Incidência , Densidade Demográfica , Chuva , Estações do Ano , TemperaturaRESUMO
NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.
Assuntos
Diferenciação Celular/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , NADH NADPH Oxirredutases/imunologia , NADPH Oxidases/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Diferenciação Celular/genética , Quimiocinas/genética , Quimiocinas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/enzimologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Monócitos/enzimologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
UNLABELLED: The cell fate determinant Numb is aberrantly expressed in cancer. Numb is alternatively spliced, with one isoform containing a long proline-rich region (PRR(L) ) compared to the other with a short PRR (PRR(S) ). Recently, PRR(L) was reported to enhance proliferation of breast and lung cancer cells. However, the importance of Numb alternative splicing in hepatocellular carcinoma (HCC) remains unexplored. We report here that Numb PRR(L) expression is increased in HCC and associated with early recurrence and reduced overall survival after surgery. In a panel of HCC cell lines, PRR(L) generally promotes and PRR(S) suppresses proliferation, migration, invasion, and colony formation. Knockdown of PRR(S) leads to increased Akt phosphorylation and c-Myc expression, and Akt inhibition or c-Myc silencing dampens the proliferative impact of Numb PRR(S) knockdown. In the cell models explored in this study, alternative splicing of Numb PRR isoforms is coordinately regulated by the splicing factor RNA-binding Fox domain containing 2 (RbFox2) and the kinase serine/arginine protein-specific kinase 2 (SRPK2). Knockdown of the former causes accumulation of PRR(L) , while SRPK2 knockdown causes accumulation of PRR(S) . The subcellular location of SRPK2 is regulated by the molecular chaperone heat shock protein 90, and heat shock protein 90 inhibition or knockdown phenocopies SRPK2 knockdown in promoting accumulation of Numb PRR(S) . Finally, HCC cell lines that predominantly express PRR(L) are differentially sensitive to heat shock protein 90 inhibition. CONCLUSION: Alternative splicing of Numb may provide a useful prognostic biomarker in HCC and is pharmacologically tractable.
Assuntos
Processamento Alternativo , Carcinoma Hepatocelular/genética , Diferenciação Celular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Humanos , Células Tumorais CultivadasRESUMO
UNLABELLED: Radiofrequency ablation (RFA) is considered a curative treatment option for hepatocellular carcinoma (HCC). Growing data have demonstrated that cryoablation represents a safe and effective alternative therapy for HCC, but no randomized controlled trial (RCT) has been reported to compare cryoablation with RFA in HCC treatment. The present study was a multicenter RCT aimed to compare the outcomes of percutaneous cryoablation with RFA for the treatment of HCC. In all, 360 patients with Child-Pugh class A or B cirrhosis and one or two HCC lesions ≤ 4 cm, treatment-naïve, without metastasis were randomly assigned to cryoablation (n = 180) or RFA (n = 180). The primary endpoints were local tumor progression at 3 years after treatment and safety. Local tumor progression rates at 1, 2, and 3 years were 3%, 7%, and 7% for cryoablation and 9%, 11%, and 11% for RFA, respectively (P = 0.043). For lesions >3 cm in diameter, the local tumor progression rate was significantly lower in the cryoablation group versus the RFA group (7.7% versus 18.2%, P = 0.041). The 1-, 3-, and 5-year overall survival rates were 97%, 67%, and 40% for cryoablation and 97%, 66%, and 38% for RFA, respectively (P = 0.747). The 1-, 3-, and 5-year tumor-free survival rates were 89%, 54%, and 35% in the cryoablation group and 84%, 50%, and 34% in the RFA group, respectively (P = 0.628). Multivariate analyses demonstrated that Child-Pugh class B and distant intrahepatic recurrence were significant negative predictors for overall survival. Major complications occurred in seven patients (3.9%) following cryoablation and in six patients (3.3%) following RFA (P = 0.776). CONCLUSION: Cryoablation resulted in a significantly lower local tumor progression than RFA, although both cryoablation and RFA were equally safe and effective, with similar 5-year survival rates.
Assuntos
Carcinoma Hepatocelular/cirurgia , Ablação por Cateter , Criocirurgia , Neoplasias Hepáticas/cirurgia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Criocirurgia/métodos , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: Epigenetic alterations are well documented in hepatocarcinogenesis. However, hypomethylation of long interspersed nuclear element 1(LINE-1) promoter and its relationship with clinicopathological features in hepatocellular carcinoma(HCC) remain unknown. METHODS: The bisulfite-specific PCR and DNA sequencing analysis was performed to assess the methylation status of LINE-1 promoter in a pilot cohort of 71 patients with HCC. Additionally,methylation levels of two hot CpG sites of LINE-1 promoter, site 7 and 18 were measured by real-time PCR and compared with clinicopathological parameters in a cohort of 172 HCC. All the patients included were in BCLC stage A or B. RESULTS: Most patients with HCC (87.3%) showed hypomethylation of LINE-1 promoter compared with HBV-related cirrhosis and normal controls (P < 0.001). The HCC patients with LINE-1 promoter hypomethylation had a median tumour-free survival (TFS) and overall survival (OS)post-resection of 22.0 (95% CI: 13.330.7) months and 35.0 (95% CI: 24.046.1) months, respectively, compared with 40 months and ~60 months for those with LINE-1 promoter hypermethylation (P < 0.05). Multivariate analyses showed that the hypomethylation level at CpG site 7 and 18 of LINE-1 promoter, along with tumour size and tumour differentiation, was independently associated with both TFS and OS for patients with HCC after resection. CONCLUSION: Promoter hypomethylation of LINE-1, especially at the CpG site 7 and 18, was associated with a poor prognosis in HCC.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirurgia , Metilação de DNA , Hepatectomia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirurgia , Elementos Nucleotídeos Longos e Dispersos/genética , Regiões Promotoras Genéticas , Adolescente , Adulto , Idoso , Sequência de Bases , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Diferenciação Celular , Distribuição de Qui-Quadrado , Ilhas de CpG , Intervalo Livre de Doença , Epigênese Genética , Feminino , Hepatectomia/efeitos adversos , Hepatectomia/mortalidade , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise Multivariada , Modelos de Riscos Proporcionais , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Análise de Sequência de DNA , Fatores de Tempo , Resultado do Tratamento , Carga Tumoral , Adulto JovemRESUMO
BACKGROUND: Cryoablation is one of the local therapies for hepatocellular carcinoma (HCC), but its safety and effect has not been studied in patients with Child class A or B and Barcelona Clinic Liver Cancer (BCLC) stage C HCC. Metastasis-associated in colon cancer-1 (MACC1) overexpression has been associated with poor prognosis of HCC, but its predictive value to post-cryoablation outcomes remains unknown in patients with BCLC stage C HCC. METHODS: This study assessed the safety and outcomes of cryoablation measured by time to progression (TTP) and overall survival (OS), and predictive value of MACC1 mRNA and protein overexpression in tumorous tissue to post-cryoablation outcomes in 120 advanced HCC patients with child-pugh class A or B by quantitative polymerase chain reaction and immunohistochemical staining. The potenial correlation of MACC1 and c-Met expression to tumor cell proliferation and apoptosis was also analyzed. RESULTS: The cryoablation in patients with advanced unresectable HCC resulted in a median TTP and OS of 5.5 (4.2- 6.7) months and 10.5 (9.0-12.0) months, respectively and no significant complications, comparable to the historical report for RFA therapy. The MACC1 mRNA and nuclear protein expression was significantly increased in tumorous tissues in these patients than that in normal liver tissue controls. Higher expression of MACC1 mRNA and nuclear protein in tumorous tissues in these patients was associated with shorter post cryoablation median TTP and OS than that with lower MACC1 expression. CONCLUSIONS: Cryoablation is a safe and effective therapeutic option for patients with advanced HCC and Child-pugh class A or B cirrhosis; and a higher intratumoral expression of MACC1 or nuclear translocation predicts poor outcomes of cryotherapy in these patients.
Assuntos
Carcinoma Hepatocelular/terapia , Criocirurgia , Neoplasias Hepáticas/terapia , Metástase Neoplásica , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Adulto , Idoso , Sequência de Bases , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Primers do DNA , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores , Fatores de Transcrição/genética , Resultado do TratamentoRESUMO
BACKGROUND: It is a challenging issue regarding the optimal antiviral treatment of children with chronic hepatitis B (CHB). The efficacy comparison of interferon (IFN) or nucleos(t)ide analogs (NAs) monotherapy with their combination could better understand this issue. METHODS: PubMed, EMBASE, Cochrane Library, Wanfang, CNKI, and abstracts of major international hepatology meetings were searched from inception to Feb 8, 2022. Randomized control trials and observational studies reporting the efficacy of combination therapy with IFN and NAs in children with CHB were eligible. RESULTS: A total of 17 studies were included. Compared with IFN monotherapy, combination therapy with IFN and NAs was significantly associated with increased rates of HBV DNA undetectable, HBeAg clearance, HBeAg seroconversion, alanine transaminase (ALT) normalization as well as the composite treatment response both at the end of treatment and during the follow-up period (RRs ranged from 1.23 to 1.75). A favorable trend for HBsAg seroconversion was found in IFN plus NAs-treated children, but not for the HBsAg clearance at the end of treatment. Although a similar trend towards the superiority of the combination therapy versus NAs monotherapy was observed (RRs ranged from 1.24 to 2.33) except for the HBV DNA undetectable rate at the end of treatment, the number of reported studies was limited. CONCLUSIONS: Combination therapy with IFN and NAs is more effective than IFN monotherapy in viral suppression and serological response for children with CHB. More studies were still needed to reveal the efficacy of this combination therapy compared with NAs monotherapy.
Assuntos
Hepatite B Crônica , Interferons , Humanos , Criança , Interferons/uso terapêutico , Nucleosídeos/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , DNA Viral , Resultado do Tratamento , Antivirais/uso terapêutico , Quimioterapia CombinadaRESUMO
BACKGROUND AND AIM: Gastric fundus perforation is a serious complication of endoscopic mucosal resection and endoscopic submucosal dissection performed for the removal of early gastric cancers or subepithelial tumors. The novel over-the-scope clip (OTSC) has recently been found to be effective for closing gastrointestinal-tract perforations and accesses for natural orifice transluminal endoscopic surgery. However, feasibility studies of OTSCs in gastric fundus perforation are still lacking. The aim of this study was therefore to demonstrate the feasibility of endoscopic closure of gastric fundus perforation using the OTSC system in a dog model. METHODS: Gastric fundus perforations were created by needle-knife electrocautery in seven dogs. The perforations were then closed using the OTSC clipping system. Stomach distension was maintained by maximum insufflation with air and methylene blue solution (500 mL) was instilled to submerge the closed perforation. Leaks were detected laparoscopically. RESULTS: Perforations were closed in all seven cases with a mean time of 18.5 ± 6.4 min (11-28 min). Twin Grasper assistance failed to release the OTSCs in two of the seven cases (2/7, 28.6%) because of difficulties associated with the J-maneuver (retroflexion of endoscope) required for the gastric fundus procedure, and OTCS were forced into place by suction. Minor leakage was observed in one case (1/7, 14.3%). No damages related to the clip system were found during postmortem examinations. CONCLUSIONS: Despite difficulties associated with the J-maneuver of the endoscope, this small series demonstrated that sufficient closure of gastric fundus perforation could be achieved using the OTSC system.
Assuntos
Fundo Gástrico/lesões , Fundo Gástrico/cirurgia , Gastroscopia/instrumentação , Técnicas de Fechamento de Ferimentos/instrumentação , Animais , Modelos Animais de Doenças , Cães , Estudos de Viabilidade , Feminino , Gastroscópios , Gastroscopia/efeitos adversos , Gastroscopia/métodos , Técnicas de Fechamento de Ferimentos/efeitos adversosRESUMO
We present the case of one 58-year-old man with advancd hepatocellular carcinoma and hepatitis-B virus-related liver cirrhosis who received hepatic cryoablation. Magnetic resonance imaging (MRI) showed multiple liver tumors and the diameter of the largest tumor was more than 10cm. The patient received 2 percutaneous cryoablations in December 2009 and January 2010. Ten months later, MRI showed that not only the treated areas underwent necrosis but also the non-treated area decreased. The a-fetoprotein (AFP) level and the frequency of circulated regulatory T cell (Treg) before treatment were 13,800ng/mL and 15.6%, respectively. Following the cryoablations they dropped to 436ng/mL and 7.6%, respectively, 10 months later. The patient remains in good condition until now.
Assuntos
Carcinoma Hepatocelular/cirurgia , Criocirurgia , Neoplasias Hepáticas/cirurgia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Contagem de Linfócitos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Necrose , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , alfa-Fetoproteínas/metabolismoRESUMO
OBJECTIVE: To clarify the expression and clinical significance of metastasis-associated in colon cancer 1 (MACC1) mRNA in hepatocellular carcinoma (HCC). METHODS: The expression and distribution of MACC1 were assessed by quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemical staining (IHC) in a cohort of hepatitis B virus-related HCC, including 138 in early (A), 96 in intermediate (B) and 120 in advanced stages (C). The association of MACC1 mRNA with disease progression and outcomes was analyzed by univariate and multivariate Cox analysis. RESULTS: The intratumoral expressions of MACC1 mRNA in HCC stage I (0.001 76, range: 0.000 54 - 0.002 47), stage II (0.002 49, range: 0.000 55 - 0.006 78) and stage III (0.008 35, range: 0.006 86 - 0.009 88) were about 3-, 4- and 14-fold higher than that in the normal liver tissue (0.000 59, range: 0.000 57 - 0.000 60), respectively. Intratumoral expression of MACC1 mRNA increased with disease progression from stage I to stage III. HCC clinical staging classification, age, portal vein invasion and tumor differentiation were significantly associated with intratumoral high expression of MACC1 mRNA (All P < 0.05). Immunohistochemical staining showed that there was an increased MACC1 expression in cytoplasm of HCC cells and positive nuclear staining in some cases. Increased MACC1 mRNA expression could predict poor outcome and recurrence in stage A and B HCC postoperatively. The median tumor-free survival and total survival of patients with high MACC1 mRNA expression were 34.0 and 40 months, respectively, significantly lower than that in those with low expression (48.0 and 48.0 months) (all P < 0.01). Cox analysis showed that Child-Pugh grading and high expression of MACC1 mRNA were independent predictive factors, and high expression of MACC1 was an independent predictive factor affecting the tumor-free survival. CONCLUSIONS: MACC1 mRNA up-regulation is a feature of disease progression in HCC. MACC1 mRNA expression in the HCC may become an independent predictive factor for recurrence and survival in postoperative HCC patients.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fatores de Transcrição/metabolismo , Adulto , Idoso , Carcinoma Hepatocelular/virologia , DNA Viral/análise , Intervalo Livre de Doença , Feminino , Seguimentos , Vírus da Hepatite B , Humanos , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Taxa de Sobrevida , Transativadores , Fatores de Transcrição/genética , Regulação para Cima , Adulto JovemRESUMO
Hepatitis B virus (HBV) may contribute to hepatocarcinogenesis by blocking p53 function. A p53 response element-like binding sequences, TGCCT...TGCCT, was found in HBV genome. To clarify whether HBV DNA can, like some other DNA viruses, bind to P53 protein and form a DNA-protein complex, we used a series of plasmids encoding full-length or mutant HBV or p53 fragments to determine the binding ability of HBV DNA after cotransfected into cells by electrophoretic mobility shift (and supershift) assay. We found that HBV DNA could bind to P53 protein and form DNA-protein complexes in human hepatoma cell lines. Cotransfection with p53 and HBV DNA increased the replication of HBV, CAT activity, tumor cell apoptosis, and cytoplasmic P53 accumulation in the hepatoma cells. In conclusions, our observations suggest that the interaction of HBV and p53 at the levels of protein-protein and DNA-protein, which resulted in inactivation of p53 transactivation.
Assuntos
Carcinoma Hepatocelular/virologia , Transformação Celular Viral , DNA Viral/metabolismo , Vírus da Hepatite B/metabolismo , Neoplasias Hepáticas/virologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA Viral/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Neoplasias Hepáticas/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/genética , Replicação ViralRESUMO
Apocynum venetum L., belonging to the family Apocynaceae, is a popular medicinal plant, which is commonly used in the treatment of hypertension, neurasthenia, and hepatitis in China. In the present study, the total flavonoids (TFs) were prepared from the leaves of A. venetum, and its protective effects on carbon tetrachloride (CCl4)-induced hepatotoxicity in a cultured HepG2 cell line and in mice were investigated. Cell exposed to 0.4% CCl4 (v/v) for 6 h led to a significant decrease in cell viability, increased LDH leakage, and intracellular reactive oxygen species (ROS). CCl4 also induced cell marked apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP). Pretreatment with TFs at concentrations of 25, 50, and 100 µg/mL effectively relieved CCl4-induced cellular damage in a dose-dependent manner. In vivo, TFs (100, 200, and 400 mg/kg BW) were administered via gavage daily for 14 days before CCl4 treatment. The high serum ALT and AST levels induced by CCl4 were dose-dependently suppressed by pretreatment of TFs (200 and 400 mg/kg BW). Histological analysis also supported the results obtained from serum assays. Furthermore, TFs could prevent CCl4-caused oxidative damage by decreasing the MDA formation and increasing antioxidant enzymes (CAT, SOD, GSH-Px) activities in liver tissues. In summary, both in vitro and in vivo data suggest that TFs, prepared from A. venetum, showed a remarkable hepatoprotective and antioxidant activity against CCl4-induced liver damage.
Assuntos
Apocynum/química , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Flavonoides/farmacologia , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Catalase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Glutationa Peroxidase/metabolismo , Células Hep G2 , Humanos , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Testes de Função Hepática , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Folhas de Planta/química , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully. rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interferon Tipo I/farmacologia , Fígado/metabolismo , Linhagem Celular Tumoral , DNA Complementar/química , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Proteínas Recombinantes , Esterol O-Aciltransferase/genética , Regulação para Cima , Esterol O-Aciltransferase 2RESUMO
Tumor necrosis factor (TNF) has a critical role in diverse cellular events including inflammation, apoptosis and necroptosis through different signaling complexes. However, little is known about how the transition from inflammatory signaling to the engagement of death pathways is modulated. Here we report that the cytoplasmic retinoic acid receptor gamma (RARγ) controls receptor-interacting protein kinase 1 (RIP1)-initiated cell death when cellular inhibitor of apoptosis (cIAP) activity is blocked. Through screening a short hairpin RNA library, we found that RARγ was essential for TNF-induced RIP1-initiated apoptosis and necroptosis. Our data suggests that RARγ initiates the formation of death signaling complexes by mediating RIP1 dissociation from TNF receptor 1. We demonstrate that RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes. In addition, we demonstrate that RARγ has a similar role in TNF-induced necroptosis in vivo. Thus, our study suggests that nuclear receptor RARγ provides a key checkpoint for the transition from life to death.The molecular switch between how tumour necrosis factor (TNF) controls inflammation versus cell death is less well defined. Here, the authors show that the nuclear receptor retinoic acid receptor gamma is released from the nucleus to disrupt TNF initiated cell death complexes in the cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do Ácido Retinoico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Citoproteção/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos Knockout , Modelos Biológicos , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
AIM: To investigate the inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma (HCC). METHODS: Recombinant sense and antisense p33(ING1b) plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33(ING1b) with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21(WAF1/CIP1). In addition, the expression and mutation rates of p33(ING1b) in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: Overexpression of p33(ING1b) inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33(ING1b) and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21(WAF1/CIP1). Immunostaining results showed co-localized P33(ING1b) with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33(ING1b) presented a low gene mutation rate (7.1%). CONCLUSION: p33(ING1b) collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33(ING1b) normal function may be an important mechanism for the development of HCC retaining wild-type p53.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Neoplasias Hepáticas/fisiopatologia , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Apoptose/fisiologia , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA , Fase G1 , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas/patologia , Proteínas Nucleares , Proteínas/metabolismo , Fase de Repouso do Ciclo Celular , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
AIM: To investigate the contribution of HBV in the development of hepatocarcinoma by examining the effects of HBV on p53 function in SMMU-7721 cell line. METHODS: Plasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild-type HBV) or PCMVHBVb (mutation type HBV) into the hepatoma cell line SMMU-7721 by lipofectamine. Apoptosis cells were labeled with annexin V-FITC and confirmed by flow cytometry. Reporter plasmid PG13-CAT or p21-luc was cotransfected, respectively, into each group to determine the transactivation activity of p53 and its effect on p21 promoter. Western blot was performed to observe p53 expression in hepatoma cell line of each group. RESULTS: The group transfected with pCMVp53 alone exhibited higher luciferase activity and higher apoptosis rate, otherwise, the p53 expression and reporter activity of PG13-CAT or P21-luc as well as cell apoptosis rate were obviously higher in the group cotransfected of pCMVp53 with pCMVHBVa, but not in the other cotransfected group. CONCLUSION: Transient transfection of HBV into the SMMU-7721 cell line can enhance p53 expression and its effects on development of hepatocarcinoma.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Hepatite B/complicações , Neoplasias Hepáticas/virologia , Proteína Supressora de Tumor p53/genética , Apoptose , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/fisiopatologia , Divisão Celular , Linhagem Celular Tumoral , Hepatite B/virologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/fisiopatologia , TransfecçãoRESUMO
OBJECTIVE: To explore the biological impact of 40 amino acid deletion at the C-terminal of hepatitis B virus X on the proliferation of hepatoma cells. METHODS: Cells of SMMC-7721 hepatoma cell line were transfected with HBx and its derivative HBx3'-40, harboring the 40 amino acid deletion at the distal C-terminal region. Cell growth curve, colony formation in soft agar plate and tumorigenesis assay in nude mice were used to observe the alterations induced by the transfection of HBx and HBx3'-40. The expression level of PCNA in tumor cells was also investigated. RESULTS: The growth rates of the cells transfected with HBx and HBx3'-40 were markedly increased as compared with that of the control group. The colony formation rates were enhanced in the cells transfected with HBx(48.7 +/- 8.1) and HBx3'-40 (82.8+/-6.0), comparing with the control (26.9 +/- 3.5) %. In the tumorigenic assay, the size and weight of tumors were significantly increased in the cells transfected with HBx (0.412 +/- 0.212, 0.395 +/- 0.159) % and HBx3'-40 (1.476 +/- 0.232, 0.987 +/- 0.279) %, as compared with the control group (0.051 +/- 0.024, 0.033 +/-0.004) %. The expression level of PCNA in tumors was increased in both HBx (59.00 +/- 2.58) % and HBx3'-40 (69.25 +/- 3.77) % transfected cells, comparing with the control (37.67 +/- 2.52) %. Overall, the cells transfected with HBx3'-40 demonstrated the highest proliferative capacity. CONCLUSION: The deletion of 40 amino acids in the C-terminal of HBx is correlated with an enhanced proliferation of hepatoma cells and may play an important role in the malignant transformation of the liver.
Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Neoplasias Hepáticas/patologia , Deleção de Sequência , Transativadores/genética , Sequência de Aminoácidos , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
OBJECTIVE: To investigate the effect of hepatitis C virus (HCV) non-structure protein NS5A on the activity of calcium-regulating protein alpha subunit of nascent polypeptide-associated complex (NACA) promoter. METHODS: HepG2 cell plasmid pCAT3-NACA, containing NACA promoter, was transfected alone or cotransfected with pcDNA3.1(-)-NS5A, and chloramphenicol acetyl transferase (CAT) enzyme activity was assayed by enzyme-linked immunoassay (ELISA). RESULTS: The CAT activity in the pcDNA3.1(-)-NS5A cotransfection group was 20.7% of the CAT activity in the pCAT3-NACA group. CONCLUSION: HCV non-structural protein NS5A has a down-regulating effect on the promoter of NACA gene.
Assuntos
Chaperonas Moleculares/biossíntese , Regiões Promotoras Genéticas/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/farmacologia , Sequência de Bases , Regulação para Baixo , Humanos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Ativação Transcricional , TransfecçãoRESUMO
OBJECTIVE: To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display. METHODS: PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis. RESULTS: Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP). CONCLUSION: Many proteins with different functions could bind with interferon alpha promoter.
Assuntos
DNA Complementar/genética , Proteínas de Ligação a DNA/biossíntese , Hepatócitos/metabolismo , Interferon-alfa/biossíntese , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Hepatócitos/citologia , Humanos , Interferon-alfa/genética , Regiões Promotoras Genéticas/genéticaRESUMO
MicroRNA-153 (miR-153) is considered to be a tumor regulator. Silencing of miR-153 expression induced apoptosis in breast cancer cells. Data on mechanism suggest that up-regulation of miR- 153 level promotes cell proliferation via the down regulation of the expression of PTEN or FOXO1, which attenuates the proliferation of cancerous cells. This study aims to identify the effect of miR-153 on the activity of chemotherapeutic and targeted agents in HCC cells and to investigate the mechanisms involved. MTT, soft agar, trans-well and flow cytometry assays were performed to examine whether miR-153 down-regulated the activity of the chemotherapeutic and targeted drugs, Sorafenib, Etoposide and Paclitaxel in HCC cells. The rate of proliferation inhibition, relative survival rates and IC50 values of each drug were calculated. Western blot and luciferase assays were performed to assess whether miR-153 modulates the expression of important genes related to cell proliferation, apoptosis or survival. Results showed that miR-153 attenuated the effect of Etoposide, Paclitaxel and Sorafenib on HepG2 cells; the IC50 value increased from 0.25±0.01µmol/L to 1.02±0.14µmol/L, 0.05±0.01µmol/L to 0.14±0.02µmol/L and from 1.09±0.15µmol/L to 5.18±0.99µmol/L, respectively. In addition, miR-153 also reduced the effect of these drugs on MHCC- 97H, MHCC-97 L and L-02 cells; and it also reduced the effects of Sorafenib, Etoposide and Paclitaxel on anchor-independent growth of HepG2 cells. Over-expression of miR-153 down-regulated the activity of Etoposide and Paclitaxel on cell cycle arrest of HepG2 cells and the effect of Sorafenib on the invasion and migration of HepG2 cells. Furthermore, overexpression of miR-153 also enhanced the growth of HepG2, MHCC-97H, MHCC-97 L and L-02 cells. Mechanisms data showed that overexpression of miR-153 down regulated the activity of luciferase reporters, p15-Luc and p21-Luc; and enhanced the protein level of pro-survival or anti-apoptosis proteins Survivin and BCL-2. These results show that overexpression of miR-153 protects HepG2 cells against the effects of these drugs via multiple mechanisms, and miR-153 may be a novel target for HCC in future diagnostic and therapeutic interventions.