RESUMO
BACKGROUND: The effect of Porphyromonas gingivalis (Pg) infection on oesophageal squamous cell carcinoma (ESCC) prognosis, chemotherapeutic efficacy, and oesophageal cancer cell apoptosis resistance and proliferation remain poorly understood. METHODS: Clinicopathological data from 312 ESCC oesophagectomy patients, along with the computed tomography imaging results and longitudinal cancerous tissue samples from a patient subset (n = 85) who received neoadjuvant chemotherapy (NACT), were analysed. Comparison of overall survival and response rate to NACT between Pg-infected and Pg-uninfected patients was made by multivariate Cox analysis and Response Evaluation Criteria in Solid Tumours v.1.1 criteria. The influence of Pg on cell proliferation and drug-induced apoptosis was examined in ESCC patients and validated in vitro and in vivo. RESULTS: The 5-year overall survival was lower in Pg-positive patients, and infection was associated with multiple clinicopathological factors and pathologic tumour, node, metastasis stage. Of the 85 patients who received NACT, Pg infection was associated with a lower response rate and 5-year overall survival. Infection with Pg resulted in apoptosis resistance in ESCC and promoted ESCC cell viability, which was confirmed in longitudinal cancerous tissue samples. Pg-induced apoptosis resistance was dependent on fimbriae and STAT3. CONCLUSIONS: Pg infection is associated with a worse ESCC prognosis, reduced chemotherapy efficacy, and can potentiate the aggressive behaviour of ESCC cells.
Assuntos
Infecções por Bacteroidaceae/epidemiologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Porphyromonas gingivalis/patogenicidade , Animais , Infecções por Bacteroidaceae/mortalidade , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Neoplasias Esofágicas/microbiologia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/microbiologia , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Masculino , Camundongos , Terapia Neoadjuvante , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Homeostasis between pro- and anti- inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, pro-inflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen Porphyromonas gingivalis enhances the activity of Janus kinase 3 (JAK3) in innate immune cells, and subsequently phospho-inactivates Nedd4-2, an ubiquitin E3 ligase. In turn, Wingless-INT (Wnt) 3 (Wnt3) ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in pro-inflammatory cytokine levels. In contrast, JAK3 or Wnt3a inhibition robustly enhances nuclear factor kappa-light-chain-enhancer of activated B cells activity and the production of pro-inflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gain- and loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and ß-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P. gingivalis-infected gingival tissues, and increases disease severity. Together, our results reveal a new anti-inflammatory role for JAK3 in innate immune cells and show that the underlying signaling pathway involves Nedd4-2-mediated Wnt3a ubiquitination.
Assuntos
Infecções por Bacteroidaceae/complicações , Reabsorção Óssea/prevenção & controle , Inflamação/prevenção & controle , Janus Quinase 3/metabolismo , Doenças Periodontais/prevenção & controle , Substâncias Protetoras , Proteína Wnt3A/metabolismo , Animais , Infecções por Bacteroidaceae/microbiologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Janus Quinase 3/genética , Camundongos , Camundongos Endogâmicos C57BL , Doenças Periodontais/etiologia , Doenças Periodontais/metabolismo , Doenças Periodontais/patologia , Porphyromonas gingivalis/patogenicidade , Transdução de Sinais , Proteína Wnt3A/genéticaRESUMO
BACKGROUND: While Syk has been shown to associate with TLR4, the immune consequences of Syk-TLR interactions and related molecular mechanisms are unclear. METHODS: Gain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting. RESULTS: Syk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNß and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3ß. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNß and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr-Akt) or constitutively active GSK3ß (GSK3 S9A). Moreover, neither inhibition of PI3K-Akt signaling nor neutralization of de novo synthesized IFNß could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKß and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses. CONCLUSIONS: Syk negatively regulates TLR4-mediated production of IFNß and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K-Akt and NF-κB signaling pathways. GENERAL SIGNIFICANCE: Syk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.
Assuntos
Células Dendríticas/imunologia , Inflamação/etiologia , Interferon beta/metabolismo , Interferon gama/biossíntese , Interleucina-10/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Células Cultivadas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição STAT/metabolismo , Quinase Syk , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although GSK3ß has been reported to have contrasting effects on the progression of different tumors, it's possible functions in esophageal squamous cell carcinoma (ESCC) and the related molecular mechanisms remain unknown. Here, we investigated the expression, function, and molecular mechanism of GSK3ß in the development of ESCC in vitro and in vivo. Though the expression of total GSK3ß was significantly increased, the phosphorylated (inactivated) form of GSK3ß (Ser9) was concurrently decreased in the cancerous tissues of patients with ESCC compared with controls, suggesting that GSK3ß activity was enhanced in cancerous tissues. Further pathological data analysis revealed that higher GSK3ß expression was associated with poorer differentiation, higher metastasis rates, and worse prognosis of ESCC. These results were confirmed in different ESCC cell lines using a pharmacological inhibitor and specific siRNA to block GSK3ß. Using a cancer phospho-antibody array, we found that STAT3 is a target of GSK3ß. GSK3 inhibition reduced STAT3 phosphorylation, and overexpression of constitutively active GSK3ß had the opposite effect. Moreover, STAT3 inhibition mimicked the effects of GSK3ß inhibition on ESCC cell migration and viability, while overexpression of a plasmid encoding mutant STAT3 (Y705F) abrogated these effects, and these results were further substantiated by clinicopathological data. In addition, a GSK3 inhibitor (LiCl) and/or STAT3 inhibitor (WP-1066) efficiently suppressed the growth of ESCC cells in a xenograft tumor model. Altogether, these results reveal that higher GSK3ß expression promotes ESCC progression through STAT3 in vitro and in vivo, and GSK3ß-STAT3 signaling could be a potential therapeutic target for ESCC treatment.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cloreto de Lítio/farmacologia , Masculino , Camundongos , Fosforilação , Prognóstico , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Tirfostinas/farmacologiaRESUMO
Serum- and glucocorticoid-regulated kinase (SGK)1 is associated with several important pathologic conditions and plays a modulatory role in adaptive immune responses. However, the involvement and functional role of SGK1 in innate immune responses remain entirely unknown. In this study, we establish that SGK1 is a novel and potent negative regulator of TLR-induced inflammation. Pharmacologic inhibition of SGK1 or suppression by small interfering RNA enhances proinflammatory cytokine (TNF, IL-12, and IL-6) production in TLR-engaged monocytes, a result confirmed in Cre-loxP-mediated SGK1-deficient cells. SGK1 inhibition or gene deficiency results in increased phosphorylation of IKK, IκBα, and NF-κB p65 in LPS-stimulated cells. Enhanced NF-κB p65 DNA binding also occurs upon SGK1 inhibition. The subsequent enhancement of proinflammatory cytokines is dependent on the phosphorylation of TGF-ß-activated kinase 1 (TAK1), as confirmed by TAK1 gene silencing. In vivo relevance was established in a murine endotoxin model, in which we found that SGK1 inhibition aggravates the severity of multiple organ damage and enhances the inflammatory response by heightening both proinflammatory cytokine levels and neutrophil infiltration. These findings have identified an anti-inflammatory function of SGK1, elucidated the underlying intracellular mechanisms, and establish, for the first time, that SGK1 holds potential as a novel target for intervention in the control of inflammatory diseases.
Assuntos
Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Lipopolissacarídeos/toxicidade , Insuficiência de Múltiplos Órgãos/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Toll-Like/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas Imediatamente Precoces/genética , Inflamação/induzido quimicamente , Inflamação/enzimologia , Inflamação/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Insuficiência de Múltiplos Órgãos/induzido quimicamente , Insuficiência de Múltiplos Órgãos/genética , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Receptores Toll-Like/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
Pathogen-induced reactive oxygen species (ROS) play a crucial role in host innate immune responses through regulating the quality and quantity of inflammatory mediators. However, the underlying molecular mechanisms of this effect have yet to be clarified. In this study, we examined the mechanism of action of ROS stimulated by Porphyromonas gingivalis in gingival epithelial cells. P. gingivalis induced the rapid production of ROS, which lead to the phosphorylation of JAK2 and increased levels of secreted proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß. Neutralization of ROS by N-acetyl-l-cysteine (NAC) abrogated the phosphorylation of JAK2 and suppressed the production of IL-6 and IL-1ß. ROS-mediated phosphorylation of JAK2 induced the phosphoactivation of c-Jun amino-terminal protein kinase (JNK) and the downstream transcriptional regulator c-Jun. Inhibition of JAK2, either pharmacologically or by small interfering RNA (siRNA), reduced both the phosphorylation of these molecules and the production of proinflammatory cytokines in response to P. gingivalis. Furthermore, pharmacological inhibition or siRNA-mediated gene silencing of JNK or c-Jun mimicked the effect of JAK2 inhibition to suppress P. gingivalis-induced IL-6 and IL-1ß levels. The results show that ROS-mediated activation of JAK2 is required for P. gingivalis-induced inflammatory cytokine production and that the JNK/c-Jun signaling axis is involved in the ROS-dependent regulation of IL-1ß and IL-6 production.
Assuntos
Células Epiteliais/imunologia , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Janus Quinase 2/metabolismo , Porphyromonas gingivalis/imunologia , Espécies Reativas de Oxigênio/toxicidade , Células Cultivadas , Células Epiteliais/microbiologia , Humanos , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Apple (Malus pumila Mill.) is one of the important economic crops in the arid areas of Xinjiang, China. For a long time, there has been a problem of high consumption but low yield in water and fertilizer management, prevent improvements in apple quality and yield. In this study, 5-year-old 'Royal Gala' apple trees in extremely arid areas of Xinjiang were used as experimental materials to carry out field experiments. considering 5 irrigation levels (W1, 30 mm; W2, 425 mm; W3, 550 mm; W4, 675 mm; W5, 800 mm) and 5 fertilization levels (F1, 280 kg·ha-1; F2, 360 kg·ha-1; F3, 440 kg·ha-1; F4, 520 kg·ha-1; F5, 600 kg·ha-1) under magnetoelectric water irrigation conditions. The results demonstrated that magnetoelectric water combined with the application of 675 mm irrigation amount and 520 kg·ha-1 fertilization amount was the most effective combination. These results occurred by increasing net photosynthetic rate of apple leaves, improved the quality of apples, increased apple yield, and promoted the improvement of water and fertilizer use efficiency. Additionally, the quadratic regression model was used to fit the response process of yield, IWUE and PFP to irrigation amount and fertilization amount, and the accuracy was greater than 0.8, indicating good fitting effects. The synergistic effect of water and fertilizer has a positive effect on optimizing apple water and fertilizer management. Principal component analysis showed that the magnetoelectric treatment combined water and fertilizer mainly affected apple yield, water and fertilizer use efficiency and vitamin C content related to quality. This study provides valuable guidance for improving water and fertilizer productivity, crop yield and quality in extreme arid areas of Xinjiang by using Magnetoelectric water irrigation.
RESUMO
Beyond the traditional use of ceria as an abrasive, the scope of nanoceria applications now extends into fuel cell manufacturing, diesel fuel additives, and for therapeutic intervention as a putative antioxidant. However, the biological effects of nanoceria exposure have yet to be fully defined, which gave us the impetus to examine its systemic biodistribution and biological responses. An extensively characterized nanoceria (5 nm) dispersion was vascularly infused into rats, which were terminated 1 h, 20 h or 30 days later. Light and electron microscopic tissue characterization was conducted and hepatic oxidative stress parameters determined. We observed acute ceria nanoparticle sequestration by Kupffer cells with subsequent bioretention in parenchymal cells as well. The internalized ceria nanoparticles appeared as spherical agglomerates of varying dimension without specific organelle penetration. In hepatocytes, the agglomerated nanoceria frequently localized to the plasma membrane facing bile canaliculi. Hepatic stellate cells also sequestered nanoceria. Within the sinusoids, sustained nanoceria bioretention was associated with granuloma formations comprised of Kupffer cells and intermingling CD3⺠T cells. A statistically significant elevation of serum aspartate aminotransferase (AST) level was seen at 1 and 20 h, but subsided by 30 days after ceria administration. Further, elevated apoptosis was observed on day 30. These findings, together with increased hepatic protein carbonyl levels on day 30, indicate ceria-induced hepatic injury and oxidative stress, respectively. Such observations suggest a single vascular infusion of nanoceria can lead to persistent hepatic retention of particles with possible implications for occupational and therapeutic exposures.
Assuntos
Cério/toxicidade , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Catalase/metabolismo , Cério/química , Glutationa Redutase/metabolismo , Granuloma/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase (Desciclizante)/análise , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The rise in infections caused by antibiotic-resistant bacteria is outpacing the development of new antibiotics. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of clinically important bacteria that have developed resistance to multiple antibiotics and are commonly referred to as multidrug resistant (MDR). The medical and research communities have recognized that, without new antimicrobials, infections by MDR bacteria will soon become a leading cause of morbidity and death. Therefore, there is an ever-growing need to expedite the development of novel antimicrobials to combat these infections. Toward this end, we set out to refine an existing mouse model of pulmonary Pseudomonas aeruginosa infection to generate a robust preclinical tool that can be used to rapidly and accurately predict novel antimicrobial efficacy. This refinement was achieved by characterizing the virulence of a panel of genetically diverse MDR P. aeruginosa strains in this model, by both 50% lethal dose (LD50) analysis and natural history studies. Further, we defined two antibiotic regimens (aztreonam and amikacin) that can be used as comparators during the future evaluation of novel antimicrobials, and we confirmed that the model can effectively differentiate between successful and unsuccessful treatments, as predicted by in vitro inhibitory data. This validated model represents an important tool in our arsenal to develop new therapies to combat MDR P. aeruginosa strains, with the ability to provide rapid preclinical evaluation of novel antimicrobials and support data from clinical studies during the investigational drug development process. IMPORTANCE The prevalence of antibiotic resistance among bacterial pathogens is a growing problem that necessitates the development of new antibiotics. Preclinical animal models are important tools to facilitate and speed the development of novel antimicrobials. Successful outcomes in animal models not only justify progression of new drugs into human clinical trials but also can support FDA decisions if clinical trial sizes are small due to a small population of infections with specific drug-resistant strains. However, in both cases the preclinical animal model needs to be well characterized and provide robust and reproducible data. Toward this goal, we have refined an existing mouse model to better predict the efficacy of novel antibiotics. This improved model provides an important tool to better predict the clinical success of new antibiotics.
Assuntos
Amicacina , Pseudomonas aeruginosa , Camundongos , Humanos , Animais , Amicacina/farmacologia , Aztreonam/farmacologia , Testes de Sensibilidade Microbiana , Drogas em Investigação/farmacologia , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , BactériasRESUMO
BACKGROUND: Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. METHODS: Gain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. RESULTS: LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces ß-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3ß, and ß-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. CONCLUSIONS: TLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3ß-ß-catenin signaling and NF-κB activity. GENERAL SIGNIFICANCE: Wnt3a-Dvl3-ß-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses.
Assuntos
Proteínas Desgrenhadas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamação/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animais , Citocinas/metabolismo , Endotoxinas/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Insuficiência de Múltiplos Órgãos/metabolismo , Fosforilação/fisiologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the effect of Ginkgo biloba extracts (EGb761) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis and its antioxidant activity in Wistar rats. METHODS: 71 Wistar rats were randomly divided into three groups: AFB1 (group A); AFB1 +EGb761 (group B), Control (group C). Rats in gurop A and B were injected with AFB, through abdomen and the doses were 100-200 microg/kg, one to three times a week. Liver biopsy were performed in all rats during 14th w, 28th w, 42th w and 55th w, and were executed at 64th w. Gammaglutamyl transpeptidase-positive hyperplastic cell foci (gamma-GT foci) and histopathology of the liver tissue were observed. The levels of malondialdehyde (MDA), as well as the activity of Glutathione peroxidase (GSH-Px) was examined. RESULTS: At 42th w and 55th w, the gamma-GT focus area (mm2/focus) and general area of foci (mm2/cm2) of group B were significantly smaller than that in group A (P = 0.000). The incidence of hepatocelluiar carcinoma (HCC) in group B (26.92%) was significantly lower than that in group A(76%) (P = 0.000). Group C didnt have HCC development. EGb 761 markedly increased GSH-Px activity, reduced MDA levels (P < 0.05). CONCLUSION: EGB761 shows effective inhibition to hepatocarcinogenesis induced by AFB1 in rats, which may be related to its antioxidant activity.
Assuntos
Antioxidantes/farmacologia , Ginkgo biloba/química , Neoplasias Hepáticas Experimentais/patologia , Extratos Vegetais/farmacologia , Aflatoxina B1/toxicidade , Animais , Glutationa Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Malondialdeído/metabolismo , Folhas de Planta/química , Plantas Medicinais/química , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
In order to explore the proteins responsible for hepatocellular carcinoma (HCC), aflatoxin B(1)-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) was analyzed with 2-DE and MS. By comparing HCC samples with their own precancerous biopsies and HCC-surrounding tissues, a group of candidate proteins that differentially expressed in HCC were obtained. Peroxiredoxin (Prx) II, one of the candidates with distinct alteration, was further investigated and validated. Western blot and RT-PCR assays confirmed the overexpression of Prx II in both tree shrew and human HCC tissues. RNA interference for silencing Prx II was employed subsequently to explore the function and underlying mechanism of Prx II on liver cancer cell line Hep3B. Results showed the cell proliferation and clone formation decreased obviously when Prx II expression was inhibited, while the flow cytometer analysis showed the percentage of cell apoptosis enhanced. Inhibition of Prx II expression also obviously increased the generation of ROS and malondialdehyde, both are the products from peroxidation. These results imply the important role of Prx II in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby to provide a favorable microenvironment for cancer cell surviving and progressing.
Assuntos
Aflatoxina B1 , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Peroxirredoxinas/isolamento & purificação , Adulto , Idoso , Animais , Eletroforese em Gel Bidimensional , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Peroxirredoxinas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TupaiaRESUMO
OBJECTIVE: To evaluate the mRNA and protein expressions of peroxiredoxin II (PrxII) in hepatocellular carcinoma (HCC) and their significance. METHODS: HCC was induced by aflatoxin B1 (AFB1) in 6 tree shrews (Tupaia belangeri chinensis). The expression levels of PrxII mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot on HCC tissues and on their surrounding liver tissues (para-HCC). Biopsied liver tissues were taken before the HCC induction (pre-HCC) from the same animals and from a group of blank controlled animals that served as controls. Liver biopsy specimens from 18 cases of human HCC and from 17 healthy human volunteers were studied using the same methods. RESULTS: The mRNA and protein expressions of PrxII in tree shrew HCC tissues were significantly higher than those in para-HCC and pre-HCC tissues, and also higher than those in the liver tissues from the control animals (all P < 0.05). The expression levels of PrxII mRNA and protein in human HCC tissues were also significantly higher than those in their para-HCC tissues and in the human normal liver tissues (P < 0.05). CONCLUSION: PrxII might play an important role in hepatocarcinogenesis and might be used as a molecular target for HCC prevention and treatment.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Peroxirredoxinas/genética , Adulto , Idoso , Animais , Carcinoma Hepatocelular/patologia , Feminino , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/patologia , Masculino , Pessoa de Meia-Idade , TupaiidaeRESUMO
AIM: To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma (HCC), the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS: Tree shrew HCC was induced with aflatoxin B1 (AFB1), and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30th-, 60th-, and 90th-wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique. RESULTS: The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION: Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.
Assuntos
Apoptose/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Aflatoxina B1/toxicidade , Animais , Expressão Gênica , Genes bcl-2 , Genes p53 , Neoplasias Hepáticas Experimentais/induzido quimicamente , Proteínas Associadas aos Microtúbulos/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tupaiidae , Proteína X Associada a bcl-2RESUMO
Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival.
Assuntos
Adenoviridae/genética , Ciclina E/genética , Vetores Genéticos/genética , Neoplasias/genética , Vírus Oncolíticos/genética , Regiões Promotoras Genéticas , Animais , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genes Reporter , Humanos , Camundongos , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: The purpose of this study was to observe the effect of fasting and feeding on (18)F-FDG uptake in a mouse model of human non-small cell lung cancer. METHODS: In in vivo studies, (18)F-FDG small-animal PET scans were acquired in 5 mice bearing non-small cell lung cancer A549 xenografts on each flank with continuous feeding and after overnight fasting to observe the changes in intratumoral distribution of (18)F-FDG and tumor (18)F-FDG standardized uptake value (SUV). In ex vivo studies, intratumoral spatial (18)F-FDG distribution assessed by autoradiography was compared with the tumor microenvironment (including hypoxia by pimonidazole and stroma by hematoxylin and eosin stain). Five overnight-fasted mice and 5 fed mice with A549 tumors were observed. RESULTS: Small-animal PET scans were obtained in fed animals on day 1 and in the same animals after overnight fasting; the lapse was approximately 14 h. Blood glucose concentration after overnight fasting was not different from fed mice (P = 0.42), but body weight loss was significant after overnight fasting (P = 0.001). Intratumoral distribution of (18)F-FDG was highly heterogeneous in all tumors examined, and change in spatial intratumoral distribution of (18)F-FDG between 2 sets of PET images from the same mouse was remarkably different in all mice. Tumor (18)F-FDG mean SUV and maximum SUV were not significantly different between fed and fasted animals (all P > 0.05, n = 10). Only tumor mean SUV weakly correlated with blood glucose concentration (R(2) = 0.17, P = 0.03). In ex vivo studies, in fasted mice, there was spatial colocalization between high levels of (18)F-FDG uptake and pimonidazole-binding hypoxic cancer cells; in contrast, pimonidazole-negative normoxic cancer cells and noncancerous stroma were associated with low (18)F-FDG uptake. However, high (18)F-FDG uptake was frequently observed in noncancerous stroma of tumors but rarely in viable cancer cells of the tumors in fed animals. CONCLUSION: Host dietary status may play a key role in intratumoral distribution of (18)F-FDG. In the fed animals, (18)F-FDG accumulated predominantly in noncancerous stroma in the tumors, that is, reverse Warburg effect. In contrast, in fasted status, (18)F-FDG uptake was found in hypoxic cancer cells component (Pasteur effect). Our findings may provide a better understanding of competing cancer glucose metabolism hypotheses: the Warburg effect, reverse Warburg effect, and Pasteur effect.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Fluordesoxiglucose F18 , Glucose/metabolismo , Neoplasias Pulmonares/diagnóstico por imagem , Animais , Glicemia/análise , Glicemia/metabolismo , Peso Corporal , Linhagem Celular Tumoral , Dieta , Feminino , Humanos , Hipóxia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Nitroimidazóis/química , Projetos Piloto , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Microambiente TumoralRESUMO
AIM: To investigate p53 mutation and p21 expression in hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B(1) (AFB(1)) in tree shrews, and to reveal the role of these genes in hepatocarcinogenesis. METHODS: Tree shrews were divided into four groups: group A, those infected with HBV and fed with AFB(1) (n = 39); group B, those infected with HBV alone (n = 28); group C, those fed with AFB(1) alone (n = 29); and group D, normal controls (n = 20). The tree shrews underwent liver biopsies once every 15 wk. Expression of p53 and p21 proteins and genes in the biopsies and tumor tissues of the experimental tree shrews was detected, respectively, by immunohistochemistry, and by Southern blotting and reverse transcription-polymerase chain reaction and sequencing. RESULTS: The incidence of hepatocellular carcinomas (HCC) was higher in group A (66.7%) than that in group B (3.57%) and C (30%). The time of HCC occurrence was also earlier in group A than that in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, respectively, P<0.01). p53 protein was not detected by immunohistochemistry in all groups before the 75(th) wk of the experiment. At the 105(th) wk, the positive rates fo p53 were 78.6%, 60% and 71.4% in groups A, B and C, respectively, which were significantly higher than that in group D (10%) (all P<0.05). An abnormal band of p53 gene was observed in groups A and C. The mutation points of p53 gene in tree shrews with HCC were at codons 275, 78 and 13. The nucleotide sequence and amino acid sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homologies with those of human p53, respectively. The immunopositivity for p21 was found before HCC development. The incidence of HCC was significantly higher in tree shrews that were positive for p21 than those negative for p21 (80.0% vs 11.0%, P<0.001). The incidence of HCC in p21 positive animals in group A was significantly higher than those positive for p21 in group C (P<0.05). CONCLUSION: A remarkable synergistic effect on HCC development exists between HBV and AFB(1). p53 mutation promotes the development of HCC. HBV and AFB(1) may synergistically induce p53 gene mutation, and stimulate ras gene expression. ras gene is activated at the earlier stage during hepatocarcinogenesis. p21 protein may be an early marker, and the alterations of p53 may be a late event in the development of HCC.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Modelos Animais de Doenças , Neoplasias Hepáticas/fisiopatologia , Proteína Oncogênica p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Tupaiidae , Animais , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Hepatite B/complicações , Incidência , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologiaRESUMO
OBJECTIVE: To detect the expression and variation of the p53 gene in hepatocarcinogenesis of tree shrews induced by hepatitis B virus (HBV) and aflatoxin B1 (AFB1). METHODS: Tree shrews were divided into four groups: group A, infected with HBV and fed with AFB1; group B, only infected with HBV; group C, fed with AFB1 alone; and group D normal control. The tree shrews underwent liver biopsy every 15 weeks. Liver and tumor tissues were detected by immunohistochemistry and molecular biotechnologies. RESULTS: The incidence of hepatocellular carcinoma (HCC) was higher in group A (66.7%) than in groups B (0) and C (30%). HCC occurrence was earlier in group A than in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, t=3.336, P<0.01). Mutated p53 protein was not found in all groups before 75 weeks of experiment. At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60.0% and 71.4% in groups A, B and C respectively, which were significantly higher than that in group D (10%) (chi2> or =5.03, P<0.05). An abnormal band of the p53 gene was detected in groups A and C. The mutational points of the p53 gene in liver cancer of tree shrews were at codon 275, 78 and 13. Nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 were 91.7% and 93.4% in homology compared with those of human p53, respectively. CONCLUSIONS: Remarkable synergistic effect on HCC exists between HBV and AFB1. Mutated p53 protein expressed before occurrence of HCC promotes the development of HCC. HBV and AFB1 may synergistically induce p53 gene mutation.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Variação Genética , Neoplasias Hepáticas Experimentais/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Distribuição de Qui-Quadrado , Cocarcinogênese , Modelos Animais de Doenças , Feminino , Masculino , Dados de Sequência Molecular , Probabilidade , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , TupaiidaeRESUMO
OBJECTIVE: To detect the expression and variation of p53 gene during tree shrews' hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B1 (AFB1). METHODS: Tree shrews were divided into four groups: the tree shrews were infected with HBV and fed with AFB1 in group A, only infected with HBV in group B, fed with AFB1 alone in group C, and normal control in group D. All the tree shrews were performed liver biopsy every 15 weeks. The tissues of liver and tumor were detected by immunohistochemistry and molecular biotechnologies. RESULTS: (1) The incidence of hepatocellular carcinoma (HCC) in group A (66.7%) was higher than that in Group B and C (30%). HCC appearance in group A was earlier than that in group C (120.0 weeks +/-16.6 weeks vs 153.3 weeks +/-5.8 weeks, t = 3.336, P<0.01). (2) Mutated p53 protein was not found before the 75th week of the experiment in each group. (3) At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60% and 71.4% in group A, B and C respectively, which were much higher than that (10%) in group D (x2 > or = 5.03, P<0.05). An abnormal band of p53 gene was detected in both group A and C. (4) The mutation points of p53 gene in liver cancer of tree shrew were at codon 275, 78 and 13. The nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homology with those of human p53 respectively. CONCLUSIONS: There is a remarkable synergistic effect between HBV and AFB1 on HCC. Mutated p53 protein is expressed before HCC occurrence, which promotes the development and progress of HCC. HBV and AFB1 may synergistically induce p53 gene mutation.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas Experimentais/genética , Mutação Puntual , Proteína Supressora de Tumor p53/genética , Aflatoxina B1/toxicidade , Animais , Cocarcinogênese , Regulação Neoplásica da Expressão Gênica , Variação Genética , Hepatite B/virologia , Vírus da Hepatite B , RNA Neoplásico/análise , TupaiidaeRESUMO
The objective of this study was to determine whether (18)F-misonidazole could detect hypoxia in macroscopic and microscopic tumors in mice. In nude mice, subcutaneous xenografts and peritoneal metastases were generated utilizing human non-small cell lung cancer A549 and HTB177 cells. Animals were co-injected with (18)F-misonidazole, pimonidazole and bromodeoxyuridine, and tumor perfusion was assessed by Hoechst 33342 injection. The intratumoral distribution of (18)F-misonidazole was determined by micro-PET scan and autoradiography. Pimonidazole, bromodeoxyuridine and Hoechst 33342 were detected by immunohistochemistry on the autoradiography sections. Submillimeter micrometastases found to be severely hypoxic. In both peritoneal metastases and subcutaneous xenografts models, PET images displayed significant (18)F-misonidazole uptake, and its distribution was non-uniform in these macroscopic subcutaneous tumors. In frozen sections, digital autoradiography and immunohistochemistry revealed similar distributions of (18)F-misonidazole, pimonidazole and glucose transporter-1, in both microscopic and macroscopic tumors. Bromodeoxyuridine stained-positive proliferative regions were well perfused, as judged by Hoechst 33342, and displayed low (18)F-misonidazole accumulation. (18)F-misonidazole uptake was low in tumor stroma and necrotic zones as well. Microscopic non-small cell lung cancer metastases are severely hypoxic. (18)F-misonidazole PET is capable to image hypoxia noninvasively not only in macroscopic tumors but also in micrometastases growing in mice. Accordingly, (18)F-misonidazole may be a promising agent to detect the burden of micrometastatic diseases.